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T T H H È È S S E E En vue de l'obtention du DOCTORAT DE LUNIVERSITÉ DE TOULOUSE Délivré par l'Université Toulouse III - Paul Sabatier Discipline ou spécialité : Gènes Cellules et Développement JURY Pr. Kerstin BYSTRICKY : Directeur de thèse Dr. Saadi KHOCHBIN : Rapporteur Dr. Eve DEVINOY : Rapporteur Dr. Laurent LACROIX : Examinateur Ecole doctorale : Biologie, Santé, Biotechnologies (BSB) Unité de recherche : UMR 5099, CNRS-LBME Directeur(s) de Thèse : Kerstin BYSTRICKY Rapporteurs : Présentée et soutenue par Luca Bellucci Le 12 mars 2013 Titre : The role of histone variant H2A.Z in the regulation of gene expression in breast cancer cells.

Transcript of THÈSE - Paul Sabatierthesesups.ups-tlse.fr/1939/1/2013TOU30011.pdfFigure 2 . Schematic...

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TTHHÈÈSSEE

En vue de l'obtention du

DDOOCCTTOORRAATT DDEE LL’’UUNNIIVVEERRSSIITTÉÉ DDEE TTOOUULLOOUUSSEE

Délivré par l'Université Toulouse III - Paul Sabatier Discipline ou spécialité : Gènes Cellules et Développement

JURY Pr. Kerstin BYSTRICKY : Directeur de thèse

Dr. Saadi KHOCHBIN : Rapporteur Dr. Eve DEVINOY : Rapporteur

Dr. Laurent LACROIX : Examinateur

Ecole doctorale : Biologie, Santé, Biotechnologies (BSB) Unité de recherche : UMR 5099, CNRS-LBME

Directeur(s) de Thèse : Kerstin BYSTRICKY

Rapporteurs :

Présentée et soutenue par Luca Bellucci Le 12 mars 2013

Titre : The role of histone variant H2A.Z in the regulation of gene expression in breast cancer

cells.

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Mots clefs : Epigénétique, H2A.Z, p21, HDACi, Cancer du sein, Acétylation

Résumé travaux.

Pendant mes trois ans de thèse, j’ai étudié les mécanismes de régulation génique

impliqués dans le cancer du sein. Je me suis intéressé en particulier à la régulation

épigénétique du gène p21 dans les cellules cancéreuses mammaires hormono-

indépendantes, ER�-négatives (MDA-MB231).Nous avons démontré que l’activation du

gène p21 dans ces cellules était p53 indépendante, stimulée en utilisant des inhibiteurs

des histones deacétylases et contrôlée par l’acétylation du variant d’histone H2A.Z. Mes

études ont permis la rédaction et publication d’un article (Bellucci et al., 2013 [1]).

Selon les cellules utilisées, l’expression du gène p21 peut toutefois être p53 dépendante

ou indépendante et le mécanisme d’activation du gène p21, via les HDACi, reste peu

compris. Par ailleurs, il a été démontré, dans autres systèmes cellulaires, que la

régulation de p21 dépendait de la localisation du variant d’histone H2A.Z. Nous avons

montré que dans les cellules MDA-MB231 (p53- négatives), H2A.Z est lié fortement au

site d’initiation de la transcription du gène p21. Un traitement à la Trichostatin A (TSA), un

inhibiteur des histone-deacétylases, augmente l’acétylation de H2A.Z favorisant ainsi

l’expression de p21. A l’opposé, une cellule déplétée en H2A.Z ne répond plus aux

HDACi. Nous avons démontré que la présence du variant d’histone H2A.Z dans sa forme

acétylé était importante pour l’expression de p21.

En utilisant comme gène modèle celui de la Cycline D1, nous avons montré que

l’acétylation de H2A.Z joue un rôle fondamental dans la régulation de la transcription en

présence et en absence du récepteur aux œstrogènes. De plus, nous avons identifié des

enzymes impliqués dans l’acétylation de H2A.Z (Tip60) et dans le remodelage de la

chromatine (Tip48), nous permettant de proposer un modèle pour l’activation de la

transcription de ce gène.

J’ai ainsi participé aux travaux décrits dans deux autres articles (Dalvai et al., 2012 [2] ;

Dalvai et al., 2013 [3]).

[1] Luca Bellucci*, Mathieu Dalvai*, Silvia Kocanova, Fatima Moutahir, and Kerstin

Bystricky. (2013) “Activation of p21 by HDAC inhibitors requires acetylation of

H2A.Z.” PLOS One doi: 10.1371/journal.pone.0054102.

[2] Dalvai M, Bellucci L, Fleury L, Lavigne AC, Moutahir F, Bystricky K. (2012) “H2A.Z-

dependent crosstalk between enhancer and promoter regulates Cyclin D1

expression.” Oncogene doi: 10.1038/onc.2012.442

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[3] Mathieu Dalvai, Laurence Fleury, Luca Bellucci, Silvia Kocanova and Kerstin

Bystricky. (2013) “TIP48/reptin and H2A.Z requirement for initiating chromatin

remodeling in estrogen activated transcription.”

PLOS genetics (in press).

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Key words: Epigenetics, H2A.Z, p21, HDACi, Breast cancer, Acetylation

Work summary.

During my PhD, I studied the mechanisms of regulation of gene expression in breast

cancer. In particular, I was interested in the epigenetic regulation of p21 gene expression

in an estrogen receptor negative breast cancer cells (MDA-MB231 cells). We found that in

these cells a TSA treatment stimulated p21 expression and increased acetylation of

H2A.Z present at the p21 promoter. H2A.Z was strongly associated with the transcription

start site of p21. Moreover, depleting the cellular pool of H2A.Z compromised p21

activation and response to HDACi. Acetylation of H2A.Z rather than its association of

regulatory element per se was important for p21 expression. My studies led to a

publication (Bellucci et al., 2013 [1]) which shows that p21 gene activation in MDA-MB231

cells is p53-independent and controlled by the H2A.Z histone variant and its acetylation.

Normally, activation of p21 expression can be p53-independent or dependent, according

to the cell system used. But the mechanism behind p21 activation, via HDACi, remains

poorly understood. Moreover, p21 regulation depends on the binding of the histone variant

H2A.Z.

Using the Cyclin D1 gene as a model, I also participated in a project, which shows how, in

this case too, H2A.Z acetylation is critical for gene regulation in estrogen receptor positive

and negative breast cancer cells. We identified the enzymes involved in H2A.Z acetylation

(Tip60) and in chromatin remodeling (Tip48), to propose a model for transcription

activation of this gene. Results have been described in two other original publications

(Dalvai et al., 2012 [2]; Dalvai et al., 2013 [2]).

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[1] Luca Bellucci*, Mathieu Dalvai*, Silvia Kocanova, Fatima Moutahir, and Kerstin

Bystricky. (2013) “Activation of p21 by HDAC inhibitors requires acetylation of

H2A.Z.” PLOS One doi: 10.1371/journal.pone.0054102.

[2] Dalvai M, Bellucci L, Fleury L, Lavigne AC, Moutahir F, Bystricky K. (2012) “H2A.Z-

dependent crosstalk between enhancer and promoter regulates Cyclin D1

expression.” Oncogene doi: 10.1038/onc.2012.442

[3] Mathieu Dalvai, Laurence Fleury, Luca Bellucci, Silvia Kocanova and Kerstin

Bystricky. (2013) “TIP48/reptin and H2A.Z requirement for initiating chromatin

remodeling in estrogen activated transcription.”

PLOS genetics (in press).

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I.� Epigenetics and regulation of gene expression. ........................ 7�

A.� Chromatin structure. ..................................................................................... 7�

B.� DNA methylation .......................................................................................... 9�

1.� DNA Methyl Transferases ................................................................................... 9�

2.� Hydroxymethylation. ...........................................................................................11�

C.� Remodeling complexes .............................................................................. 11�

D.� Histone modifications. ................................................................................ 14�

1.� Histone methylation. ...........................................................................................15�

2.� Histone phosphorylation. ....................................................................................17�

3.� Histone acetylation. ............................................................................................17�

4.� Other histone modifications. ...............................................................................19�

5.� Histone modification networks. ...........................................................................20�

E.� Histone variants. ......................................................................................... 21�

1.� Histone H3 family. ..............................................................................................22�

2.� Histone H2A family. ............................................................................................23�

3.� Histone H2B family. ............................................................................................24�

4.� Histones H1........................................................................................................25�

II.� Histone H2A.Z ............................................................................. 26�

A.� H2A.Z localization in the genome. .............................................................. 27�

B.� H2A.Z deposition. ....................................................................................... 28�

C.� H2A.Z on gene promoters. ......................................................................... 30�

D.� H2A.Z and regulation of gene expression. ................................................. 32�

E.� H2A.Z acetylation. ...................................................................................... 33�

III.�HDAC inhibitors. ......................................................................... 34�

A.� Inhibitors for HDAC class I/II. ..................................................................... 37�

B.� Inhibitors for class III HDACs...................................................................... 37�

IV.�Estrogen-dependent gene regulation. ...................................... 38�

V.� Breast cancer. ............................................................................. 43�

A.� Breast cancer treatments. .......................................................................... 44�

B.� Biomarkers. ................................................................................................ 44�

VI.�P21 ............................................................................................... 46�

A.� The p21 promoter and HDACi activation of p21. ........................................ 47�

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VII.� Thesis project. ......................................................................... 49�

VIII.� Results. ................................................................................. 51�

IX.�Supplementary data. ................................................................ 101�

A.� Materials and Methods. ............................................................................ 104�

X.� Discussion and outlook. .......................................................... 105�

References ..................................................................................... 110�

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I. Epigenetics and regulation of gene expression.

Epigenetics relate to heritable modifications and changes in chromatin composition, which

occur without DNA sequence modifications. Every cell in an organism has the same

genome, the same genotype, but at the same time, each cell (or group of cells) maintains

unique phenotypical characteristics and biological functions. Different states of chromatin

compaction, conformation and modifications ensure the plasticity of cellular and tissue

specific gene expression. They are fundamental during differentiation and development of

an organism and can be heritable from one generation to the next.

Epigenetic mechanisms play an important role in diseases related to lifestyle, early life

experience and environmental exposure to toxins. In fact, they are responsible for the

integration of environmental cues at the cellular level. For these reasons, epigenetics

have acquired a therapeutic relevance in multiple diseases such as inflammation,

metabolic diseases and cancer (Arrowsmith, Bountra et al. 2012).

Since the first data obtained by Barbara McClintock in the 1940s-1960s, in parallel to the

work of Brink (Mc 1950; Brink 1958; Brink 1958; Brink 1958; Brink 1958; McClintock

1958), the research about epigenetics has greatly progressed. Looking for an explanation

for non-Mendelian gene regulation in the past 20 years, many mechanisms, pathways,

actors, complexes and substrates of epigenetics have been uncovered (Wolffe and

Matzke 1999).

The targets of epigenetic regulation are DNA and, especially, nucleosomes. Nucleosomes

are the basic unit of chromatin and are substrates for the “deposition” of the epigenetic

marks which, once “read” by specific complexes charged to “interpret” these marks, can

modulate the transcriptional activity (Egger, Liang et al. 2004; Hake, Xiao et al. 2004).

Before developing epigenetics and its implication in gene regulation, I will describe

chromatin and its multiple facets.

A. Chromatin structure.

The mechanisms of epigenetic regulation rely on the structure and the composition of

chromatin. Genomic DNA in eukaryotic cells ranging from yeast to humans is packaged

into chromatin via DNA –protein interactions. Depending on the compaction state of

chromatin, two different states of chromatin, heterochromatin and euchromatin, have been

described. The heterochromatic state represents a compacted and “closed” form of the

genetic material. It is observable during the Barr body formation, at centromeres and is, in

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general, correlated to gene silencing. Heterochromatic compaction is achieved by different

mechanisms such as DNA methylation, histone methylation and other modifications. On

the opposite, euchromatin is the “open” conformation of the chromatin. Also due to histone

modifications (acetylation for example), euchromatin supports gene transcription. In fact,

this low compacted chromatin conformation combined with distinct histone modifications

allows DNA to be more accessible to the transcriptional machinery.

DNA is packaged and organized inside the nuclear volume in eucaryotes. The repeating

unit of chromatin organization is the nucleosome. The nucleosome is an assembly of 147

base pairs of DNA turned two times around a histone octamer composed of two copies

each of the histones H2A, H2B, H3 and H4 (Fig.1). Numerous amino acids of the N-

terminal tail of each histone are available for modification. Indeed, post-translational

modifications of histones are the marks to preserve the balance between hetero- and

euchromatin, and thus between silencing and transcription (Saha, Wittmeyer et al. 2006;

Luger, Dechassa et al. 2012).

Figure 1. Levels of chromatin compaction between double helix DNA and the metaphasic chromosome.

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B. DNA methylation

DNA methylation is important during development and adult life, and plays a central role in

gene expression, parental genomic imprinting or X chromosome inactivation. DNA

methylation patterns are established very early during embryonic development. Such

epigenetic modifications characterize extended areas of transcriptionally silent chromatin.

The four bases of DNA can be methylated, yet only cytosine usually is in eukaryotic cells.

To perform methylation, a methyl group must be exchanged between S-adenosyl

methionine (SAM) and the cytosine carbon 5 (Figure 2). This reaction is done by an

enzyme called DNA Methyl Transferase, or DNMT. Methylated cytosines are preferentially

found in CpG dinucleotides. The CpG dinucleotides do not have a uniform distribution:

series of CpGs are found in short regions of the genome, called “CpG islands”, located on

gene promoters and/or in the first exon of about 60% of human genes (Bird 2002).

Figure 2 . Schematic representation of DNA methylation.

1. DNA Methyl Transferases

There are two classes of DNMTs. The first class, DNMT1 in mammalian cells (Yen,

Vertino et al. 1992), is involved in the maintenance of methylation patterns during

replication. DNMT1 recognizes hemi-methylated DNA and ensures that the same

methylation pattern is conserved between mother and daughter cells.The second class of

enzymes comprises the mammalian DNMT3a and DNMT3b proteins. These proteins are

involved in establishing new methylation patterns during early embryonic development

(Okano, Takebayashi et al. 1999). The deletion of one of these three DNMTs is lethal for

the organism (Okano, Bell et al. 1999).

This important modification has a strong impact not only on DNA conformation but also on

the DNA-protein interactions. In fact, changing DNA structure changes most proteins’

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affinity for DNA, as for example histones and transcription factors. CpG islands are

recognized by a 70 amino acid domain found in some proteins, called MBD (methyl-CpG

Binding Domain). This domain is characteristic of a protein family, including for example

MeCP2, MBD1, MBD2, MBD3 and MBD4. Not all of these proteins bind methylated DNA

(Filion and Defossez 2004). Some authors even describe a DNA-demethylase activity for

MBD2, but this point is not very clear and is still under discussion. On the other hand, we

have the example of Kaiso, a transcription factor, which, although it has no MBD, is able

to bind methylated CpG islands. Another characteristic of these proteins is their capacity

to recruit other enzymes, in particular histone methyltransferases. These enzymes are for

example implicated in lysine 9 methylation on histone H3 (Fuks, Hurd et al. 2003; Bowen,

Palmer et al. 2004). These mechanisms synergize to repress transcription. This

cooperation is the basis of the maintenance of the X chromosome inactivation for example

(Heard, Rougeulle et al. 2001).

Transcription can also be repressed via the interaction between DNMT1 and HDACs

(histone deacetylases) which act synergistically (Dobosy and Selker 2001; Robertson

2002), (Rountree, Bachman et al. 2000). MeCP2 is also implicated in the recruitment, on

DNA methylated promoters, of a corepressor histone methyltransferase complex (mSin3)

and histone deacetylases (HDAC1) (Figure 3).

Figure 3. Crosstalk between DNA methylation and histone deacetylation.

This repression is relieved, in vivo, by the deacetylase inhibitor Trichostatin A (see page

34 for more information), indicating that deacetylation of histones (and/or of other

proteins) is an essential actor of this repression mechanism. So, two global mechanisms

of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2

(Jones, Thomas et al. 1998; Nan, Ng et al. 1998; Fuks, Hurd et al. 2003).

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2. Hydroxymethylation.

In addition, another DNA modification has recently been described, 5-

hydroxymethylcytosine (5-hmC). This modification is catalyzed by the ten-eleven

translocation (TET) family of DNA hydroxylases. 5-hmC, interestingly, seems to be

associated with gene activation. In fact, recent studies in embryonic stem (ES) cells

revealed that (i) 5-hmC is enriched in gene bodies where it correlates with gene

expression levels, (ii) 5-hmC is associated with low gene expression when present at the

transcription start site (TSS) and (iii) 5-hmC is found at binding sites for pluripotency

transcription factors in undifferentiated ES cells. Therefore, Tet enzymes could be

implicated in the initiation of active DNA demethylation. In particular, some links between

cytosine hydroxymethylation and cancer have been demonstrated. 5-hmC levels are

greatly reduced in most cultured, immortalized tumor cells (Haffner, Chaux et al. 2011;

Yang, Liu et al. 2012). TET mutational inactivation has been reported to be associated

with decreased 5-hmC levels in various myeloid leukemias (Delhommeau, Dupont et al.

2009). Recently, Lian and collaborators have shown that “loss of 5-hmC” is an epigenetic

hallmark of melanoma. Genome-wide mapping reveals loss of the 5-hmC landscape in the

melanoma epigenome (Lian, Xu et al. 2012). In conclusion, DNA methylation, nucleosome

remodeling and several histone N-terminal tail modifications cooperate to create active or

inactive chromatin, which is more or less accessible to the transcriptional machinery.

C. Remodeling complexes

To change between an open, active and a closed, inactive chromatin state remodeling

complexes are required. These complexes are responsible for changes in nucleosome

deposition and chromatin structure in order to facilitate or hinder access to DNA.

Therefore, gene expression, DNA repair, replication and other biological processes also

depend on the modulation and recruitment of these complexes.

Chromatin remodeling complexes use ATP to control nucleosome positioning. They are

able to evict nucleosomes and are involved in exchanging canonical core histones with

histone variants (Clapier and Cairns 2009). The ability to reposition nucleosomes play an

important role in regulating gene expression as well as mediating access to DNA during

replication and repair (Hartley and Madhani 2009; Jiang and Pugh 2009; Segal and

Widom 2009). Remodeling complexes typically contain an ATPase domain and can be

associated with different accessory proteins (Figure 4). The ATPase subunits of these

enzymes belong to the SNF2 superfamily of helicase-related proteins and contain a

common core of two RecA helicase domains. These couple ATP hydrolysis to protein

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conformational changes. In addition to the helicase domain, proteins of a given family

share other characteristic domains that define their biological function (Figure 4) (Flaus,

Martin et al. 2006). SWI/SNF members contain a bromodomain, ISWI members contain a

HAND-SANT-SLIDE domain, and CHD members contain a double chromodomain.

ATPase of the INO80 group are characterized by a “split” helicase, which has a long

insertion separating the DExx and HELICc domain (Flaus, Martin et al. 2006). Using

ATPase domains and/or associated subunits, these complexes create specific interactions

with their modified or unmodified nucleosomal substrate (Figure 4). Each domain interacts

and recognizes specific nucleosome modifications: Bromodomain (BRD/bromo) for

acetylated histones, chromodomain (CHD/chromo) for methylated histones, the plant

homeodomain (PHD) for unmodified/acetylated/methylated histone tails and globular

domain of histones, and finally, HAND/SANT/SLIDE domains recognize nucleosomes and

nucleosomal DNA. Moreover, these complexes can read DNA sequences, structure or

methylation, recognize histone modifications, detect the presence of histone variants and

can interact with chromatin-associated proteins such as transcription factors to identify

specific target nucleosomes in the nucleus. An additional level of regulation is the

alternative splicing of remodeling complex components that result in different isoforms

with distinct properties. Therefore, these complexes contain DNA-binding motifs that are

present in the ATPase or in accessory subunits (Figure 4) (Fyodorov and Kadonaga 2002;

Grune, Brzeski et al. 2003). But the direct effects of DNA sequence are difficult to define,

because their activity (on nucleosomes position) depends on a complex interplay between

numerous factors: DNA sequence specificity of nucleosomes, competitive binding of

transcription factors and histone octamer composition (Jiang and Pugh 2009; Bai and

Morozov 2010). In addition, special conformational features of DNA could play an

important role. This is the case for the ISWI-type complex ACF, which can be directed by

an intrinsically curved DNA sequence element (Rippe, Schrader et al. 2007).

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Figure 4. Protein domain structure of chromatin remodeling complexes. Chromatin remodeling complexes consist of a catalytic ATPase, which belongs to the SF2 helicase superfamily, and accessory regulatory subunits. (A) In the CHD, ISWI and SWI/SNF families the ATPase domain (blue color) is interrupted by only a small protein sequence, whereas the ATPase domain of the INO80 family is “split” by a long insertion. In the scheme the ATP binding site is indicated by a star, the DExx motif is depicted in dark blue and the helicase C terminal domain (HELICc) in light blue. Although the families of ATPases share a common catalytic domain they contain unique flanking regions for interactions with (i) DNA via SLIDE, AT hook (AT), DBINO and possibly Myb-like domains, (ii) chromatin via SANT, bromo- (bromo) or chromodomains (chromo), and (iii) with other proteins as mediated for example by the helicase-SANT-associated (HSA) domain. The interaction domains present in all family members are indicated in orange, while those that are present in only a subset of the family members are colored in green. (B) Noncatalytic subunits of chromatin remodeling complexes can regulate the activity and the substrate recognition of the complex. Via the homeobox and DDT domains they interact with the ATPase domain and bind DNA. DNA interaction modules are also present in MBDs AT hooks (AT), WAC motifs, Zinc fingers, ARID and high mobility group (HMG) domains. Finally, histones or histone modifications are recognized by subunits with bromo, chromo, SANT and PHD motifs while interactions with other proteins or protein modifications are mediated by WD, SH2, SH3 and ELM2 domains or the LXXLL motif. Moreover, some associated proteins contain catalytic activity as for example WSTF, Tip60 and histone deacetylase 1/2 (HDAC1/2). The functions of the bromo-associated homology domain (BAH) are currently unknown. (Erdel, Krug et al. 2011)

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Since a number of subunits of remodeling complexes contain MBD domains capable of

recognizing methylated DNA, this modification might also play a critical role for targeting

chromatin remodelers (Zhang, Ng et al. 1999; Feng and Zhang 2001). The DNA-

sequence dependent targeting of remodelers is not necessarily mediated by the

remodeling complex subunits but can also occur via interaction with other proteins. The

most interesting is that these complexes can recognize distinct histone modifications (see

next section), via bromodomain interactions with acetylated histones or chromodomain

and PHD finger interactions with methylated lysines (Taverna, Li et al. 2007). These

complexes have both ATPase and interactions domains, as is the case for RSC, Chd1,

ISWI and Acf1 that contain bromodomains recognizing histone acetylation states to

promote or inhibit their own activity (Goodwin and Nicolas 2001; Corona, Clapier et al.

2002; Ferreira, Flaus et al. 2007). For histone methylation, it was found that human Chd1

binds to H3K4me2/3 stronger than to H3K4me1 via its double chromodomain (Sims, Chen

et al. 2005). PHD fingers can also recognize methylated lysine residues on H3 histone

tails in a protein context dependent manner (Taverna, Li et al. 2007).

Another important and interesting characteristic of these complexes is their involvement in

non-replicative incorporation and stabilization of histone variants H3.3 (Chd1 or ATRX)

(see page 18 for more information about histone variants), H2A.Z (Swr1 (Mizuguchi, Shen

et al. 2004)) (see page 20 and Chapter II) and CenH3/CENP6A (Chd1 and RSF). For this

process, it is difficult to distinguish if the histone variant represents only the product of the

remodeler-driven incorporation reaction or whether already incorporated H3.3, H2A.Z or

CenH3 recruits additional remodeling complexes to enhance the reaction. In any case,

histone variants have been identified as signals that change the activity of chromatin

remodelers. The histone H2A.Z, which is often found in nucleosomes at transcriptional

control regions, increases the activity of the human ISWI remodeling complex in vitro

(Goldman, Garlick et al. 2010).

D. Histone modifications.

Within the fundamental chromatin unit, the nucleosome, N-terminus and, in some cases,

the core and the C-termini of histones are subject to a multitude of covalent modifications.

In this manner, the histone-modifying enzymes can alter the chromatin structure and/or

influence the binding of effector molecules that affect patterns of gene expression.

Combinations of modifications generate the so-called “histone code”. On a given gene

promoter can be found several histone marks (activating or/and repressive), which as a

result of the balance between these modifications (sometimes more than one on the same

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histone) and their reading by protein complexes, will influence gene activation or

repression (Strahl and Allis 2000). Principal histone modifications are lysine acetylation,

lysine and arginine methylation, serine and threonine phosphorylation, ADP-ribosylation,

ubiquitination, sumoylation, propionylaiton, butyrylation, crotonylation and other yet

unknown or poorly appreciated chemical modifications.

A plethora of specific enzymes catalyzes these modifications. In fact, each histone

modification is created by a specific enzyme: histone methyl transferases (HMT) catalyze

histone methylation, histone acetyl transferases (HAT) are responsible for histone

acetylation, etc. Most modifications are reversible and specific enzymes are required for

demethylating (HDM), deacetylating (HDAC) histones (see below for more explanations).

1. Histone methylation.

Methylation of histones can occur on lysine and arginine residues. Lysines can be mono,

di- , or tri-methylated, whereas arginines can be mono- or di-methylated, thus greatly

extending the complexity of histone modification-dependent gene regulation. Some

evidence suggests that arginine methylation on H3 and H4 histones is more dynamic than

lysine methylation and marks domains of active or inactive gene expression (Stallcup

2001; Bannister, Schneider et al. 2002). In fact, arginine methylation and its involvement

in transcriptional regulation seems to be complex. It depends on the enzyme “employed”

by the cell; in particular, arginines methylated by PRMT1 (Protein Arginine Methyl-

Trasferase) and CARM1 (Coactivator-associated Arginine Methyl-Trasferase) are found

on transcriptionally active genes. In contrast, di-methylation of arginines due to the action

of PRMT5 protein is involved in repression (Wysocka, Allis et al. 2006).

Lysine methylation seems to be a quite stable mark, with what appears to be a more

complicated readout (Zhang and Reinberg 2001). Moreover, some lysine methylations are

involved specifically in gene activation whereas others are in gene silencing. In the case

of lysine 4 and lysine 9 and 27 on H3 histone, lysine 4 methylation is a gene activation

mark whereas lysine 9 and 27 methylation correlates with gene repression (Bannister,

Schneider et al. 2002; Fischle, Wang et al. 2003). These marks are “written” by histone

methyltransferases (HMTs) and “read” by chromodomain-containing proteins, such as

heterochromatin protein 1 (HP1) and Polycomb (Pc). These proteins specifically recognize

methyl marks, depending on their location on the histones (Fischle, Wang et al. 2003)

(Figure 5). Several studies demonstrate the dynamic character of lysine methylation and

tear down the old “dogma” which proposed that this mark is irreversible (Dillon and

Festenstein 2002). Several evidences show that active demethylation exists even for

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lysine 4 and/or 9 of H3 histone (Ma, Baumann et al. 2001; Ghosh and Harter 2003;

Nicolas, Roumillac et al. 2003; Janicki, Tsukamoto et al. 2004).

Two main explanations for demethylation: the first supposes nucleosome disassembly

and the substitution of methylated histones by a newly synthesized, unmodified histone

(Ahmad and Henikoff 2002). The second is due to the discovery of a new class of

enzymes. The first protein described is the human enzyme PAD4 (peptidylarginine

deiminase 4), which converts the methyl group in citrulline (Cuthbert, Daujat et al. 2004;

Denman 2005). In 2004, the enzyme LSD1 (lysine-specific histone demethylase) was

described. It is able to demethylate the mono- and di-methylated lysine 4 of H3 in vivo

(Shi, Lan et al. 2004). Metzger showed in 2005 that the LSD1 protein is able to

demethylate lysine 9 of H3, too (Metzger, Wissmann et al. 2005). More recently another

histone lysine demethylase, JHDM1 (Jumonji domain-containing histone demethylation)

was discovered (Tsukada, Fang et al. 2006). This protein needs Fe2+ and alpha-

ketoglutarate as cofactor to demethylate lysine 36 of the histone H3 specifically, either

mono- or di-methylated (Figure 4).

Two other enzymes are important for the demethylation process: Histone Demethylase

Jumonji D3 (JMJD3) and Jumonji D2 (JMJD2). These enzymes have a role in breast

cancer in particular. JMJD2B is highly expressed in human breast cancer. 17-beta-

estradiol (E2) induces JMJD2B expression in an ER� (Estrogen Receptor �) -dependent

manner. This enzyme interacts with ER� and components of the SWI/SNF-B chromatin-

remodeling complex. JMJD2B is recruited to ER� target sites to demethylate H3K9me3,

allowing the transcription of ER-regulated genes (MYB, MYC and CCDN1) leading to cell

proliferation and tumor development. As a consequence, knockdown of JMJD2B severely

impairs estrogen-induced cell proliferation and the tumor formation capacity of breast

cancer cells (Kawazu, Saso et al. 2011).

JMJD3 is specifically required to demethylate H3K27me3 (Xiang, Zhu et al. 2007) on the

anti-apoptotic ER� target gene BCL2. In hormone responsive breast cancer cell such as

MCF-7, JMJD3 and ER� work in concert following estradiol (E2) stimulation. In fact, these

two proteins colocalize on the Estrogen Responsive Elements present on the BCL2 gene

promoter to remove the H3K27me3 repressive mark. By removing this mark from the

poised (yet silent) enhancer region, they permit appropriate gene activation and anti-

apoptotic effects following E2 stimulation. Interestingly, the depletion of JMJD3 in ER�-

positive cells (MFC-7) dramatically increases apoptosis in E2-treated cells. In contrast,

ER�-negative cells (MDA-MB231) are insensible to JMJD3 depletion, suggesting that

JMJD3 effects are specific to E2-responsive cells. In conclusion, JMJD3 seems to play an

important role in apoptosis control in ER�-dependent breast cancer cells (Svotelis, Bianco

et al. 2011).

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2. Histone phosphorylation.

Histone phosphorylation involves threonine and serine residues. It is a dynamic

modification; in fact, its regulation depends on the “balance” between kinase and

phosphatase activity. H1 phosphorylation, for example, leads to the condensation and

segregation of chromosomes during mitosis and meiosis (Gurley, Walters et al. 1978).

The phosphorylation of serine 10 of H3 histone is involved in chromosome condensation

and has a role in mitosis. Recent work revealed a new role of this modification in

transcription activation. This modification inhibits the interaction between the HP1 protein

and the methyl-lysine 9 of H3 histone (Eissenberg and Elgin 2005).

Histone phosphorylation seems to be involved in apoptosis as well (Lee, Nakatsuma et al.

1999). In particular, the phosphorylation of serine 32 on the H2B histone seems to be a

good marker of apoptotic cells (Ajiro 2000). Moreover, phosphorylation of the histone

variant H2A.X (�-H2A.X) is the canonical mark for DNA double strand breaks (Rogakou,

Pilch et al. 1998; Ismail and Hendzel 2008) (see below).

3. Histone acetylation.

Histone acetylation is canonically associated to transcriptional activation (Allfrey, Faulkner

et al. 1964; Hebbes, Thorne et al. 1988). In fact, this modification helps histone

disassembly by decreasing the electrostatic interactions occurring between DNA

(negatively charged) and the histones (positively charged) (Travers and Thompson 2004).

In this manner, histone acetylation “opens” chromatin and allows transcriptional factors to

access the DNA template (Kuo, Zhou et al. 1998; Strahl and Allis 2000). Conversely,

histone deacetylation mainly contributes to a “closed” chromatin state and transcriptional

repression.

Two classes of enzymes, Histone Acetyl Transferases (HAT) and Histone Deacetylases

(HDAC), assure the balance between acetylation and deacetylation. There are three

families of HAT : the GNAT (Gcn5 related N-Acetyltransferases, (Georgakopoulos T

1992)) family, the MYST family (Tbf2/Sas3 Sas2 Tip60, (Utley and Cote 2003)) and the

p300/CBP proteins (Bannister and Kouzarides 1996). The majority of HAT is active in

multiproteic complexes (Yang 2004) and the associated factors at these complexes seem

control the substrate specificity and increase the catalytic activity (Grant, Duggan et al.

1997). The histone acetylation is recognized by bromo-domain proteins (Dhalluin, Carlson

et al. 1999; Loyola and Almouzni 2004) (Figure 5).

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The first member of HDAC family discovered was HDAC1 (Taunton, Hassig et al. 1996).

Up to now, eighteen enzymes have been discovered, in human, and classified in four

groups (I to IV). The first group has a strong homology with the yeast protein Rpd3

(Reduced Potassium Dependency), these enzymes are called class I (HDAC 1,2,3 and 8).

Class II (HDAC 4, 5, 6, 7, 9 and 10) proteins display some homologies with the yeast

protein Hda1 (Histone Deacetylase1). HDAC11 is the only member of the class IV. These

three groups have a zinc-dependent enzymatic activity whereas the HDACs of class III,

called “sirtuins”, homologs of Sir2 (Silent Information Regulator 2) of Saccharomyces

cerevisiae, form a separate family and their deacetylase activity is NAD+ dependent

(Figure 5).

It is important to note that the HDACs are characterized from their activity on histones, but

they have other substrates: they regulate the acetylation of other proteins, which have an

important role in cells like p53, Bcl-6, E2Fs factors, tubulin, HSP90 (Heat Shock Protein

90), �-catenin and some others (Dokmanovic, Perez et al. 2007). Maybe “lysine

deacetylases” would be the best name for these enzymes but it is rarely used and HDAC

remains the most commonly used name (Yang and Seto 2008).

Figure 5. Schematic representation of histone acetylation and methylation pathways complexity: chromatin marks and epi-enzyme deregulation in cancer. Chromatin enzymes able to deposit or erase an epigenetic mark are indicated as writers (teal blue) and erasers (purple), respectively. Epi-drugs

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able to inhibit the activity of chromatin enzymes are indicated as blockers (brown). (Conte and Altucci 2012).

4. Other histone modifications.

Acetylation, methylation and phosphorylation are the most studied histone modifications,

but other modifications exist that play important roles in cellular processes. The mono-

ADP-ribosylation for example, involves arginine and glutamate residues of H1 and H2B

histones (Burzio, Riquelme et al. 1979; Golderer and Grobner 1991). From a general point

of view, the ADP-ribosylation seems to be involved in DNA damage repair (Kreimeyer,

Wielckens et al. 1984).

Mono-ubiquitination is the covalent addition of an ubiquitin peptide on a lysine residue

(Jason, Moore et al. 2002). Generally, the addition of a poly-ubiquitine leads to protein

degradation; but histone mono-ubiquitination seems to correlate with transcription

activation (Thorne, Sautiere et al. 1987). In fact, it has been shown that the H2B mono-

ubiquitination can promote the H3 lysine4 methylation in yeast, contributing to

transcriptional regulation (Dover, Schneider et al. 2002; Sun and Allis 2002).

Sumoylation, the addition of a SUMO peptide -normally involved in protein degradation-

has been described and studied on the H4 histone. This modification seems to be

involved in gene silencing. It recruits histone deacetylase (HDAC1) and the

heterochromatin protein1 (HP1) to sumoylated H4 (Shiio and Eisenman 2003).

Biotinylation of lysine residues through the addition of a biotine peptide leads to a

competition with other modifications on the same residue (Camporeale, Shubert et al.

2004) and seems to be involved in cellular proliferation (Stanley, Griffin et al. 2001;

Narang, Dumas et al. 2004).

Butyrylation and propionylation are structurally similar to acetylation. These histone

modification, were found for the first time in vivo on the histone H4 and share with

acetylation the same enzymes, the acetyltransferases CBP and p300 (Chen, Sprung et al.

2007). These enzymes can use propionyl-CoA and butyryl-CoA as precursors. Moreover,

in the case of propionylation, the NAD+ dependent deacetylase Sir2 is involved in the

removal of the propionyl group. Like acetyl-CoA, propionyl- and buryryl-CoA are

intermediates in biosynthesis and energy production. It has been proposed that

propionylation is used as an alternative to the acetylation, depending on the metabolic

pattern of the cell. Studying the propionylation of the lysine 23 of the H3 histone, Liu et al;

suggest that the propionylation might be a stage-specific marker in hematopoiesis and

leukemogenesis (Liu, Lin et al. 2009).

Recently, using different approaches based on mass spectrometry, 67 histone post-

translational modification sites were identified. Among these, 28 sites are characterized by

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a new histone modification: lysine crotonylation (Kcr). Crotonylation differs structurally

from methylation and acetylation. It may thus require specific enzymes for recognition,

addition and removal. Despite the fact that the co-enzyme involved (Crotonyl-CoA) is

generated from acetyl-CoA, the enzymes responsible of histone acetylation are not

involved in crotonylation. In fact, overexpression of canonical histone acetyl-transferases

and the exposure to histone deacetylases do not change the amount of Kcr. This histone

modification is conserved during evolution and has a characteristic genomic pattern. Kcr is

enriched at enhancers and the TSS of active genes. Moreover, in mouse male germ cells,

the histone Kcr marks the postmeiotically expressed X-linked genes. Indicating that Kcr

may be involved in male postmeiotic gene expression pattern. Similar to histone

hyperacetylation, Kcr seems to be associated with histone removal that follows germ cell

maturation. So, Kcr may affect chromatin structure and therefore facilitate histone

replacement. In this manner, this newly characterized histone modification could be an

important histone mark in the establishment of a region-specific male epigenome

organization (Tan, Luo et al. 2011; Montellier, Rousseaux et al. 2012).

5. Histone modification networks.

Histone modifications are not independent of each other. The first evidence concerns the

modifications on H3 lysine 9. This lysine can be methylated or acetylated; these two

modifications are totally exclusive because they happen on the same lysine NH2 group.

Another example: in yeast, the phosphorylation of the serine 10 promotes H3-K14

acetylation by Gnc5 (Cheung, Briggs et al. 2000) and, at the same time, inhibits the

methylation of H3-K9 by Suv39H1 (Ahringer 2000). In this manner, serine 10

phosphorylation has a positive effect on transcription since blocking H3-K9 methylation

(repressive mark) and promoting H3-K4 acetylation increases transcription activation. In

contrast, H3-K9 methylation inhibits H3-S10 phosphorylation by Aurora kinase in vitro

(Ahringer 2000). In addition, the same methylation (H3-K9) inhibits, in vitro, H3-K4

methylation (activation mark) by Set7 (Decristofaro, Betz et al. 2001). Therefore, the

transcriptional repressive state is due to lysine 9 deacetylation followed by H3-K9

methylation, which keeps the domain in an inactive state blocking H3-S10 phosphorylation

and H3-K4 methylation.

A similar situation involves the H4 histone and its modifications. H4-R3 methylation

promotes H4-K8 and K12 acetylation (in vitro by p300); but it is a negative feedback,

because this acetylation inhibits H4-R3 methylation by PRMT1, suggesting that H4-R3

methylation might come before acetylation for transcriptional activation (Decristofaro, Betz

et al. 2001). The network is more complex, in fact it can involve different tails of different

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histones. For example, ubiquitination of lysine 123 of H2B histone, which promotes and is

essential for H3-K4 methylation in S. cerevisiae (Briggs, Xiao et al. 2002; Dover,

Schneider et al. 2002). In the same manner, but in vitro, H3-K4 methylation by Set7

promotes H4 acetylation by p300, and is inhibited by H3-K9 methylation (Decristofaro,

Betz et al. 2001). Moreover, H2B ubiquitination is essential for H3 methylation. In

particular the ubiquitination of H2B catalyzed by the ubiquitin-conjugating enzyme Rad6,

is required for K4 and K79 methylations of H3 histone. The precise mechanism remains

poorly understood, but several studies support the idea that H2Bub may act as a bridge to

recruit Set1-COMPASS (methylation complex, required for silencing of genes located near

telomeres or within rDNA) and Dot1, involved in K4 and K79 methylations, respectively.

This process results in gene silencing (Dover, Schneider et al. 2002; Chandrasekharan,

Huang et al. 2010).

Therefore, post-translational modifications of histones have a fundamental role in the

transcription process. The cell defines gene fate (transcribed or not) through stable marks,

such as methylation or acetylation, inherited during meiosis, or through other marks

induced by signaling, such as phosphorylation. Moreover, histone modifications allow

chromatin remodeling and therefore affect recruitment and processing of transcriptional

machinery.

E. Histone variants.

Canonical histone genes are intronless and occur in clusters, facilitating coordinated gene

regulation (Albig and Doenecke 1997). Their mRNAs lack a polyA tail, instead having a

dyad sequence that forms a 3’ stem loop structure. Their expression is replication-

dependent. The number of canonical genes that code for each histone family member can

exceed ten genes (Marzluff, Gongidi et al. 2002).

In contrast to canonical histone genes, expression of most known histone variants is

replication-independent. Genes encoding histone variants typically lay outside of clusters,

can contain introns, and occur in low copy numbers. Like standard genes, mRNAs

produced from histone variant genes possess a polyA tail (Doenecke, Albig et al. 1997).

These variants have distinct genomic localizations and post-translational modifications

(PTM), thus increasing the complexity of chromatin architecture. Several studies about

histone variants indicate that they play a role in many biological processes such as

transcription, DNA damage response, and cell cycle. For all these reasons they are

proposed to form an extra layer of the “histone code” (Hake and Allis 2006; Arnaudo,

Molden et al. 2011).

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All canonical histones have a family of variants except histone H4. These variants have

different roles in cell processes (Figure 6).

Figure 6. Canonical core histones and their variants. The major core histones contain a conserved histone-fold domain (HFD). In addition, they contain N- and C-terminal tails that harbor sites for various post-translational modifications. For simplicity, only well-established sites for lysine methylation (red flags) and serine phosphorylation (green circles) are shown (other types of modifications, such as ubiquitylation, are not shown). In the histone H3.3 variant, the residues that differ from the major histone H3 (also known as H3.1) are highlighted in yellow. H1 variants are not shown, see text. (Sarma and Reinberg 2005)

1. Histone H3 family.

Histone H3 (known as H3.1) has three major variants as well as a testis specific variant

5H3t and a variant that localizes to centromeres (CENP-A) which plays a role in

chromosome segregation during mitosis (Howman, Fowler et al. 2000). The other three

variants of H3 are surprisingly similar. In fact H3.1 only differs from H3.2 by a change in

Cysteine 96 to Serine, and H3.3 differs from H3.1 by only 5 residues. However, their

expression levels, localization in chromatin and modification states vary significantly. H3.3

is expressed in a replication independent manner, while canonical variants (H3.1 and

H3.2) are only expressed during S phase (Stein and Stein 1984). Moreover, different

chaperones recognize and assemble H3.1 and H3.3 into nucleosome in a replication

dependent and independent manner respectively (Tagami, Ray-Gallet et al. 2004). H3.3 is

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localized to heterochromatin and enriched for histone modifications that are associated

with gene activation (Ahmad and Henikoff 2002; McKittrick, Gafken et al. 2004) (Figure 6).

H3.1 and H3.2 do not show specific localization in genome and are considered identical;

however, one study suggests that H3.2 is modified with marks associated with gene

repression while H3.1 shows both activating and repressive marks (Ahmad and Henikoff

2002; Hake, Garcia et al. 2006).

The presence of these different H3 variants and their involvement in specific processes,

led some authors to propose an intriguing hypothesis. Hake and Allis proposed the “H3

barcode hypothesis”. Mammalian H3 variants, although similar in amino-acid sequence,

are able, through different and specific post-translational “signatures”, to create distinct

chromosomal domains and territories, which influence epigenetic states during cellular

differentiation and development (Hake and Allis 2006).

2. Histone H2A family.

H2A family is one of the most sequence divergent families, with 20 unique sequences in

humans. Canonical human H2A is encoded by 16 genes within gene clusters and has 12

unique sequences. Sequence diversity comes from divergent C-terminal tails, but the

biological relevance of this diversity remains unknown. H2A family variants include H2A.X,

H2A.Bbd, macroH2A and H2A.Z; these variants differ in sequence and have different

functions. (Figure 6).

H2A.X is most similar in sequence to its canonical version but has divergent C-terminal

tail. H2A.X mRNA can have either a polyA tail or the stem and loop dyad structure,

indicating that it can undergo both replication dependent and independent transcription

(Mannironi, Bonner et al. 1989). This variant can be acetylated, ubiquitinated and

phosphorylated. The phosphorylation occurs in response to DNA damage at Serine 129;

H2A.X phosphorylated localizes to DNA double strand breaks, helping to recruit proteins

for DNA repair (Rogakou, Pilch et al. 1998; Ikura, Tashiro et al. 2007). Acetylation and

ubiquitination also play a role in this process; in particular, acetylation at lysine 5 is a

prerequisite for ubiquitination and subsequent release of H2A.X from DNA damage sites

(Ikura, Tashiro et al. 2007).

The second variant is H2A.Bbd, his name derives from the fact that this variant is

excluded from inactive X (Barr body deficient). H2A.Bbd is associated with acetylated H4,

and therefore implicated in transcriptional activation (Chadwick and Willard 2001). The

sequence of this variant is short and rich in arginine in comparison to canonical H2A.

Nucleosomes containing H2A.Bbd contain 128bp of DNA as opposed to the traditional

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146, indicating that the incorporation of this variant impacts chromatin structure (Doyen,

Montel et al. 2006).

MacroH2A is the largest H2A variant containing a 30kDa macro domain at its C-terminus

which can recognize ribosylated protein residues (Karras, Kustatscher et al. 2005).

Human macroH2A is encoded by two genes macroH2A2 and macroH2A1. MacroH2A1

has two splicing variants: macroH2A1.1 and macroH2A1.2. macroH2A1.1 but not 1.2 is

able to bind metabolites of NAD+ and may have unique, yet to be discovered, functions.

Interestingly, the macroH2A1 splicing isoforms are differentially expressed in primary

tumors that either give rise to metastases or do not. In fact, it has been shown that the

splicing factors Ddx7 and Ddx5 are overexpressed in invasive tumor cells and participate

in tumor-cell migration and invasion. These splicing factors control alternative-splicing

networks, including macroH2A1 histone genes. macroH2A1 splicing isoforms differentially

regulate the transcription of a set of genes involved in redox metabolism. In this manner,

the splicing choice of an epigenetic factor, as macroH2A1, plays a critical role in tumor

progression (Dardenne, Pierredon et al. 2012). macroH2A is generally enriched in silent

areas both on autosomes and on inactive X. However, recent ChIP-on-Chip experiments

have shown that macroH2A1 can be found at a subset of active genes. Moreover,

macroH2A can be ubiquitinated, acetylated and phosphorylated. Ubiquitination at K115 is

implicated in X-inactivation, while phosphorylation of S137 is enriched during mitosis

(Chu, Nusinow et al. 2006; Bernstein, Muratore-Schroeder et al. 2008).

Finally, the histone H2A.Z variant is highly conserved during evolution and is essential for

development in higher eukaryotes (Draker and Cheung 2009). H2A.Z localizes to the

promoters of genes and, in mammals, its localization to the transcription starting site

correlates with gene activation (Barski, Cuddapah et al. 2007). In yeast, H2A.Z is found at

transcriptionally active areas near the telomeres and silent mating loci to prevent

spreading of heterochromatin. These findings suggest that H2A.Z is important for

maintaining active chromatin in regions near silent chromatin (Meneghini, Wu et al. 2003).

Acetylation of H2A.Z is linked to gene activation. However, there is evidence that H2A.Z

can also act in gene repression (Millar, Xu et al. 2006; Draker and Cheung 2009; Mehta,

Braberg et al. 2010) – see below for more details.

3. Histone H2B family.

Human H2B has 17 isoforms, which are encoded by 25 genes. Most H2B sequences vary

by just a few amino acids and are encoded by genes located in the main cluster. There

are two proteins that differ significantly from other H2B, which are the testis-specific H2B

(H2B1A and H2BFWT) (Zalensky, Siino et al. 2002; Churikov, Siino et al. 2004). H2B

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variants are poorly studied, maybe because of their sequence similarity, so their biological

function is not well understood. Moreover, these variants are not extensively modified as

H3 or H4, and aside from a few lysine acetylation sites, other modified forms are sparsely

abundant.

4. Histones H1.

Histone H1 is commonly referred to as the linker histone. A single copy of this histone has

been proposed to bind near the entry/exit site of DNA on the nucleosome, thus stabilizing

chromatin and DNA structure (Bustin, Catez et al. 2005; Woodcock, Skoultchi et al. 2006).

Most H1 proteins contain a globular domain, a short N-terminal tail and a longer lysine rich

C-terminal tail. Sequence divergence between histone H1 isoforms is high, and occurs

mainly in the N- and C- terminal regions of the protein. There are 11 isoforms in

mammals. These isoforms are generally classified based on the timing of expression or

tissue specificity (Izzo, Kamieniarz et al. 2008). The replication-dependent isoforms

include histone H1.1 – H1.5 and H1.t, while replication-independent variants are H1.X,

H1.t2, HILS1, H1oo and H1.0. Histone H1.0 – H1.5 are expressed in somatic cells, with

H1.1 restricted to certain tissue types (Daujat, Zeissler et al. 2005; Godde and Ura 2008).

Histone H1.t, H1.t2 and HILS1 are expressed in testis specific tissues and H1oo is oocyte

specific (Godde and Ura 2008). Histone H1.X has mainly been reported in tissue culture

cells (Wisniewski, Zougman et al. 2007). Moreover, a large number of post-translational

modifications have been shown for the H1 family, including lysine methylation,

phosphorylation, acetylation, ubiquitination, formylation, and ADP ribosylation (Poirier and

Savard 1980; Garcia, Busby et al. 2004; Wisniewski, Zougman et al. 2007; Talasz, Sarg

et al. 2009).

Several studies have supported the fact that H1 variants are redundant, lacking specific

functions in chromatin organization and gene expression control (Fan, Sirotkin et al.

2001). However, it seems that it is not true concerning H1.4 and H1.2. In fact, H1.4 is

involved in a heterochromatization process. This variant is deacetylated at lysine 26 and

methylated, facilitating recruitment of Polycomb complexes and HP1, whereas

simultaneous phosphorylation of serine 27 blocks HP1 binding (Vaquero, Scher et al.

2004; Daujat, Zeissler et al. 2005). H1.2 is involved in apoptosis induced by double-strand

breaks (Konishi, Shimizu et al. 2003). A H1.2 containing complex involved in repression of

p53-mediated transcription has also been isolated (Kim, Choi et al. 2008). These variants

seem to have a role in breast cancer. In fact, the depletion of H1.4 in the human breast

cancer cell line T47D leads to cellular death. Moreover, in the same cell type, the

depletion of H1.2 leads to a stop in G1 of the cell cycle, a defect in chromatin structure

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and changes in expression of specific genes involved in the cell cycle (Sancho, Diani et al.

2008).

The high number of isoforms and their modification patterns may thus result in a linker-

code similar to the histone code (Godde and Ura 2008).

II. Histone H2A.Z

As we have seen, gene regulation is based on a complex crosstalk between different

actors: DNA modifications, chromatin composition, histone post-translational

modifications, histone variant incorporation and chromatin remodeling complexes interplay

to an accurate and fine gene regulation. This regulation has an impact on all the biological

processes. Our particular interest is the involvement of the histone variant H2A.Z in gene

regulation.

H2A.Z is a highly conserved histone variant of H2A. It is essential for viability in different

organisms such as Tetrahymena thermophila (Liu, Li et al. 1996), Drosophila

melanogaster (Clarkson, Wells et al. 1999), Xenopus laevis (Ridgway, Brown et al. 2004),

and mice (Faast, Thonglairoam et al. 2001). In contrast, loss of H2A.Z is tolerated in

Saccharomyces cerevisiae, suggesting that this variant may have different roles in

different organisms. H2A.Z variants share about 90% similarity among the various higher

eukaryotes. H2A and H2A.Z differ in amino acids sequence but share about 60% of

identity (Jackson, Falciano et al. 1996). These changes are sufficient to create a unique

chromatin conformation in H2A.Z enriched regions. H2A.Z differs from H2A by a C-

terminus containing an alternative docking domain and an extended specific acidic patch.

This acidic patch and C-terminus contain some residues that are important for H2A.Z

deposition into chromatin (Jensen, Santisteban et al. 2011; Wang, Aristizabal et al. 2011).

This replacement creates a different interface in the nucleosome that can recruit specific

factors/modulators (Figure 7).

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Figure 7. Schematic representation of histone H2A.Z role in gene activation. (A) Incorporation of H2A.Z in chromatin by the replacement of H2A–H2B dimers with H2A.Z–H2B dimers. ATP-dependent exchange by Swr1-related complexes is assisted by H2A.Z–H2B chaperones. H2A.Z-containing nucleosomes are less stable than canonical nucleosomes. (B) Promoter of yeast inactive gene has a nucleosome-free region (NFR) surrounded by two well positioned nucleosomes. (C) Specific incorporation of H2A.Z at nucleosomes −1 and +1 poises genes for transcriptional activation. (D) Binding of activators leads to H2A.Z acetylation, eviction of H2A.Z nucleosomes and transcription initiation. (Billon and Cote 2012)

A. H2A.Z localization in the genome.

Nevertheless, the relevance of H2A.Z in biological processes is due to its localization

within the genome. In Tetrahymena, the equivalent of H2A.Z, hv1, is found exclusively in

the transcriptionally active macronucleus, but is absent in the transcriptionally inert

micronucleus, suggesting that this variant has a function in transcription (Allis, Glover et

al. 1980). In Drosophila's H2A.Z variant, H2AvD, has a nonrandom distribution on

polytene chromosomes, and antibodies against H2AvD stain both euchromatin and

heterochromatin regions of the genome (Leach, Mazzeo et al. 2000). Moreover, in flies,

H2AvD variant is unique, in that it is a functional fusion between H2A.Z and H2A.X in

other organisms. So, the distinctive distribution of H2AvD may have been adopted to

accommodate the dual functions of H2A.Z and H2A.X in this organism.

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Immunofluorescence examination of differentiated mouse fibroblasts showed that H2A.Z

is distributed across the entire interphase nucleus, but is excluded from transcriptionally

silent and HP1�-enriched pericentric heterochromatin (Sarcinella, Zuzarte et al. 2007).

Staining of mouse metaphase chromosomes also showed that H2A.Z is enriched on

chromosome arms and depleted from the constitutive centromeric heterochromatin.

Based on these observations, distribution between H2A.Z and H3K9me3 (mark of

constitutive pericentric heterochromatin) in mouse embryonic fibroblasts was proposed to

be mutually exclusive (Bulynko, Hsing et al. 2006). In contrast, in trophoblast cells of the

developing mouse embryo, H2A.Z is concentrated at pericentric heterochromatin and

colocalizes with HP1� (Rangasamy et al., 2003). At present, it is unclear why H2A.Z

genomic distribution changes with developmental stages, but it is possible that H2A.Z’s

function switches at different stages of development or differentiation.

Using 2D and 3D immunofluorescence analysis to examine the inactive X chromosome in

female mouse cells, H2A.Z was found to localize to only one side of the centromere of

each sister chromatid (Rangasamy et al., 2003). Additional studies support the idea that a

specific fraction of H2A.Z may have centromere-related functions. Firstly, H2A.Z binds to

CENP-A (the H3 variant specific to centromere) in a nucleosomal purification fraction

(Foltz, Jansen et al. 2006). Second, RNAi-mediated depletion of H2A.Z in mouse L929

and monkey Cos-7 cells resulted in chromosome segregation defects characterized by

formation of chromatin bridges between separating nuclei (Rangasamy, Greaves et al.

2004). Finally, in yeast, colony-sectoring studies showed that H2A.Z-deletion strain

(htz1�) has increased rates of chromosome loss, suggesting that a chromosome

segregation function of H2A.Z is conserved between yeast and human cells (Krogan,

Baetz et al. 2004).

B. H2A.Z deposition.

It is now believed that SWR1-related ATP-dependent chromatin remodelers that

specifically exchange canonical H2A-H2B for H2A.Z-H2B dimers within nucleosomes

(Morrison and Shen 2009) perform the majority of H2A.Z incorporation into chromatin.

To replace canonical dimers by variants dimers, SWR1-related complexes evict them from

the nucleosome and replace them by an H2A.Z-containing nucleosome. It has been

proposed that this process is catalyzed by two families of large ATP-dependent

complexes, both highly conserved during evolution.

Using the energy from ATP hydrolysis, these complexes are able to slide, disrupt, evict

nucleosomes or exchange histone-dimers (Clapier and Cairns 2009). A subclass of the

SWI2/SNF2 superfamily of enzymes is responsible for H2A.Z incorporation in chromatin

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(Morrison and Shen 2009). In yeast, the complex responsible for H2A.Z deposition is

SWR1; it removes H2A-H2B dimers to replace it by H2A.Z-H2B dimers. For to perform

this action, the SWI2/SNF2 ATPase/helicase domain of the SWR1 subunit is necessary

(Figure 8). Recent studies in higher eukaryotes identified p400 and SRCAP (SWI2/SNF2-

related CBP activator protein) complexes as homologous to the SWR1 complex (Figure 8)

(Kusch, Florens et al. 2004; Ruhl, Jin et al. 2006; Wong, Cox et al. 2007). The purification

of these complexes showed functional conservation, as they were able to catalyze

exchange activity similar to the yeast SWR1 complex. These different complexes may be

involved in different types of H2A.Z exchange depending on cellular stimuli and chromatin

context.

Another important chromatin-remodeling complex, involved in H2A.Z deposition/eviction is

INO80. It belongs to the same SWI2/SNF2 subfamily as Swr1 and plays a role in

transcription, DNA repair and replication (Morrison and Shen 2009; Watanabe and

Peterson 2010). Under stress conditions, the INO80 complex seems to be recruited to the

coding regions for elongation of transcription as well as replication forks (Papamichos-

Chronakis and Peterson 2008; Klopf, Paskova et al. 2009). H2A.Z eviction in yeast

suggests a role for this complex in the relocalization of H2A.Z under stress conditions. It

remains to be determinated if the described mammalian INO80 complex has similar roles

in higher eukaryotes (Conaway and Conaway 2009). In addition, it has recently been

shown that eviction of H2A.Z by INO80, from an in vitro exchange, is ATP-dependent

(Papamichos-Chronakis, Watanabe et al. 2011).

Some other complexes are involved in H2A.Z deposition: TIP48/TIP49 containing

SWR1/SRCAP (Cai, Jin et al. 2005) and/or TIP60/P400 (Ikura, Ogryzko et al. 2000),

(Kusch, Florens et al. 2004). It has been shown that p400 is responsible for H2A.Z

incorporation into the TFF1/pS2 gene when the estrogen receptor binds the promoter

(Gevry, Hardy et al. 2009). Moreover, was reported that the AAA+ family (ATPases

Associated with various cellular Activities) members TIP48/TIP49 participate in the

exchange H2A – H2A.Z (Choi, Heo et al. 2009). The replacement is facilitated by TIP60-

mediated H2A acetylation. TIP48/TIP49 proteins (also known as TIP49b/TIP49a,

Rvb2/Rvb1 or even reptin/pontin) are important for assembly and activity of the histone

acetyltransferase TIP60 complex (Jha, Shibata et al. 2008)(Dalvai et al., 2013 PLOS

Genetics, in press).

In higher eukaryotes, the p400 protein is associated with Tip60, a MYST-family

acetyltransferase, creating a physical merge of the yeast SWR1 remodeler and NuA4

acetyltransferase complexes (Figure 8). Drosophila's Tip60 is able to acetylate and

exchange H2A.Z and has a p400/domino subunit homologous to SWR1 (Eissenberg and

Elgin 2005). In human cells, p400 is implicated in the p53/p21 senescence pathway by

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inhibiting p21 and stimulating p53 toward apoptosis (Fuchs, Gerber et al. 2001; Chan,

Narita et al. 2005; Samuelson, Narita et al. 2005). This complex is involved in H2A.Z and

responsible for incorporation of H2A.Z at specific promoters in vivo (Gevry, Chan et al.

2007; Gevry, Hardy et al. 2009; Cuadrado, Corrado et al. 2010).

Figure 8. SWR1-related complexes in mammalian cells. (Billon and Cote 2012)

C. H2A.Z on gene promoters.

Several studies using chromatin immunoprecipitation (ChIP) in S. cerevisiae have shown

that H2A.Z maps preferentially to the 5’ ends of genes within euchromatic regions

(Guillemette, Bataille et al. 2005; Li, Pattenden et al. 2005; Zhang, Roberts et al. 2005),

(Dalvai, Bellucci et al. 2012). In fact, the common feature of yeast RNA polymerase II

promoters is the presence of an approximately 150bp-long nucleosome-free region

located about 200 bp upstream of the translation initiation codon (Yuan, Liu et al. 2005).

ChIP-microarray analysis shows that H2A.Z is specifically found in the nucleosomes that

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flank these nucleosome-free regions (Raisner, Hartley et al. 2005). Moreover, some of

these yeast studies show that H2A.Z is associated with inducible genes under repressed

or basal-expression conditions (Guillemette, Bataille et al. 2005; Millar, Xu et al. 2006).

Interestingly, depleting H2A.Z in yeast did not repress genes but activation of transcription

was reduced. Therefore, H2A.Z localization at the 5’ of genes may act to set up a

chromatin architecture compatible with gene regulation at promoters.

Similar studies in mammalian cells show that H2A.Z preferentially localizes to gene

promoters (Barski, Cuddapah et al. 2007; John, Sabo et al. 2008; Dalvai, Bellucci et al.

2012). However, there are some distinctive characteristics of H2A.Z distribution in

mammalian cells. First, H2A.Z deposition spreads over several nucleosomes upstream

and downstream of the transcription starting site (Schones, Cui et al. 2008). Second, in

human T cells, H2A.Z enrichment is correlated with gene activity, since H2A.Z is often

associated with actively expressed genes (Barski, Cuddapah et al. 2007). Additional

studies show that H2A.Z can be found at promoters of inducible genes. In fact, genome

wide analysis of chromatin at binding sites of a nuclear hormone receptor, the

glucocorticoid receptor (GR), revealed an enrichment of H2A.Z at constitutive and

hormone-induced GR-binding sites (John, Sabo et al. 2008). Similarly, a recent study

shows that H2A.Z can recruit Brd2 to Androgen-Receptor-regulated genes. Brd2 seems to

be a coactivator of transcription of the BET family of proteins, containing tandem

bromodomains to bind acetyl-lysines. The primary site of interaction between Brd2 and

H2A.Z is acetylated H4, a mark of gene activation (Draker, Ng et al. 2012). In the same

manner, different studies in our laboratory show a characteristic localization and behavior

of H2A.Z on the promoters of inducible genes. H2A.Z localizes to the starting site (in the

case of Cyclin D1 gene also at the 3’ end of the gene) and, after gene induction, its

enrichment decreases but the rate of H2A.Z acetylation increases, which finally activates

gene expression (Dalvai, Bellucci et al. 2012). We will have the possibility to discuss these

H2A.Z characteristics later. Similarly, H2A.Z is enriched at the promoter of the p53-

regulated p21 gene in U2OS osteosarcoma cells and target p53 binding sites (Gevry,

Chan et al. 2007). In this case, H2A.Z depletion activates p21. On the other hand, we

demonstrated that for the same p21 gene in another cellular context (ER-negative, p53-

negative breast cancer cells), the depletion of H2A.Z was not sufficient to activate p21.

We needed to increase H2A.Z acetylation for p21 expression. Hence, H2A.Z acetylation

seems to be more important than the presence of the non-acetylated form of H2A.Z for

gene activation (Bellucci et al., 2012).

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D. H2A.Z and regulation of gene expression.

The localization of H2A.Z at gene promoters close to the TSS suggests that this histone

variant has a role in regulating gene expression. For example, in yeast, GAL1/GAL10

genes are activated upon switching from glucose to galactose-containing growth media,

and PHO5 gene is induced upon growth under low phosphate conditions. Some studies

have shown that H2A.Z is required for the induction of these genes. In fact, when H2A.Z is

depleted their activation is greatly impaired (Santisteban, Kalashnikova et al. 2000; Adam,

Robert et al. 2001). In the same manner, H2A.Z is enriched at the promoters of heat

shock activated genes. Only an attenuated activation of these genes occurs after H2A.Z

depletion (Zhang, Roberts et al. 2005).

Deletion of H2A.Z in S. cerevisiae results in downregulation of 214 genes, the majority of

which are clustered at regions adjacent to telomeres (Meneghini, Wu et al. 2003). The

authors suppose that downregulation is a consequence of heterochromatin spreading into

adjacent euchromatin. Sir2/3 (the heterochromatin-associated proteins) are found in the

telomere-proximal euchromatin regions in the mutant strain htz1�. Interestingly, deletion

of Sir2 in the htz1� background restored expression of many downregulated genes. This

study defines a role for H2A.Z in regulating expression of the genes located near

heterochromatin-euchromatin boundaries, and suggests that H2A.Z antagonizes the

physical spreading of heterochromatin. A similar function is conserved in higher

eukaryotes. H2A.Z is distributed throughout euchromatin regions of the mouse cell

nucleus, and is excluded from the pericentric heterochromatin (Sarcinella, Zuzarte et al.

2007). ChIP analysis in chicken and human T cells show that H2A.Z is enriched at

insulator regions (Bruce, Myers et al. 2005; Barski, Cuddapah et al. 2007). If insulators

are specialized structures that define heterochromatin-euchromatin boundaries, the

presence of H2A.Z at these structures supports a role in mediating a boundary function for

H2A.Z.

Finally, many studies suggest a repressive role of H2A.Z on inducible genes. It is the case

of p21, upon DNA damage, in U2OS and in a p53-dependent manner (Gevry, Chan et al.

2007). In fact, depletion of H2A.Z results in the derepression on the p21 gene. This is not

always the same case: changing cellular context and p53 status changes the role of

H2A.Z. Our studies on inducible genes show that depleting H2A.Z activates gene

expression, and this activation is accompanied by an increase in H2A.Z acetylation

(Dalvai, Bellucci et al. 2012). The same phenomenon was observed on the p21 gene in

ER, p53-negative cells (MDA-MB231). Here, however, depletion of H2A.Z was not

sufficient to activate p21. To activate p21, in fact, it was necessary to increase H2A.Z

acetylation, using for example a treatment with HDACi (Bellucci, Dalvai et al. 2013).

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Thus, it becomes clear that post-translational modifications of H2A.Z, as of all the other

histones and their variants, are key for regulating gene expression.

E. H2A.Z acetylation.

The N-terminal tail of H2A.Z is acetylated in different species (Bruce, Myers et al. 2005;

Mehta, Braberg et al. 2010; Dalvai, Bellucci et al. 2012). Human H2A.Z is acetylated at

residues K4, K7, K11 and K13 while yeast H2A.Z is at K3, K8, K10 and K14 residues

(Raisner, Hartley et al. 2005; Samuelson, Narita et al. 2005; Halley, Kaplan et al. 2010). In

yeast, the predominant acetylation site is the K14 and several groups have reported that

Esa1 (the histone acetyltransferase found within the NuA4 HAT complex) and Gcn5 (the

HAT part of the SAGA complex) are responsible for H2A.Z acetylation in vivo (Keogh,

Mennella et al. 2006; Millar, Xu et al. 2006). H2A.Z K14 acetyl is found on the promoters

of actively transcribed genes, whereas the non-acetylated form is associated with

inducible but repressed genes (Millar, Xu et al. 2006). Similar studies in chicken cells have

shown that the acetylated form is concentrated at the 5’ end of active genes but absent

from inactive gene (Bruce, Myers et al. 2005). In addition, activation of genes in

mammalian cells is accompanied by loss of H2A.Z at the 5’ end of genes: this is the case

for c-myc gene upon activation by interleukin-2 (Farris, Rubio et al. 2005), p53-regulated

p21 upon DNA damage (Gevry, Chan et al. 2007) and GR-regulated genes upon hormone

activation (John, Sabo et al. 2008). Valdés-Mora et al. however, suggested in a genome-

wide analysis that in prostate cancer cells, acetylation of H2A.Z at the promoter of genes

correlated with their activity and with tumor development (Valdes-Mora, Song et al. 2012).

We further developed this notion by demonstrating that acetylation of H2A.Z is a key

player for gene activation in breast cancer cells. While the total amount of H2A.Z usually

decreases after induction, the ratio of acetylated H2A.Z/total H2A.Z increases and leads

to gene activation (Dalvai, Bellucci et al. 2012; Bellucci, Dalvai et al. 2013). (Figure 9)

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Figure 9. H2A.Z deposition by the SWR1 complex and the connection with histone acetylation. (1) Canonical H2A, H4 and variant H2A.Z histones are acetylated by yeast NuA4 acetyltransferase. (2) Acetylation of H2A and H4 N-terminal tails by NuA4 stimulates the SWR1 complex through interaction with its bromodomain subunit Bdf1. This leads to enhanced ATP-dependent dimer exchange activity which replaces H2A–H2B dimers with H2A.Z–H2B dimers. (3) NuA4 might acetylate H2A.Z N-terminal tails to promote local chromatin remodeling and gene activation. (Billon and Cote 2012)

III. HDAC inhibitors.

We have seen in the preceding chapters that histone acetylation directs regulation of gene

expression. The balance between histone acetylation (performed by histone

acetyltransferases (HAT)) and histone deacetylation (performed by histone deacetylases

(HDAC)) is important for this process. The ability to move this balance in favor of hyper- or

hypo-acetylation of histones allows modulation of gene expression.

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HDAC enzymes, in fact, play an important role in cell proliferation and are involved in

several pathologies, in particular in cancer.

Purification and characterization of HDAC enzymes lead to the development of specific

inhibitors for the different classes of HDAC (HDACi). HDAC inhibition is now considered

for experimentally modifying gene expression and, as a consequence, also for therapeutic

treatments. It is possible to propose therapeutic trials adapted to different cancer types,

depending on the HDAC involved.

Histone deacetylase enzymes can be inhibited by different means. First, using RNAi to

inhibit the mRNA translation; second, by using molecules, such as MC1568

(Scognamiglio, Nebbioso et al. 2008) or the DIM (3,3’-diindolylmethane) (Li, Li et al.

2010), which allow protein degradation. Moreover, using for example calyculine A, protein

complexes formation which are important for HDAC function can be blocked. This

compound inhibits all cellular phosphorylation phenomena and the action of some HDAC.

However, several compounds that inhibit the catalytic activity are available. They

specifically block or compete with access to the active site of the enzymes. Among these,

there are Trichostatin A (TSA) and Sodium Butyrate (from natural sources), and SAHA,

Tubacin and others (which are synthetic) (Figure 10).

One of the most powerful HDAC inhibitors is TSA. This compound is produced by

Streptomyces during fermentation. TSA is, naturally, an antifungal agent, but in 1990,

Yoshida showed that it is able to inhibit HDACs (Yoshida, Hoshikawa et al. 1990). TSA is

able to inhibit the HDAC enzymes of class I, II and IV.

In general, there are six classes of HDACi: the carboxylates, the benzoamides, the epoxy

acetones, the cyclic peptides, the hybrid molecules and the hydroxamic acids and their

derivatives (Figure 10). Most known HDACi are pan-inhibitors of one or more classes of

HDAC enzymes.

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Figure 10. Structural classes of HDAC inhibitors. Six basic classes of HDAC inhibitors are shown: a)

small-molecular-weight carboxylates, including sodium butyrate, valproic acid, and sodium

phenylbutyrate; b) hydroxamic acids, including CBHA, TSA, SAHA, and LAQ824; c) benzamides,

including MS-275 and CI-994; d) epoxyketones, including AOE and trapoxin B; e) cyclic peptides,

including depsipeptide and lapicidi; and f) hybrid molecules, such as CHAP31 and CHAP50

(Drummond, Marx et al. 2005).

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A. Inhibitors for HDAC class I/II.

The inhibitors of the HDAC class I and II can regulate gene expression directly via histone

hyper acetylation or indirectly through the acetylation of transcription factors controlled by

this modification type. Generally, the HDACi regulated genes are involved in proliferation

and cellular cycle progression (oncogenes or oncosuppressors), cell survival, DNA

replication or repair, or protein degradation by the ubiquitin/proteasome system

(Drummond et al., 2005). Natural molecules like Sodium Butyrate (NaBu), the

phenylbutyrate (PB), the phenylacetate (PA) and valproic acid (VPA) are carboxylates.

These compounds have a lower efficiency than hydroxamic acids (Gottlicher, Minucci et

al. 2001). VPA is used as an anti-epileptic treatment. The NaBu can be obtained from

fermentation of an anaerobic bacteria present in the human intestinal lumen. It is known to

be a non-competitive inhibitor of HDAC but it is less specific and powerful than TSA (Kruh

1982).

MS275 and N-acetyldinaline are benzamides and act like the hydroxamates on the active

site of the HDAC (see below). MS275 has a strong inhibitory activity (Saito, Yamashita et

al. 1999) and has entered clinical trial phase under the brand name Etunostat. The

cyclopeptides are characterized by their structure and their strong inhibitory action of

HDAC. They have a bacterial or fungal origin. Trapoxine, for example, at nanomolar

concentration induces histones hyperacetylated accumulation in mammalian cells and

inhibits in an irreversible manner the purified HDAC deacetylation (Kijima, Yoshida et al.

1993). The CHAPs inhibitors (Cyclic hydroxamic acid-containing peptides) are TSA and

Trapoxine hybrids and have a reversible action on HDAC (Furumai, Komatsu et al. 2001).

Since the 1990s, TSA remains the HDAC inhibitor of reference. The inhibitory activity of

TSA in the catalytic pocket of HDAC has been a model for the synthesis of other

inhibitors. For example, SAHA, also called Vorinostat, is a HPC (Hybrid polar compounds)

synthetic compound, which is a hydroxamic acid inhibitor of second generation (Richon,

Emiliani et al. 1998).

B. Inhibitors for class III HDACs

Different type of compounds, synthetic or natural, can inhibit Sirtuin deacetylase activity.

Nocotinamide, for example, a product of the deacetylation reaction, is a non-competitive

inhibitor for HDACs of class III. There is also the splitomycine, found by screening

(Bedalov, Gatbonton et al. 2001). But, the most commonly used is sirtinol (Sir 2 inhibitor

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naphtol) (Grozinger, Chao et al. 2001). To study sirtuins, activator compounds can be

used to stimulate their activity. One of them, Reveratrol, is also a SERM (Selective

Estrogen Receptor Modulator). Such compound is of interest since it emphasizes the link

between estrogen receptor and HDAC activities (Bhat, Lantvit et al. 2001).

Increasingly often, the HDAC inhibition is used in fundamental or clinical research to

modulate gene regulation. In particular, our group is interested in estrogen-dependent

gene regulation in breast cancer cell lines. Therefore, we decided to use the characteristic

effects of HDAC inhibitors to investigate changes in gene expression and regulation (see

Results). Here, the aim is to introduce and explain the basis of estrogen-dependent gene

regulation: the mechanisms, the actors, the “exceptions” and the aberrations.

IV. Estrogen-dependent gene regulation.

Four pathways controlling estrogen-activated transcription have been described (Hall,

Couse et al. 2001) (Figure 11)

• The classic pathway (genomic pathway): ER binds estradiol, dimerizes and

associates with EREs (estrogen Responsive Elements) within regulatory

sequences of estrogen-regulated genes.

• The growth factor dependent pathway: growth factors activate kinase function to

phosphorylate a variety of transcription factors, including AP1 and ER. Activation

of ER-target genes can occur in the absence of estradiol, but still requires ER.

• The ERE-independent pathway. In this pathway, the ER interacts with other

transcription factors and does not need to bind ERE sequences on DNA.

• The non-genomic pathway. ER plays no direct role in gene expression.

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Figure 11. The different mechanisms of estradiol and estrogen receptor signaling. The biological

effects of estradiol (E2) are mediated through four ER pathways. 1, classical ligand-dependent, E2-ER

complexes bind to EREs in target promoter leading to an up- or down-regulation of gene transcription.

2, ligand-independent. Growth factors (GF) activate intracellular kinase pathways, leading to

phosphorylation (P) and activation of ER at ERE-containing promoters in a ligand-independent

manner. 3, ERE-independent, E2-ER complexes alter transcription of genes containing alternative

response elements such as AP-1 through association with other DNA-bound transcription factors

(Fos/Jun), which tether the activated ER to DNA, resulting in an up-regulation of gene expression. 4,

Cell-surface (nongenomic) signaling, E2 activates a putative membrane-associated binding site,

possibly a form of ER linked to intracellular signal transduction pathways that generate rapid tissue

responses. (Hall, Couse et al. 2001)

When ER is inactive, it is present in cell as a monomeric form aporeceptor. This monomer

shuttles between the cytosol and the nucleus, and is associated with a multiprotein

complex: dimers of heat shock protein hsp90 (Catelli, Binart et al. 1985), a monomer of

hsp70 (Ratajczak, Carrello et al. 1993), the p59 protein (Renoir, Radanyi et al. 1990) and

many other proteins (Segnitz and Gehring 1995). Hsp90, hsp70 and the other proteins,

bind the receptor to keep it in a “standby” conformation, ready to associate with ligands

(Bohen 1995).

Estradiol binds to the ER in the cytosol; activated ER dissociates from the chaperon

protein complex. The hormone binds the E domain of ER� (Figure 11). The ligand-binding

domain (LBD) changes conformation. This conformational change allows the passage at a

phosphorylate and transcriptionally active form of ER�. This phosphorylation is performed

after E2 binding and increases the affinity of the ER�/E2 complex (Denton, Koszewski et

al. 1992).

E2 binding induces ER� dimerization, essential for the normal functions of ER�. Mutations

that block the dimerization inactivate ER� (Lees, Fawell et al. 1990). In all the cases, the

agonists as well as the antagonists of ER�, stabilize the dimer (Tamrazi, Carlson et al.

2002). ER� dimers, in the nucleus, bind DNA on the ERE sequences which are minimal

palindromic sequence of 13bp: 5’-GGTCAnnnTGACC-3’ (Klein-Hitpass, Ryffel et al.

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1988). In fact, the majority of EREs are imperfect palindromes; this is the case of the ERE

on pS2/TFF1 promoter (Berry, Nunez et al. 1989), of the half ERE on the cathepsin D

promoter and it’s the same (half ERE) for the progesterone receptor (PR) promoter

(Augereau, Miralles et al. 1994). ER� in complex with an agonist or antagonist has the

same affinity for the ERE sequences (Cheskis, Karathanasis et al. 1997). Once

associated with EREs, ER recruits many cofactors, which can be transcription activators

or repressors depending on the target gene (Klinge, Jernigan et al. 2001) (Table 1-2). The

majority of the phosphorylation sites of ER� localize in the AF-1 domain on the protein

(Figure 12). The Cdk2/cyclineA complex phosphorylates the serines 104 and 106

(Rogatsky, Trowbridge et al. 1999). Serine 118 can be phosphorylated in two manners: in

an estrogen-dependent manner, via the cdk7 subunit of TFIIH complex (Chen, Riedl et al.

2000) or in an estrogen-independent manner via MAP kinases activity (Kato, Endoh et al.

1995; Bunone, Briand et al. 1996). RSK1, a kinase of the MAPKs pathway,

phosphorylates the serine 167 (Joel, Smith et al. 1998). The same serine can be

phosphorylated also by AKT (Campbell, Bhat-Nakshatri et al. 2001). PKA phosphorylates

the serine 236, which localizes in the DNA binding domain (Chen, Pace et al. 1999).

Finally, the Src tyrosine-kinases family can phosphorylate ER� in the E region on the

tyrosine 537 (Migliaccio, Wetsel et al. 1993; Arnold, Vorojeikina et al. 1995).

Figure 12. Schematic diagram of the human estrogen receptor, ER�. The estrogen receptor consists of

six functional domains, including the DNA-binding domain (DBD), the ligand-binding domain (LBD),

the ligand-independent activation function AF-1, and the ligand-dependent activation function AF-2.

(Adapted from (Shao and Brown 2004)).

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Table 1-2. Adapted from (Hall and McDonnell 2005).

In the majority of the cases, the ER� phosphorylation has a positive effect on the

transcriptional activity. The phosphorylated AF-1 domain is a docking platform for the

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recruitment of chromatin modification and remodeling complexes. In addition, the AF-2

domain can also be phosphorylated (Figure 12). The Src kinases are able to

phosphorylate ER� on the tyrosine 537. This phosphorylation can control, maybe through

a conformational change, the ER�/E2 binding, the ER� dimerization (Migliaccio, Wetsel et

al. 1993; Arnold, Vorojeikina et al. 1995) and/or cofactor recruitment. The PKA (Protein

kinase A) can negatively regulate ER� in absence of hormone. In fact, this enzyme

phosphorylates ER� on serine 236 and prevents its dimerization (Chen, Pace et al. 1999).

ER� is a target for other post-translational modifications. Certain lysine residues can be

acetylated by p300 (Wang, Fu et al. 2001). In addition, ER� acetylation is ligand- and

p160-dependent. Lysines 266 and 268 are targeted by p300. The acetylation of these

residues increases the ER� binding to the DNA and its ligand-dependent transcriptional

activity (Kim, Woo et al. 2006). ER� can be sumoylated by SUMO-1; this modification has

an impact on the transcriptional activity of ER� (Sentis, Le Romancer et al. 2005). Finally

ER� can be ubiquitinated, the ubiquitination plays a role in ER� degradation via the

proteasome (Nawaz, Lonard et al. 1999) and in ER� activity regulation (Reid, Hubner et

al. 2003).

Finally, ER� can bind DNA in an ERE independent manner. When bound by ligands, the

ER� can behave as a cofactor for other transcriptional factors: fos/jun (AP-1), Sp1, NF-kB

or ATF-2/c-jun (Nilsson, Makela et al. 2001; Baron, Escande et al. 2007). ER� forms

different complexes that are able to recognize different promoter elements (responding to

AP-1, to SP1, to NF-kB) and modulate the transcription by “docking”. In fact, ER� after E2

binding can interact with fos/jun transcription factors to modulate their transcriptional

activity when they bind DNA via the AP-1 sites (Gaub, Bellard et al. 1990). Moreover, the

complex ER�/E2 can bind physically with Sp-1 protein modulating its transcriptional

activity (Porter, Saville et al. 1997; Saville, Wormke et al. 2000). The effects of this

interaction on the transcription are cell type dependent (Saville, Wormke et al. 2000; Safe

2001). In addition, several studies have shown that the ER�/E2 complex inhibits the NF-

kB activity (Stein and Yang 1995; Harnish, Scicchitano et al. 2000). A study shows that in

U2OS and MFC-7 cell lines, the ER�/E2 complex interacts with NF-kB and prevents its

binding at the response elements present on the IL6 gene promoter (Stein and Yang

1995). In conclusion, ER�/E2 binds ATF-2/c-jun increasing the transcriptional activity on

the CRE (cAMPC response element) sites. This induction, described for the CCND1

(Cyclin D1) expression regulation, is hormone-dependent and needs the functions of AF-1

and AF-2 domains on ER� (Sabbah, Courilleau et al. 1999).

In 1994, Lieberherr and Gross have shown the increase of the intracellular concentration

of calcium, inositol triphosphate (IP3) and diacylglycerol (DAG), after estradiol induction.

In addition, they showed that using pertussis toxin (which blocks the receptor binding G

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proteins), it is possible to prevent these effects. These observations suggest the existence

of a cell membrane receptor, involved in estrogen response, coupled to phospholipases

and proteins (Lieberherr and Grosse 1994). G-proteins coupled receptors represent a

large receptor family with seven transmembrane domains. They recognize stimuli in

heterodimeric form (G���) to dissociate the complex in G� G�� subunits (Gatherer 2000).

The best candidate to translate estrogen activation in this manner is the G-proteins

coupled receptor G30 (GPR30). Thomas and collaborators have shown a strong affinity of

E2 for GPR30 in ER�-negative but GPR30-positive cells (Filardo and Thomas 2005).

Moreover, they have shown that GPR30 activation induces calcic mobilization and MAP

Kinases pathway activation (Revankar, Cimino et al. 2005). In addition, this membrane

receptor seems to be involved in anti-estrogens resistance effects.

V. Breast cancer.

Cancer is a major health problem, which is actually the first cause of death in men (33%)

and the second cause of death in women (23%). In the past 20 years, the mortality rate

was diminished by 22% with an acceleration in decrease during the last 10 years for men.

For women, mortality also decreased but not with the same trend as for men in the same

period.

Breast cancer in particular, is a pathology with a slow evolution. The main risk is

development of metastasis, which represents the first cause of death for this disease.

There are different types of breast cancer, among which adenocarcinomas are the most

common (95%). The breast cancer evolves from channels (canal cancers) or from lobes

(lobular cancers) of mammary glands. They are called in situ if the cancer cells are

confined to the canals or lobes, and infiltrating if the cancer cells are present in

neighboring tissues. In this latter case, the malignant cells spread into the ganglions in the

armpits, but also in the rest of organism.

Upon diagnosis, a breast cancer is classified for its morphology, size, invasive capacity,

and for the presence of hormone receptors or for hyper-expression of HER2 protein

(Human epidermal growth factor receptor). Recently, this classification has been

improved, thanks to genomic analysis that permits distinction into luminal, basal or HER2+

type cancers. The basal cancers (or cell lines) are the most aggressive ones.

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A. Breast cancer treatments.

The treatment depends on the patient. Depending on the tumor characteristics, five

different treatments are possible and frequently combined: surgery, radiotherapy,

chemotherapy, hormone therapy and targeted treatments.

Our group is interested in the gene regulation program induced upon hormone treatments.

Important differences characterize tumors that express the Estrogen Receptor and tumors

that do not express this receptor. The first one can be treated by hormone-therapy, while

the second type is insensitive to all hormonal treatments. Hormone therapy works by

blocking the activity of the estrogens, which are the steroid hormones involved in the

development of breast cancer. There are different strategies: the suppression of the

estrogens production via oophorectomy; the inhibition of the steroidal precursor

conversion in estrogen using aromatase inhibitors (AI) or, finally, the direct inhibition of the

estrogen receptors (ER) activity. This last approach uses anti-estrogen compounds as

competitive inhibitors of estrogens such as SERMs (Selective estrogen receptor

modulator): Tamoxifen or Raloxifen for example. Alternatively, pure anti-estrogen

compounds such as ICI182780 (Fulvestrant) which bind to ER and induce its degradation

and are therefore called SERD (Selective estrogen receptor disruptor) (Howell 2000) can

be administered.

These treatments are helpful for the cancer type called "hormone-dependent". Clinically,

there are some biological markers used to predict the cancer hormone-therapy response

(Osborne and Schiff 2005; Nguyen, Taghian et al. 2008). The key biomarkers are

expression levels of ER�, PR (progesterone receptor) or HER2 in cancer biopsies.

B. Biomarkers.

The majority of breast cancers (60 – 70%) are hormone-dependent. The analysis of ER�

expression level consents to classify clinically the cancer in two categories: breast

cancers called ER+ (Estrogen receptor positive, hormone-dependent) and the cancers

called ER- (Estrogen receptor negative, hormone-independent). Actually, the presence of

ER is associated with a better vital prognosis.

The progesterone receptor has two isoforms (PRA and PRB), which are under the control

of E2 and reflect the good functioning of ER. In fact, a study has shown that 53% of

patients ER+ respond to hormonal therapy. This proportion increases to 69% if the tumor

is ER+ PR+, and decreases to 32% if the cancer is ER+ PR- (Horwitz 1988).

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HER2 expression is also used for the choice of the therapy (Skliris, Leygue et al. 2008). In

fact, HER2 hyper-expression allows erbB2 (Erythroblastic leukemia viraloncogene

homolog 2) expression, which plays a major role in the development of the tumors called

HER2+ (Slamon, Clark et al. 1987). Recently, Arteaga et al. have shown that 30% of

breast cancers overexpress HER2 and that it is a negative indicator for survival (Arteaga,

O'Neill et al. 2008). Based on these markers we can distinguish four therapeutic

categories: ER+/HER2-, ER+/HER2+, ER-/HER2+, ER-/HER2-.

Hormonal therapies are based on the use of SERMs and SERDs. SERMs are divided into

two families: the triphenyllethylenic derived, the most used being Tamoxifen and the

benzothiophene derivative, Raloxifen (Jordan, Gapstur et al. 2001). Tamoxifen has been

one of the first and most effective therapy for the treatment and prevention of breast

cancer (Jordan, Osipo et al. 2003). This molecule can behave like estrogens or anti-

estrogens depending on the tissue (Jordan, Gapstur et al. 2001). In some cell lines where

this molecule acts like an antagonist, it induces the recruitment of several transcriptional

corepressors by ER. In endometrial cells, where it acts like an agonist, tamoxifen induces

the recruitment of transcriptional coactivators (Shang and Brown 2002).

The secondary effects of SERMs have induced the development of another class of

modulators: SERDs (Miller, Bartlett et al. 2007). The compound ICI182780 is the SERD's

most employed in therapy (Osborne, Wakeling et al. 2004). Unlike SERMs, which stabilize

ER� and induce an increase in protein accumulation, SERDs induce rapid degradation of

the ER� via the proteasome (Callige, Kieffer et al. 2005). In fact, SERDs bind the ER�

and direct it on the ERE present on the promoters of ER regulated genes allowing the

recruitment of the corepressor N-CoR (Nuclear receptor corepressor) (Webb, Valentine et

al. 2003). SERDs can block breast cancer cell proliferation of MCF-7 (ER� + cell line)

(Pink and Jordan 1996). The ICI182780 is an interesting treatment used for the

Tamoxifen-resistant breast cancers (Fan, Wang et al. 2007).

In contrast with SERMs and SERDs, Aromatase Inhibitors (AI) do not interact directly with

the ER, but prevent estrogens synthesis by inhibiting the aromatase enzyme (CYP19A1),

which converts androgens into estrogens in tumors (Bulun, Lin et al. 2009). In fact, AI

inhibits the aromatase activity and diminishes estrogens concentration until almost

undetectable levels (Ali and Coombes 2002).

All these compounds and treatments target the normal estrogenic regulation. To better

understand how the treatments or/and therapy work, we need investigate the mechanisms

that underlie canonical estrogen-regulation.

Tumors and cells that are non-responsive to estrogen and hormone-treatments are, as

said, more aggressive and difficult to treat. One of the major characteristics of these cells

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is the loss of the canonical cell cycle regulation. ER-negative MDA-MB231 cells, for

example, have a non-functional p53 protein. This loss leads to the deregulation of the

normal p53 pathway, which is involved in several processes, as well as DNA damage

response, apoptosis, cell survival, etc. (Gartel and Radhakrishnan 2005). One of the most

important effector of p53 signaling is p21 (Waldman, Kinzler et al. 1995). The loss of a

functional p53, is frequently also associated with loss of p21 expression. As a

consequence, cell cycle control is lost and cells become hyperproliferative and more

aggressive.

VI. P21

p21CIP1/waf1 is a cyclin-dependent kinase inhibitor (cdki) belonging to the Cip/kip family.

This transcription factor negatively modulates cell cycle progression by mainly inhibiting

the activity of cyclin/Cdk2 complexes during G1 phase, and cyclin/Cdk1 during G2 phase.

Overexpression of p21 results in G1 or S phase arrest (Ogryzko, Wong et al. 1997).

Similarly, cells lacking p21 cannot properly block cell cycle in response to p53 activation

following DNA damage (Waldman, Kinzler et al. 1995).

Various mechanisms exist to regulate the levels of p21 in a cell. There is a transcriptional

regulation throughout the cell cycle, following DNA damage, and during differentiation and

senescence (Gartel and Radhakrishnan 2005). c-Myc is a proto-oncogene which

represses p21 transcription. The exact mechanism of repression is not clear and still

controversial but c-Myc may employ a multitude of pathways to repress p21 (Caldon, Daly

et al. 2006). Likewise, epigenetic silencing or modifications of mRNA stability are known to

modify p21 activity. In addition, the stability of p21 is regulated by proteasomal

degradation. An ubiquitin ligase, Skp2, triggers p21 degradation by ubiquitination of the N-

terminal part of the protein (Bloom and Pagano 2004). P21 can also be inactivated by

PKB/Akt phosphorylation that leads to a cytoplasmic localization (Liang and Slingerland

2003).

This protein mediates growth arrest after a transcriptional activation by p53 when cells are

exposed to DNA damaging agents such as Doxorubicin or �-irradiation. In addition, p21

can bind the proliferating cell nuclear antigen (PCNA) and thereby block DNA synthesis

(Brugarolas, Moberg et al. 1999) (Waga, Hannon et al. 1994). Apart from p53, a variety of

other factors including Sp1/Sp3, Smads, Ap2, signal transducers and activators of

transcription (STAT), BRCA1, E2F-1/E2F-3, and CAAT/enhancer binding protein � and �

are known to activate p21 transcription (Gartel and Tyner 1999).

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A. The p21 promoter and HDACi activation of p21.

p21 expression can be modulated via promoter composition changes. In fact, the proximal

p21 promoter contains six Sp1 binding sites (Gartel, Goufman et al. 2000). Both Sp1 and

Sp3 factors were found to regulate promoters of several cell cycle regulating genes,

whereby Sp1 is a potent trans-activator and Sp3 rather a repressor (Birnbaum, van

Wijnen et al. 1995). Co-factors such as p300/CBP and the p300/CBP-associated factors

(PCAF) that possess intrinsic HAT activity cooperate with Sp1/Sp3 to induce expression

of the p21 promoter in response to different stimuli (Owen, Richer et al. 1998; Billon,

Carlisi et al. 1999; Kardassis, Papakosta et al. 1999). P300/CBP can be stimulated by

p21-induced inhibition of their repressor domains in the amino-terminal end of the protein

(Snowden, Anderson et al. 2000).

Many reports indicate that HDACi induce p21 expression mainly by activating the Sp1/Sp3

pathway independently of p53 (Nakano, Mizuno et al. 1997; Gartel and Tyner 1999).

Recent studies clearly show that multiple factors, such as ATM (Ju and Muller 2003) and

c-Myc (Li and Wu 2004) are involved in HDACi-induced p21 expression in several human

cancer cell lines. A direct role for p53 in HDAC-associated p21 expression has also been

reported (Lagger, Doetzlhofer et al. 2003).

HDACi induce p53-independent expression of p21 via Sp1-binding sites in the p21

promoter. In fact, it seems that HDAC1 competes with p53 to bind a specific region on the

p21 promoter. After HDACi treatment, HDAC1 Is released from the Sp1-binding site at the

p21 promoter leading to loss of repression and an induction of transcription (Figure 13).

Furthermore, p300 is recruited to the p21 promoter (Zhao, Subramanian et al. 2006).

Although Trichostatin A effectively induces p21 expression, cell cycle arrest and apoptosis

in human gastric and oral carcinoma cell lines (Suzuki, Yokozaki et al. 2000), it has a

lower ability to induce p53 acetylation but promotes the recruitment of co-activators to the

p21 promoter and enhances histone acetylation around it (Barlev, Liu et al. 2001). The

HDACi butyrate induces p21 and apoptosis in colorectal cancer cells (Mahyar-Roemer

and Roemer 2001), and the absence of p21 increases butyrate-induced apoptosis in

HCT116 colon cancer cells. Varinostat (SAHA) induces an up to nine-fold increase in p21

mRNA and p21 protein in T24 bladder carcinoma cells (Richon, Sandhoff et al. 2000)

mainly due to enhancement of acetylation of the histones H3 and H4 around the promoter

region (Gui, Ngo et al. 2004). Moreover Lin et al. showed that HDAC1 and HDAC2 are

recruited to the Sp1/Sp3 sites to repress p21 expression. Lovastatin expels HDAC1/2

from the p21 promoter and recruits CBP, resulting in histone-H3 acetylation (Lin, Lin et al.

2008).

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The dissociation of HDAC and binding of p300/CBP to the gene promoter other than p21

were only found in transforming growth factor-� type II receptor induced by TSA (Huang,

Zhao et al. 2005). The HDACi-induced p21 expression may depend on cellular context,

p53 expression pattern, p21 promoter composition and other factors. In fact, we show that

in ER- p53- negative breast cancer cells HDACi-induced p21 expression seems to depend

on H2A.Z acetylation. In this cellular context the depletion of this histone variant prevented

p21 activation by TSA treatments (see Results and Discussion) (Bellucci, Dalvai et al.

2013).

Figure 13. Schematic model of p21cip1/waf1 regulation. (A) Repression of the p21cip1/waf1 promoter

by different factors acting at the transcriptional and the post-transcriptional (blue filled) level. Whereas

some can bind at the p21cip1/waf1 promoter directly (Sp1, p53, p300, HDAC1, E2F), others interfere

with these regulators (c-Myc, DNMT3a, DNMT1, PCNA). (B) Putative p21cip1/waf1 promoter status after

HDACi treatment. Acetylated histones around the p21cip1/waf1 promoter facilitate the access of

transcription factors and secondly, HDAC1 is released from the Sp1 site (where it competes with p53

for binding at the p21cip1/waf1 promoter) leading to a loss of repression and induction of

p21cip1/waf1 expression. Acetylated p53 has a higher binding efficacy and is recruited to the

p21cip1/waf1 promoter together with co-activators such as p300 or P/CAF. (Ocker and Schneider-

Stock 2007)

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VII. Thesis project.

As we have seen, epigenetic mechanisms play a major role in regulating gene expression.

Recent advances in experimental and computational technologies (genome wide, ChIP-

seq, ChIP on Chip etc etc) rendered gathering genome-wide information on the presence

and possible role of chromatin components possible, without, however, understanding the

mechanisms that act at the level of a single gene, a precise protein and its modification in

specific cell types.

Using breast cancer cells as a model system, I was interested in the involvement of the

histone variant H2A.Z in the transcriptional gene regulation. We decided to study the

expression control of a non-ER target gene, p21. In the laboratory, we had two principal

breast cancer cell types. MCF-7, which are ER� positive, respond to antiestrogen

treatment, in which ER�-regulated genes are activated by addition of estradiol. MCF7

cells express a wild type p53 protein. In contrast, the MDA-MB-231 cell line is ER�-

negative and p53 is mutated. Their growth is insensitive to estradiol or antiestrogen

treatments. In fact, some genes are constitutively silenced and some others escape to the

canonical ER�-dependent activation. Although epigenetic modifications are associated

with their transcriptional states, the mechanisms by which these genes escape hormonal

regulation durably are largely unknown.

I focused on regulation of p21 gene expression in MDA-MB231 cells. P21 is

transcriptionally repressed in MDA-MB231 and the findings on how p21 would be

activated were controversial. A recent study had linked H2A.Z recruitment to p21 gene

expression (Gevry, Chan et al. 2007). This study was performed in osteosarcoma cells,

which are p53-positive. In this study, H2A.Z was shown to bind the p53-binding sites at

the p21 promoter. For p21 activation, during DNA damage response, H2A.Z dissociates

from the p53 binding sites to allow p53 to bind and to activate p21 transcription. At the

same time, p21 expression is known to be stimulated by inhibitors of histone deacetylases

(HDACi). Normally, this activation can be p53-independent or dependent, according to the

cell system used (Nakano, Mizuno et al. 1997; Gartel and Tyner 1999; Lagger,

Doetzlhofer et al. 2003). But the mechanism behind p21 activation, via HDACi, remains

poorly understood.

My challenge was to understand the implication of H2A.Z in p21 activation in MDA-

MB231 cells (ER�-negative, p53 -/-). Would HDACi modulate this activation?

My studies show that p21 gene activation in MDA-MB231 cells is p53-independent and

controlled by the H2A.Z histone variant and its acetylation (Bellucci, Dalvai et al. 2013).

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We found that H2A.Z was strongly associated with the transcription start site of p21. A

TSA treatment stimulated p21 expression and increased acetylation of H2A.Z present at

the p21 promoter. Depleting the cellular pool of H2A.Z compromised p21 activation and

response to HDACi. Acetylation of H2A.Z rather than its association of regulatory element

per se was important for p21 expression.

It had been also shown that H2A.Z plays a role in Estrogen Receptor-dependent gene

activation (Gevry, Hardy et al. 2009). In this study, using MCF-7 (ER�-positive cell line)

and the TFF1 gene, they show that H2A.Z is recruited with ER� in a cyclic pattern for

gene activation.

In our laboratory, we had preliminary data showing that H2A.Z behavior on gene

promoters and enhancers could not be generalized although a subset of genes was

regulated in a similar fashion, distinct from what had been proposed for TFF1. In

particular, following gene activation until estradiol treatment, H2A.Z was lost from several

ER-target genes, including CCND1, PGR and ESR1 in MCF7 and MDA-MB231 cells. In

addition, the posttranslational status of H2A.Z, in particular its acetylation appeared to

correlate with regulation of gene expression. We decided to use an ER� inducible gene in

MCF-7, which in MDA-MB231 was constitutively expressed at very low levels. We

demonstrated that H2A.Z acetylation was critical for Cyclin D1 regulation in estrogen

receptor positive and negative breast cancer cells. We identified the enzymes involved in

H2A.Z acetylation (Tip60) and in chromatin remodeling (Tip48), to propose a model for

priming chromatin to prepare for transcription activation of this gene (Dalvai, Bellucci et al.

2012) Dalvai et al., PLOS Genetics, in press.)

Here, I would like to emphasize that breast cancer cell lines are used as a model for

studying molecular mechanisms. Our research is mechanistical in nature and aims at

understanding the mechanism of H2A.Z involvement in gene regulation. Our findings will

have some repercussions on the comprehension of breast cancer physiology, but it is not

our principal aim. The immediate aim was to clarify the role of H2A.Z and its acetylation in

gene expression; to give, in this manner, our contribution to better understanding how

chromatin structure and composition can affect the biology of a cell. It is notable that

depleting a single histone variant can reverse aberrant gene expression independently of

expression of canonical transcription factors, here the ER. Thus, our findings open new

avenues for acting upon deregulated gene expression with the aim to better know how

cancer cells function, or rather, disfunction, or evolve.

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VIII. Results.

a) Luca Bellucci*, Mathieu Dalvai*, Silvia Kocanova, Fatima Moutahir, and Kerstin Bystricky. (2013) “Activation of p21 by HDAC inhibitors requires acetylation of H2A.Z.” PLOS One doi: 10.1371/journal.pone.0054102. �

b) Dalvai M, Bellucci L, Fleury L, Lavigne AC, Moutahir F, Bystricky K. (2012) “H2A.Z-dependent crosstalk between enhancer and promoter regulates Cyclin D1 expression.” Oncogene doi: 10.1038/onc.2012.442

c) Mathieu Dalvai, Laurence Fleury, Luca Bellucci, Silvia Kocanova and Kerstin Bystricky. (2013) “TIP48/reptin and H2A.Z requirement for initiating chromatin remodeling in estrogen activated transcription.” PLOS genetics (in press).

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Activation of p21 by HDAC Inhibitors RequiresAcetylation of H2A.Z

Luca Bellucci1,2., Mathieu Dalvai1,2., Silvia Kocanova1,2, Fatima Moutahir1,2, Kerstin Bystricky1,2*

1 Laboratoire de Biologie Moleculaire Eucaryote, Universite de Toulouse, Toulouse, France, 2 LBME-UMR5099, CNRS, Toulouse, France

Abstract

Differential positioning of the histone variant H2A.Z in a p53 dependent manner was shown to regulate p21 transcription.Whether H2A.Z is involved in p21 activity in the absence of p53 is not known. The p21 gene is repressed in estrogenreceptor (ER) negative cell lines that are p532/2 and hormone independent for their growth. Here we demonstrate thatclass I and II pan Histone deacetylase inhibitors (HDACi) induce p21 transcription and reduce cell proliferation of MDA-MB231, an ERa-negative mammary tumor cell line, in a H2A.Z dependent manner. H2A.Z is associated with the transcriptionstart site (TSS) of the repressed p21 gene. Depleting H2A.Z did not lead to transcription of p21 but annihilated thestimulating effect of HDACi on this gene. Acetylation of H2A.Z but not of H3K9 at the p21 promoter correlated with p21

activation. We further show that HDACi treatment reduced the presence of the p400 chromatin remodeler at the p21 TSS.We propose a model in which association of p400 negatively affects p21 transcription by interfering with acetylation ofH2A.Z.

Citation: Bellucci L, Dalvai M, Kocanova S, Moutahir F, Bystricky K (2013) Activation of p21 by HDAC Inhibitors Requires Acetylation of H2A.Z. PLoS ONE 8(1):e54102. doi:10.1371/journal.pone.0054102

Editor: Wei-Guo Zhu, Peking University Health Science Center, China

Received August 29, 2012; Accepted December 6, 2012; Published January 18, 2013

Copyright: ß 2013 Bellucci et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the Ligue Nationale Contre le Cancer (fellowship to LB), the Institut National du Cancer (INCa grant #34696) and theFondation pour la Recherche Medicale (FRM, fellowship to MD). The funders had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

. These authors contributed equally to this work.

Introduction

Estrogen receptor negative breast cancer types are generally

more aggressive and prone to metastasize. The absence of

Estrogen receptor-alpha (ERa) correlates with hormone-indepen-

dent growth of these mammary tumor cells and causes un-

controlled proliferation and insensitivity to anti-hormonal treat-

ments. In ERa-negative cell lines, a subset of genes is

epigenetically silenced [1,2], while the majority of genes involved

in cell cycle control and proliferation are constitutively expressed

[1,3,4]. Aberrant gene expression is frequently the result of

chromatin modifications and composition, including histone post-

translational modifications and/or incorporation of histone

variants [5–9]. In particular, deregulation of enzyme complexes

responsible for histone acetylation and deacetylation can be

associated with breast cancer progression and an increase in tumor

malignancy [5]. Thus, compounds that change chromatin

modifications are a promising anti-cancer approach. Histone

deacetylase (HDAC) inhibitors, such as Trichostatin A (TSA),

Suberoylanilide hydroxamic acid (SAHA), Panobinostat (LBH598)

and sodium butyrate (NaB) can inhibit cancer cell growth in vitro

and in vivo [10–12] as a result of selective induction of

endogenous genes that play significant roles in G1-S progression

[5]. One of the major regulators of cell cycle progression is the

cyclin-dependent kinase inhibitor p21 CIP1/WAF1, a gene of the

CIP/KIP family, which inhibits CDK activity. p21 can be

stimulated by p53 and its activity results in cell cycle arrest and/

or apoptosis. Much of research on HDAC inhibitors has focused

on the upregulation of p21. Activation of p21 involves acetylation

of promoter chromatin, but the mechanism remains poorly

understood [13,14].

The histone variant H2A.Z has been shown to bind to the

promoter of p21 at the p53 binding sites in p53+/+ cells (U2oS)

[15]. In response to stress, H2A.Z is evicted to allow p53 to bind

which leads to p21 expression [15]. The p400 complex takes part

in this pathway and was proposed to be responsible for H2A.Z

deposition into the p21 promoter. Depleting p400 by siRNA

increases p21 expression in a p53 dependent manner and induces

premature senescence [16]. The mechanism of this activation is

unclear.

In the ERa-negative breast cancer cell line MDA-MB231 p53 is

mutated and non-functional. Here we show that activation of p21

in response to HDACi treatment of these ERa-negative cells

requires H2A.Z acetylation and exchange at its transcription start

site.

Materials and Methods

Cell Lines, Transfection and Western BlottingMDA-MB231, Hs-578T and HeLa cells were purchased from

ATCC (used up to 15 passages). All cell lines were maintained in

Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax

containing 50 mg/ml gentamicin, 1 mM sodium pyruvate and

10% heat-inactivated fetal calf serum (FCS) (Invitrogen). MDA-

MB231 cells were treated with 50 or 100 ng/ml TSA (Sigma-

Aldrich) and with LBH589 561028 or 561029 M for the

indicated times. 46106 MDA-MB231 cells where mock-trans-

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fected (pcDNA3.1) or transfected with 2mg of pcDNA3.1/Tip60

(gifts from Dr. Didier Trouche) with AmaxaH Cell line

Nucleofactor Kit V program X-013 according to the manufac-

turer’s protocol. MDA-MB231 cells were mock-transfected or

transfected with H2A.Z siRNA ON-TARGET plus SMARTpool

or scrambled (scr) siRNA (Dharmacon Thermo Scientific) with

Interferine (Ozyme) according to the manufacturer’s protocol.

Tip60 siRNA [17] was purchased from Eurogentec, and

transfected with Interferine (Ozyme) according to the manufac-

turer’s protocol. For western blotting total cell extracts were

isolated and proteins levels of p21 and GAPDH was analyzed by

immunoblotting on gel SDS-page 15% with anti-p21 (SantaCruz

Biotechnology, sc-397), anti-Tubulin (SantaCruz Biotechnology,

sc-5286) and with anti-GAPDH (Millipore, mab374).

RNA AnalysisTotal RNA was extracted using an RNeasy mini-kit (Qiagen)

and eluted with 35 ml of RNAase-free water. First strand cDNA

was generated using 1 or 2 mg of total RNA in a reaction

containing random oligonucleotides as primers with the Thermo-

Script RT-PCR system (Invitrogen). Real-time PCR was per-

formed on a MastercyclerH ep realplex (Eppendorf) using the

platinum SYBR Green q-PCR SuperMix (Invitrogen) according to

the manufacturer’s instructions. Amplification conditions: 1 min at

50uC, 3 min at 95uC followed by 40 cycles (20 s at 95uC, 20 s at

60uC, 20 s at 72uC). q-PCR for RPLP0 mRNA was used as an

internal control. The primers used in q-PCR: CDKN1A (p21, Cip1):

59-GGAAGACCATGTGGACCTGT-39 and 59-GGAT-

TAGGGCTTCCTCTTGG-39, H2AFZ: 59-

CCTTTTCTCTGCCTTGCTTG-39 and 59-CGGTGAGG-

TACTCCAGGATG-39, RPLP0: 59-TGGCAGCATCTA-

CAACCCTGAA-39 and 59- CACTGGCAACATTGCGGACA-

39, TIP60: 59-CAGGACAGCTCTGATGGAATAC-39 and 59-

AGAGGACAGGCAATGTGGTGAG-39, p400: 59-TGGCAGA-

GACTTGCTAAGGA-39 and 59-AGCGTCAACATCAGCT-

CACT-39 [18].

ChIP AssaysChIP analyses were performed as described previously [19]

from MDA-MB231 cells. Samples were sonicated to generate

DNA fragments between 500 and 700 bp. Chromatin fragments

were immunoprecipitated using antibodies against H2A.Z

(ab4174, ABCAM), Acetyl H2A.Z (ab18262, ABCAM), RNA

pol II (N20X, Santa Cruz), H3 (ab1791, ABCAM), Acetyl H3K9

(06–942, Millipore), TIP60 [20], p400 (ab70301, ABCAM), p300

(SC-584, SantaCruz Biotechnology) or an irrelevant HA antibody

(H6908, Sigma) as control. The precipitated DNA was amplified

by real-time PCR, with primer sets designed to amplify the

promoter and the coding region of the p21 gene. The primers used

in q-PCR are listed here: Primers 1: 59-TCAATGCCACCACCT-

TAACA-39 and 59-AGAGAGGCATCCTCCAGACA-39, Primers

2: 59-CTGTGGCTCTGATTGGCTTT-39 and 59-CTCCTAC-

CATCCCCTTCCTC-39, Primers 3: 59-GAAATGCCTGAAAG-

CAGAGG-39 and 59-GTCTGCACCTTCGCTCCTAT-39, Pri-

mers 4 (TSS): 59-ACTGGGGGAGGAGGGAAGT-39 and 59-

AGCTGAGCCTGGCCGAGT-39, Primers 5: 59-CCAG-

GAAGGGCGAGGAAA-39 and 59-GGGACCGATCCTAGAC-

GAACTT-39, Primers 6: 59-AGCCGGAGTGGAAGCAGA-39

and 59-AGTGATGAGTCAGTTTCCTGCAAG-39, Primers 7:

59-GCACCATCCTGGACTCAAGTAGT-39 and 59-

CGGTTACTTGGGAGGCTGAA-39.

Proliferation and Cell Death AssaysMTT assay was performed using CellTiter 96H AQueous One

Solution Cell Proliferation Assay (Promega) according to the

manufacturer’s instructions. For Trypan blue assay, 26105 cells

seeded in 35 mm dishes. Cells were harvested and counted by

trypan blue staining at indicated times following the different

treatments.

Results

Histone Deacetylase Inhibitors Block Proliferation andActivate p21 ExpressionProliferation of estrogen receptor alpha-negative (ERa-) breast

cancer cell lines is estrogen-independent. A priority in breast

cancer treatment is the development of agents able to contain or

reduce growth of these cell types, which are more aggressive and

insensitive to antiestrogens used to control estrogen responsive

tumors. ERa- cells, MDA-MB231, were grown in standard

medium and treated with the histone deacetylase inhibitors

(HDACi) Trichostatin A (TSA) or panobinostat (LBH589). Cell

growth was arrested by 50 or 100 ng/ml TSA (Fig. 1a) and 50 nM

LBH589 (Fig. S1) during the first 48 h of treatment. At later time

points, cell density decreased suggesting that prolonged exposure

to HDACi induced cell death. Growth arrest and apoptosis are

under the control of metabolic sensors, in particular the p21 gene

and its regulatory pathway. As previously described for pan

HDACi [21–23] p21 transcription was greatly activated in cells

exposed to TSA for 24 h in which we detected .3-fold increase in

p21 mRNA levels (Fig. 1b) and a massive augmentation in p21

protein expression compared to untreated cells (Fig. 1c, S1d).

Acetylated Histone H2A.Z Associates with theTranscription Start Site of p21Transcription regulation of the p21 gene is mediated by

dynamic binding of the histone variant H2A.Z to p53 binding

sites within the p21 promoter region in p53 positive cells, such as

ERa-positive MCF-7 (data not shown) or U2OS cells [15]. The

ERa- MDA-MB231 cell line bears a mutated, non-functional

p53 gene. We thus asked whether H2A.Z was also associated

with the p21 promoter in these cells as was shown to be the case

in the p53- osteosarcoma, SaOS cell line [15]. Using chromatin

immunoprecipitation (ChIP), we determined that H2A.Z was

present at the transcription start site (TSS) of the p21 gene. The

amplified fragment (#4, Fig. 2) is adjacent to a set of six putative

Sp1 binding sites that were shown to mediate p21 transcription

in a reporter assay [13]. The amount of H2A.Z detected at

upstream sequences, including the p53 recognition elements

(fragment #2), was significantly less abundant than at the TSS

(Fig. 2b). According to the hypothesis that H2A.Z containing

nucleosomes direct p53 binding [15], it was not surprising that

H2A.Z was absent from these sites. As previously described in

yeast [24], [25], decondensed chromatin is found at promoters of

inactive but inducible genes. Based on this observation, we

reduced the cellular pool of available H2A.Z by small interfering

RNAs directed against H2A.Z and assessed p21 mRNA

expression (Fig. 2c). p21 transcription remained insignificant

(Fig. 2c). ChIP experiments further confirmed that despite

a reduction in H2A.Z association with the TSS, polymerase II

(pol II) was not recruited to this inactive gene (Fig 2d). Thus, p21

transcriptional regulation does not only depend on the amount of

H2A.Z associated with its TSS. Interestingly, treatment with

50 ng/ml TSA reduced H2A.Z binding to the activated p21

(Fig. 2e, 1b). Release of H2A.Z was accompanied by recruitment

of pol II and, strikingly, by an increase in the presence of

p21 Activation by HDACi and H2A.Z Acetylation

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acetylated H2A.Z at the p21 TSS (Fig. 2f). This increase in

acetylation was not seen for histone H3K9 (Fig. 2g). Moreover,

the amount of acetylated H2A.Z and acetylated H3K9 present at

the TSS did not vary in siH2A.Z transfected cells in which p21

remained repressed (Fig. 2h–2i). We propose that acetylation of

H2A.Z rather than its presence correlates with p21 transcription

activation in ERa- breast cancers. We postulated that the anti-

proliferative effect of HDACi via p21 expression depends on

acetylation of H2A.Z. We thus asked whether this effect would

be abolished in siH2A.Z treated cells.

H2A.Z Controls HDACi Induced Growth Arrest via p21ExpressionMDA-MB231 cells were grown in standard medium, treated or

not with siH2A.Z for 24 hours before adding TSA or LBH.

Surviving cells (MTT test, Fig. 3a) and dead cells (trypan blue,

Fig. 3b) were counted before treatment as well as 24 h and 48 h

following HDACi addition. Reduced cell growth in TSA treated

cells was partly rescued in cells previously transfected with

siH2A.Z (Fig. 3a). In particular, significant counts of dead cells

were determined as soon as 24 h post treatment (Fig. 3b). While

siH2A.Z transfected cells also showed a 2-fold increase in cell

death compared to untreated cells, these cells were much less

sensitive to TSA (Fig. 3b). Cell death at 48 h was almost similar in

untreated cells compared to siH2A.Z transfected and TSA

exposed cultures. Furthermore, p21 mRNA levels did not vary

in siH2A.Z transfected cells upon HDACi treatment compared to

control cells treated only with TSA or LBH (Fig. 3c, S1c). H2A.Z

is also required for p21 activation upon TSA treatment in ER-

negative, p532/2 Hs-598T cells (Fig. S2c). In contrast, knock-

down of H2A.Z had no effect on p21 activation in HeLa cells (Fig.

S2a). Thus, H2A.Z specifically regulates p21 in ERa-negative

breast cancers following HDAC inhibitor treatment. We further

found that acetylation of H2A.Z bound to the p21 TSS was greatly

reduced in siH2A.Z treated cells exposed to TSA (Fig. 3d). Pol II

recruitment and elongation was abolished in TSA treated MDA-

MB231 cells, from which H2A.Z was depleted (Fig. 3e). Thus,

H2A.Z appears essential to mediate the anti-proliferative effect of

HDACi by regulating p21 expression. Notably, acetylation of

H2A.Z was necessary for this regulation. We next wanted to gain

insight into the mechanisms of H2A.Z acetylation at the p21

promoter in ERa- cells.

A Role for p400 but not Tip60 in p21 TranscriptionRegulationWe first tested the impact of depleting or overexpressing Tip60,

a histone acetyltransferase frequently found in complex with p400

and known to participate in H2A.Z mediated transcription

regulation [15,26]. Modulation of Tip60 mRNA levels did not

alter p21 expression levels, which remained almost undetectable

(Fig. 4a). Accordingly, association of H2A.Z, acetylated H2A.Z

and pol II did not vary at the p21 TSS in Tip60 depleted cells

(Fig. 4b). We next investigated which cofactor could be responsible

for TSA induced activation of p21. Tip60 did not seem to be

associated with the p21 TSS in MDA-MB231 cells treated or not

with TSA (Fig. 4c). In contrast, we detected significant amounts of

the p400 remodeler at the p21 TSS. Association of p400 decreased

in TSA treated cells in which p21 was activated (Fig. 4c). Reducing

the available pool of p400 alone was able to activate p21

expression (Fig. 4d) suggesting that the presence of p400 at the

p21 TSS represses this gene. Reduction of p400 allowed

recruitment of the p300 acetyltransferase (Fig. 4e). Concomitantly,

acetylation levels of H2A.Z markedly increased, due to eviction of

a fraction of H2A.Z and an increase in acetylated H2A.Z at the

p21 TSS (Fig. 4e). We propose that TSA controls cell growth by

modulating p21 expression. p21 activation requires release of p400

and H2A.Z, and an increase in acetylation of H2A.Z.

Discussion

Lack of regulation of the p21 gene whose expression is needed

for cells to respond to insults by arresting proliferation is frequently

observed in cancer. This loss is exacerbated by the absence of the

functional tumor suppressor p53 protein in more aggressive tumor

types. Histone acetyltransferase inhibitors have been shown to

activate p21 independently of p53. TSA (500 ng/ml) reduced

growth of MG63 osteosarcoma, p532/2 cells and activated

various p21 promoter constructs driving a luciferase reporter gene

[27]. We demonstrate that the endogenous p21 gene is also

activated by TSA or LBH589 in ERa- mammary tumor cells

whose growth rate is insensitive to hormones and antihormones. In

this study we provide a comprehensive analysis of the activation of

the p21 gene in ERa- MDA-MB231 cells which provides

a mechanistic link between histone acetylation, H2A.Z variant

incorporation and p21 mediated growth arrest.

Unlike in p53 positive cells, U2OS osteosarcoma or MCF-7

cells, where the histone variant H2A.Z is associated with the p53

binding sites of the inactive p21 gene, H2A.Z was present at the

Figure 1. HDAC inhibitors reduce proliferation and activate p21 transcription in ERa- negative/p53 mutated mammary tumor cells.a) MTT assay to quantify proliferation rates in the presence of Trichostatin A of MDA-MB231 cells. Two different concentrations (50 ng/ml and 100 ng/ml) were used. b-c) q-PCR and western blot analysis of p21 mRNA expression and protein levels. Experiments were performed three times.doi:10.1371/journal.pone.0054102.g001

p21 Activation by HDACi and H2A.Z Acetylation

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TSS in ERa- cells. H2A.Z binding to the TSS was frequently been

observed in yeast [25] and human cells [28] where is thought to

create a chromatin structure that is responsive to stimuli and co-

factor binding. Its presence in promoter chromatin was also shown

Figure 2. Acetylation of H2A.Z at the p21 promoter is necessary for its transcription. a) Schematic representation of the p21 promoterregion showing PCR amplified fragments (1–4). b) Binding of H2A.Z to the p21 promoter in MDA-MB231 cells. c) mRNA expression of H2AFZ (left) andp21 (right) in MDA-MB231 cells transfected with a smartpool siH2A.Z for 72h. d) H2A.Z and polymerase II (pol II) binding to the p21 TSS (fragment#4)in cells transfected with siH2A.Z or scramble siRNAs. e) H2A.Z binding to the p21 TSS (fragment#4) in cells treated with TSA (50 ng/ml) for 48 h. f–g)acetylated H2A.Z, polymerase II (pol II) (f) and acetylated H3K9 (g) binding to the p21 TSS (fragment #4) in cells treated with TSA. h-i) acetylatedH2A.Z (h) and H3K9 (i) amount at p21 TSS in cells transfected with siH2A.Z or scramble siRNA.doi:10.1371/journal.pone.0054102.g002

p21 Activation by HDACi and H2A.Z Acetylation

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to be important for activation of ERa target genes such as TFF1,

PGR or CCND1 [29], [30]. Association of H2A.Z with promoter

sequences could thus be related to an alternative pathway of gene

activation in the absence of cognate transcription factors, including

p53 or ERa. However, loss of H2A.Z binding was not sufficient to

stimulate p21 expression in MDA-MB231 cells. This observation

correlates with previous studies showing that a 50% reduction

H2A.Z association was not sufficient to induce changes in gene

regulation [31].

In 2007 Gevry et al. showed that p400 and H2A.Z associate

with the repressed p21 gene in U2OS cells [15]. In response to

DNA damage, p400 and H2A.Z were evicted to allow TIP60

recruitment and subsequent p21 activation [15]. More recently

Park et al., however, demonstrated that p400 inhibits TIP60

activity by direct binding to TIP60 via its SANT domain [26].

In ER-negative, p53 mutant breast cancer cell lines, TSA

treatment activates p21. Under these conditions, binding of p400

and H2A.Z to the p21 promoter was reduced, but the concomitant

increase in H2A.Z acetylation was independent of TIP60.

Figure 3. H2A.Z is required for the antiproliferative effect of HDACi. a) Cell growth assay b) Trypan blue assay c) qPCR analysis of p21mRNAexpression. d) ChIP analysis of Acetyl-H2A.Z. e) ChIP analysis of RNA pol II. MDA-MB231cells were treated or not with TSA (50 ng/ml) for 24 h/48 hand/or transfected with a smartpool siH2A.Z (72 h) as indicated.doi:10.1371/journal.pone.0054102.g003

p21 Activation by HDACi and H2A.Z Acetylation

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Furthermore, depleting p400 also stimulated p21 expression

independently of TIP60 suggesting that p400 exerts a repressive

effect. In contrast, depleting H2A.Z was not sufficient to mediate

p21 repression under normal conditions but it played a central role

in p21 activation upon TSA treatment. We propose that

acetylation of H2A.Z rather than H2A.Z per se is important to

drive proper p21 gene expression. A ratio in favor of acetylated

H2A.Z was associated with p21 activation upon TSA treatment.

This observation corroborates findings by Valdes-Mora and

colleagues who recently correlated aberrant gene expression in

prostate cancer cells with H2A.Z acetylation at specific promoters

[32]. It is tempting to speculate that p400 favors p21 activation by

catalysing H2A.Z eviction and allowing the recruitment of HATs

such as p300.

The histone-acetyltransferase CBP/p300 has been shown to act

on the p21 promoter at several Sp1 sites and independently of p53

as part of a multiprotein complex which also contains PR and Sp1

[33] in T47 mammary tumor cells. Here, in triple negative MDA-

MB231 cells (ER-, PR-, HER-), p300 was also present at the p21

promoter at levels proportional to transcriptional activity. Hence,

the cofactors required for p21 activation are distinct in p53

negative compared to positive cells [34]. These alternative

pathways in cells in which regulation of p21 does not obey to

the classical pathways, open new avenues for growth control

therapies.

Hua et al. [35] identified the histone variant H2A.Z as

a potential epigenetic marker since its hormone-dependent

expression correlates with increased probability of metastasis and

decreased patient survival in a large scale study. For it to serve as

a prognostic factor in breast cancer, the mechanisms unraveled by

our study are relevant in ER- p532/2 cells that are otherwise

difficult to act upon.

Supporting Information

Figure S1 p21 is activated in response to pan HDAC

class I and II inhibitors. a) MTT assay to quantify

proliferation rates in the presence of LBH589 of MDA-MB231

cells cultured in rich medium. Two different concentrations

(5*1029 M and 5*1028 M) were used. b) q-PCR analysis of p21

mRNA expression levels. c) p21 mRNA expression level in MDA-

MB231 treated or not with LBH589 (5*1029 M) for 48 h and/or

transfected with a smartpool siH2A.Z (72 h) as indicated. d)

Western blot analysis of p21 protein levels after 24 h of TSA

treatment at the indicated doses.

(PDF)

Figure S2 H2A.Z specifically regulates p21 in ERa-

negative breast cancers following HDAC inhibitor

treatment. a, b, c, d) q-PCR analysis of p21 and H2A.Z mRNA

expression in Hela (a, b) and in Hs-578T (c, d) cells. Cells were

treated with siH2A.Z or scramble siRNA and treated for 24 h with

two different concentrations of TSA.

(PDF)

Figure 4. p400 but not Tip60 functions in p21 expression in the absence of p53. a) Tip60 and p21mRNA expression. b) H2A.Z, Acetyl-H2A.Zand RNA pol II binding to the p21 TSS in cell transfected with siTip60 or scramble siRNA. c) ChIP analysis of Tip60 and p400 recruitment to the p21 TSS(fragment #4) in cells treated with TSA. d) p21 mRNA expression in MDA-MB231 transfected with siRNA against p400 and treated or not with TSA. e)H2A.Z, Acetyl-H2A.Z and p300 enrichment at p21 TSS (fragment #4) in MDA-MB231 transfected with si p400.doi:10.1371/journal.pone.0054102.g004

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Acknowledgments

We would like to thank Dr. Fabrice Escaffit for critical reading of the

manuscript.

Author Contributions

Conceived and designed the experiments: LB MD KB. Performed the

experiments: LB MD FM SK. Analyzed the data: LB MD KB. Wrote the

paper: LB MD KB.

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11. Wharton W, Savell J, Cress WD, Seto E, Pledger WJ (2000) Inhibition ofmitogenesis in Balb/c-3T3 cells by Trichostatin A. Multiple alterations in theinduction and activation of cyclin-cyclin-dependent kinase complexes. J BiolChem 275: 33981–33987.

12. Fortunati N, Catalano MG, Marano F, Mugoni V, Pugliese M, et al. (2010) Thepan-DAC inhibitor LBH589 is a multi-functional agent in breast cancer cells:cytotoxic drug and inducer of sodium-iodide symporter (NIS). Breast CancerRes Treat 124: 667–675.

13. Huang L, Sowa Y, Sakai T, Pardee AB (2000) Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitorsuberoylanilide hydroxamic acid (SAHA) through the Sp1 sites. Oncogene 19:5712–5719.

14. Gartel AL, Tyner AL (1999) Transcriptional regulation of the p21((WAF1/CIP1)) gene. Exp Cell Res 246: 280–289.

15. Gevry N, Chan HM, Laflamme L, Livingston DM, Gaudreau L (2007) p21transcription is regulated by differential localization of histone H2A.Z. GenesDev 21: 1869–1881.

16. Chan HM, Narita M, Lowe SW, Livingston DM (2005) The p400 E1A-associated protein is a novel component of the p53–.p21 senescence pathway.Genes Dev 19: 196–201.

17. Mattera L, Escaffit F, Pillaire MJ, Selves J, Tyteca S, et al. (2009) The p400/Tip60 ratio is critical for colorectal cancer cell proliferation through DNAdamage response pathways. Oncogene 28: 1506–1517.

18. Tyteca S, Vandromme M, Legube G, Chevillard-Briet M, Trouche D (2006)Tip60 and p400 are both required for UV-induced apoptosis but playantagonistic roles in cell cycle progression. EMBO J 25: 1680–1689.

19. Iacovoni JS, Caron P, Lassadi I, Nicolas E, Massip L, et al. (2010) High-resolution profiling of gammaH2AX around DNA double strand breaks in themammalian genome. EMBO J 29: 1446–1457.

20. Legube G LL, Tyteca S, Caron C, Scheffner M, Chevillard-Briet M, et al. (2004)Role of the histone acetyl transferase Tip60 in the p53 pathway. J Biol Chem279: 44825–44833.

21. Barlev NA LL, Chehab NH, Mansfield K, Harris KG, Halazonetis TD, et al.(2001) Acetylation of p53 activates transcription through recruitment ofcoactivators/histone acetyltransferases. Molecular Cell 8: 1243–1254.

22. Ocker M S-SR (2007) Histone deacetylase inhibitors: signalling towardsp21cip1/waf1. Int J Biochem Cell Biol 39: 1367–1374.

23. Suzuki T YH, Kuniyasu H, Hayashi K, Naka K, Ono S, et al. (2000) Effect oftrichostatin A on cell growth and expression of cell cycle- and apoptosis-relatedmolecules in human gastric and oral carcinoma cell lines. Int J Cancer 88: 992–997.

24. Guillemette B, Bataille AR, Gevry N, Adam M, Blanchette M, et al. (2005)Variant histone H2A.Z is globally localized to the promoters of inactive yeastgenes and regulates nucleosome positioning. PLoS Biol 3: e384.

25. Millar CB, Xu F, Zhang K, Grunstein M (2006) Acetylation of H2AZ Lys 14 isassociated with genome-wide gene activity in yeast. Genes Dev 20: 711–722.

26. Park JH, Sun XJ, Roeder RG (2010) The SANT domain of p400 ATPaserepresses acetyltransferase activity and coactivator function of TIP60 in basalp21 gene expression. Mol Cell Biol 30: 2750–2761.

27. Sowa Y, Orita T, Hiranabe-Minamikawa S, Nakano K, Mizuno T, et al. (1999)Histone deacetylase inhibitor activates the p21/WAF1/Cip1 gene promoterthrough the Sp1 sites. Ann N Y Acad Sci 886: 195–199.

28. Jin C, Zang C, Wei G, Cui K, Peng W, et al. (2009) H3.3/H2A.Z doublevariant-containing nucleosomes mark ‘nucleosome-free regions’ of activepromoters and other regulatory regions. Nat Genet 41: 941–945.

29. Gevry N, Hardy S, Jacques PE, Laflamme L, Svotelis A, et al. (2009) HistoneH2A.Z is essential for estrogen receptor signaling. Genes Dev 23: 1522–1533.

30. Dalvai M BL, Fleury L., Lavigne A.C., Moutahir F. and Bystricky K. (2012)H2A.Z-dependent crosstalk between enhancer and promoter regulates CyclinD1 expression. Oncogene in press.

31. Bowman TA, Wong MM, Cox LK, Baldassare JJ, Chrivia JC (2011) Loss ofH2A.Z Is Not Sufficient to Determine Transcriptional Activity of Snf2-RelatedCBP Activator Protein or p400 Complexes. Int J Cell Biol 2011: 715642.

32. Valdes-Mora F, Song JZ, Statham AL, Strbenac D, Robinson MD, et al. (2012)Acetylation of H2A.Z is a key epigenetic modification associated with genederegulation and epigenetic remodeling in cancer. Genome Res 22: 307–321.

33. Owen GI, Richer JK, Tung L, Takimoto G, Horwitz KB (1998) Progesteroneregulates transcription of the p21(WAF1) cyclin- dependent kinase inhibitor genethrough Sp1 and CBP/p300. J Biol Chem 273: 10696–10701.

34. Love IM, Sekaric P, Shi D, Grossman SR, Androphy EJ (2012) The histoneacetyltransferase PCAF regulates p21 transcription through stress-inducedacetylation of histone H3. Cell Cycle 11.

35. Hua S, Kallen CB, Dhar R, Baquero MT, Mason CE, et al. (2008) Genomicanalysis of estrogen cascade reveals histone variant H2A.Z associated with breastcancer progression. Mol Syst Biol 4: 188.

p21 Activation by HDACi and H2A.Z Acetylation

PLOS ONE | www.plosone.org 7 January 2013 | Volume 8 | Issue 1 | e54102

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ORIGINAL ARTICLE

H2A.Z-dependent crosstalk between enhancer and promoter

regulates Cyclin D1 expressionM Dalvai1,2, L Bellucci1,2, L Fleury1,2, A-C Lavigne1,2, F Moutahir1,2 and K Bystricky1,2

H2A.Z association with specific genomic loci is thought to contribute to a chromatin structure that promotes transcription

activation. Acetylation of H2A.Z at promoters of oncogenes has been linked to tumorigenesis. The mechanism is unknown. Here, we

show that in triple negative breast cancer cells, H2A.Z bound to the promoter of the constitutively, weakly expressed cyclin D1

oncogene (CCND1), a key regulator of cellular proliferation. Depleting the pool of H2A.Z stimulated transcription of CCND1 in the

absence of its cognate transcription factor, the estrogen receptor (ER). During activation of CCND1, H2A.Z was released from the

transcription start site (TSS) and downstream enhancer (enh2) sequences. Concurrently, acetylation of H2A.Z, H3 and H4 at the TSS

was increased but only H2A.Z was acetylated at enh2. Acetylation of H2A.Z required the Tip60 acetyltransferase to be associated

with the activated CCND1 on both TSS and enh2 sites. Depletion of Tip60 prevented CCND1 activation. Chromosome conformation

capture experiments (3C) revealed specific contacts between the TSS and enh2 chromatin regions. These results suggest that

release of a histone H2A.Z-mediated repression loop activates CCND1 for transcription. Our findings open new avenues for

controlling and understanding aberrant gene expression associated with tumorigenesis.

Oncogene advance online publication, 29 October 2012; doi:10.1038/onc.2012.442

Keywords: H2A.Z; chromatin; acetylation; enhancer; intragenic loop; cyclin D1

INTRODUCTION

Estrogens are intimately linked to hormone-dependent breastcancer progression by promoting G1–S transition throughregulation of key cell cycle regulatory factors including inductionof cyclin D1, MYC, HER2 and c-SRC and repression of cyclin G2,caspase 9 and p21.1,2 17b-estradiol (E2) functions are mediated byspecific estrogen receptors (ERs), which are transcription factorsbelonging to the nuclear receptor family.3 Two ERs, ERa and ERb,each expressed as several isoforms, have been characterized.4–6

A majority of breast tumors (about 80%) express both receptorsand are hormone-dependent for growth. These tumors are lessinvasive and generally not metastatic.7 In contrast, ERa-negativebreast tumors (ERaÿ ) are hormone-independent for proliferation,more invasive and prone to metastasize.8 In ERaÿ cell lines, bothESR1 and ESR2 encoding ERa and ERb respectively, are silencedtogether with many ER-target genes including pS2/TFF1 and theprogesterone receptor gene (PGR). On the other hand, anothergroup of ER target genes including cathepsin D (CTSD), VEGFor cyclin D1 (CCND1) escape transcriptional regulation.9,10

Cyclin D1, encoded by the CCND1 gene, is a D-type cyclinwhose growth factor-induced expression and accumulation aretightly controlled.11–13 Cyclin D1 regulates G1–S progression andgene expression via chromatin modifications and a plethora ofcellular functions (for a review see14). Its relative abundance hasmajor effects on cell growth and metabolism via cell cycle regu-lated dosage of its expression.15 CCND1 transcription is regulatedby an estrogen-regulated cell-type-specific enhancer and is cellcycle regulated in ER-positive breast cancer cells (ERaþ , MCF-7cells).15,16 Downregulation of Cyclin D1 has nevertheless beenshown to increase migratory capacity and is associated with poorprognosis.17,18 CCND1 expression is low, and hormone- and cell

cycle-independent in ERaÿ MDA-MB231 cells9,19 (unpublishedobservations). The CCND1 ORF spans 14 kb and is flanked by50 and 30 enhancers identified by Eeckhoute et al.16 as DNAse Ihypersensitive site, to which transcription factors are recruited inestrogen responsive ERaþ cells.16,19–21 The 30 enhancer (enh2)defined as a major site of CCND1 regulation in these cells, wasthought to be dysfunctional in ERaÿ MDA-MB231 cells. It isplausible that the lack of enh2 function relates to poor CCND1transcription. To date the mechanisms responsible for the lossof hormonal regulation of gene expression, and subsequentlyof proliferation and differentiation processes, are not fullyunderstood. In addition to aberrant DNA methylation, anunidentified epigenetic mark imposes formation of a chromatinstate that forms a barrier to ERa binding in hormone-independentcell lines.22 Epigenetic or chromatin marks are thought to mediatea functional dialog with DNA-binding factors to coordinate theactivity of cis-regulatory elements in space and time. Chromatinproperties can be regulated by DNA methylation and histonepost-translational modifications, but also by replacement ofhistones with variants.23–29 Incorporation of histone H2A.Z intothe chromatin of inactive gene promoters and its removalaccording to physiological signals participates in regulating geneexpression.30–33 H2A.Z is principally found at the promoters ofpoised genes and released during gene activation in both yeastand human cells.31,34–36 In euchromatin, a sharp peak of H2A.Z isseen at the 50 end of numerous genes and at enhancers.37 Arecent genome-wide analysis38 showed that acetylated H2A.Z isgenerally found near the transcription start site (TSS) of activegenes in prostate cancer cells.Here, we investigate the role of the histone variant H2A.Z in the

transcriptional regulation of the ER-target gene in ERaÿ breast

1Laboratoire de Biologie Moleculaire Eucaryote (LBME), University of Toulouse, Toulouse, France and 2UMR 5099 CNRS; LBME, Toulouse, France. Correspondence:

Professor K Bystricky, Laboratoire de Biologie Moleculaire Eucaryote (LBME), University of Toulouse, 118 route de Narbonne, Toulouse F-31062, France.

E-mail: [email protected]

Received 20 March 2012; revised 9 August 2012; accepted 10 August 2012

Oncogene (2012), 1–9

& 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12

www.nature.com/onc

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cancer cells. Our results demonstrate that stimulation of CCND1transcription requires release of H2A.Z from both the TSS anddownstream enhancer (enh2) sequences of CCND1. Acetylation ofH2A.Z and Tip60 acetyltransferase regulate TSS-enhancer crosstalkvia chromatin looping.

RESULTS

Depletion of H2A.Z activates CCND1 transcription

We first investigated whether H2A.Z was present at ER-target geneloci in ERÿ cells. Chromatin immunoprecipitation (ChIP) experi-ments revealed that H2A.Z bound to sequences flanking theCCND1 ORF, to the TSS and the downstream enhancer defined byEeckhoute et al.16 but not to distant upstream sites in two distinctcell lines, MDA-MB231 (Figure 1a) and MDA-MB436 (Supplemen-tary Figure S1A). H2A.Z was also present at the promoters of thesilent ER-target genes, PGR, TFF1 or ESR1 (Supplementary FigureS1B). ChIP-on-chip experiments confirmed that H2A.Z associatedexclusively with flanking regions of all tested genes and, inparticular, the enh2 sites of CCND1 (J Eeckhoute, data not shown).The presence of H2A.Z at these regulatory elements in ERaÿ

MDA-MB231 cells, prompted us to assess its potential role intranscriptional repression of these genes. To this end, weselectively depleted H2A.Z by small interference RNA (siRNA)(Figure 1b, Supplementary Figures S1C, D). Seventy-two hoursfollowing siRNA transfection, CCND1 mRNA levels increased four-fold compared with mock-transfected cells (Figure 1b), whereasPGR, TFF1 and ESR1 remained silent (Figure 1b). Under theseconditions, a potential role of the ER in stimulating CCND1expression could thus be excluded (Figure 1b). ChIP confirmedthat H2A.Z siRNA treatment lead to markedly reduced binding ofH2A.Z to the CCND1 TSS (Figure 1c left panel). Concomitant toincreased mRNA levels, polymerase II (pol II) recruitment to the

CCND1 TSS slightly increased. In contrast, decreased H2A.Zbinding to the PGR promoter did not allow polymerase IIrecruitment to this silent gene (Supplementary Figure S1E). Thus,H2A.Z eviction is sufficient to stimulate CCND1 transcription inERaÿ breast cancer cells. Yet, its depletion alone cannot restorethe expression of silenced genes as PGR, TFF1 or ESR1.We next asked whether activation of CCND1 following release of

H2A.Z from the TSS also altered the chromatin structure of thedownstream enhancer enh2, previously shown to be inactive inthese cells.16 In H2A.Z siRNA transfected cells, H2A.Z binding toenh2 was significantly diminished (Figure 1c, right panel). Inaddition, CCND1 activation in siH2A.Z-transfected MDA-MB231cells was accompanied by a strong recruitment of pol II to enh2(Figure 1c, right panel), as described previously for E2 stimulatedMCF-7 cells.16 We conclude that enh2 has an active role inER-independent CCND1 activation. H2A.Z seemed to interfere withstimulation of transcription when bound to TSS and enhancersequences of CCND1. We investigates how modulatingincorporation of this histone variant regulates gene activity.

H2A.Z acetylation correlates with activation of CCND1transcription

Recently, Valdes-Mora et al.38 demonstrated in a genome-widestudy that H2A.Z acetylation marks chromatin at the TSS of activegenes, which are frequently deregulated in prostate cancer cells.Thus, we examined whether acetylation of H2A.Z was involved inCCND1 function in breast cancer cells. A variety of inhibitors allowmodulation of histone post-translational modifications with theaim to alter gene expression patterns. Exposure of mammarytumor cell lines to trichostatin A (TSA), a pan HDACi of class 2HDACs, reduces their growth rate39 and induces changes in geneexpression patterns.40–44 We tested whether TSA would stimulateCCND1 expression. MDA-MB231 cells were cultivated in normalmedia and exposed to 50 ng/ml TSA for 24 h before mRNAquantification. CCND1 mRNA increased four-fold in treatedcompared with untreated cells (Figure 2a). We also noticed thatTSA treatment negatively affected H2AFZ mRNA levels, whichwere reduced B2-fold (Figure 2a). The ESR1 gene remained silentas previously shown22 excluding the possibility that stimulation ofCCND1 was due to binding of ERa in these TSA treated ERaÿ cells.These results prompted us to further investigate the mechanismby which transcription of CCND1 was stimulated.As expected, concomitant to increased transcription in the

presence of TSA, acetylation of histone H3 at the CCND1 TSSincreased (Figure 2b). In these cells, H3 was also acetylated atenh2 suggesting that this enhancer participates in CCND1activation (Figure 2b). We show that H2A.Z was associated withthe CCND1 TSS and that a fraction of this histone variant wasevicted from the TSS in response to TSA treatment of MDA-MB231cells (Figure 2c). Interestingly, upon eviction from the TSS, H2A.Zwas also lost from enh2 (Figure 2d). Under the same conditions,we monitored binding of acetylated H2A.Z (Ac-H2A.Z) to theCCND1 TSS and enh2 using an antibody that specificallyrecognizes H2A.Z acetylated at K4, K7 and K14 (see Materialsand methods). The amount of Ac-H2A.Z present at the CCND1 TSSor enh2 did not vary following TSA treatment. However, the ratioof Ac-H2A.Z was greatly augmented relative to the total, sharplyreduced amount of H2A.Z detected at these sequences (Figures 2cand d right panel).Based on the fact that TSA negatively affected H2AFZ mRNA

levels (Figure 2a) we wondered whether stimulation of CCND1 wasthe result of increased histone acetylation or of a reduction ofH2A.Z abundance. We then analyzed if depletion of the cellularpool of H2A.Z also altered Ac-H2A.Z binding at the stimulatedCCND1 locus. We found nearly three-fold greater amounts ofacetylated H2A.Z at the TSS (Figure 3a, left panel) and six-foldgreater amounts (Figure 3b, left panel) at the enh2 in

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Figure 1. Depletion of H2A.Z correlates with CCND1 transcriptionalactivation. (a) The recruitment of H2A.Z to the CCND1 gene atÿ 2000, TSS and enh2 was analyzed by ChIP. (b) Expression levels ofH2AFZ, CCND1, ESR1, PGR and TFF1 in ERaÿ MDA-MB231 cellstransfected with SMARTpool H2A.Z siRNA (þ ) or scramble siRNA(ÿ ) were analyzed by q-PCR. mRNA expression levels werenormalized against expression levels of the RPLP0 ribosomal gene.(c) The recruitment of H2A.Z and RNA pol II was analyzed by ChIP atthe CCND1 promoter (left panel) or enh2 enhancer (right panel).

H2A.Z-dependent intragenic looping

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siH2A.Z-transfected cells (þ ) compared with scrambled siRNA(ÿ ) treated MDA-MB231 cells. As the total amount of H2A.Z wasreduced three-fold at these sites upon H2A.Z depletion, therelative ratio of Ac-H2A.Z to total H2A.Z increased significantly(Figures 3a and b, right panels).In conclusion, these results reveal that the presence of hypo-

acetylated H2A.Z at the proximal promoter around the TSS and atthe 30 enhancer negatively controls CCND1 expression in triplenegative mammary tumor cells. Reducing the pool of free H2A.Zled to an increase in the amount of Ac-H2A.Z present at bothregulatory sequences at the expense of unmodified H2A.Z. Wetherefore attempted to identify the factors involved in acetylationof H2A.Z and chromatin modifications associated with CCND1transcriptional activation.

Recruitment of Tip60 and H2A.Z acetylation regulates enhancerchromatin structure

Histone acetylation has long been associated with activetranscription45,46 and thus the increase in acetylation of H2A.Zbound to the CCND1 locus may be part of general acetylation ofchromatin in MDA-MB231 cells. Indeed, acetylation of H3K9 andH4 increased upon depletion of H2A.Z by siRNA at the proximalpromoter (Figures 4a and b, upper panel). Global acetylation wasaccompanied by the recruitment of elongating pol II phosphory-lated at serine 5 (P-Ser 5; Figure 4d, upper panel). In contrast,

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Figure 2. TSA modulates gene expression and increases H2A.Z acetylation at the CCND1 promoter and enhancer. MDA-MB231 cells werecultivated in DMEM medium and treated (þ ) or not (ÿ ) with 50 ng/ml TSA for 24 h. (a) Gene expression was quantified by qRT–-PCR andnormalized against expression levels of the RPLP0 ribosomal gene. (b) ChIP of histone H3 acetylation (Ac-H3K9) after TSA treatment on TSS(left panel) or enh2 (right panel) sites. (c, d) The recruitment of H2A.Z and Ac-H2A.Z to the CCND1 promoter (c) and enhancer (d) sites wasanalyzed by ChIP and shown as percent input.

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H2A.Z-dependent intragenic looping

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acetylation of H3K9 was low at the downstream enhancerenh2 and did not vary with transcription activation (Figure 4a,lower panel). Acetylation of H4 also remained unchanged(Figure 4b, lower panel). In addition, H3 binding to the CCND130 enhancer but not to the TSS varied following H2A.Z evictionin these ERaÿ cells (Figure 4c). In contrast to previousobservations in prostate cancer cells,38 nucleosome density didnot vary at the promoter of the activated CCND1 gene. However,decreased nucleosome density at enh2 points to chromatindecondensation usually associated with DNAse I hypersensitivesites, which reinforces the notion that the enh2 site is a keyelement for CCND1 gene activation.16 P-ser 5 binding did notvary at the 30 end of the gene as expected47 (Figure 4d, lowerpanel). Hence, acetylation of chromatin is linked to transcriptioninitiation and elongation near the 50 end of CCND1 whileacetylation of H2A.Z characterized the active state at both theTSS and the 30 enh2.Several studies in yeast have shown that the NuA4 (an ortholog

of the human Tip60/p400 complex) and the SAGA histoneacetyltransferase complexes acetylate multiple lysine residues ofH2A.Z.48–50 Tip60 could then be one of the HATs capable ofacetylating H2A.Z in mammalian cells. Indeed, we found thatTip60 was recruited to both the TSS and enh2 of the activatedCCND1 gene in MDA-MB231 cells transfected with siH2A.Z(Figure 5a). Upon overexpression of Tip60 in MDA-MB231 cells(Supplementary Figure S2A) CCND1 expression increased nearlytwo-fold (Figure 5b), whereas transcription levels of ESR1 andH2AFZ remained unchanged (Supplementary Figure S2B). Theselective knockdown of Tip60 did not affect the basal, uninducedexpression level of CCND1 (Figure 5c, Supplementary Figure S2C),but prevented gene activation upon H2A.Z depletion (Figure 5c,Supplementary Figure S2D). In the absence of Tip60, the amountof H2A.Z and pol II bound to the TSS and enh2 of CCND1 did notvary (Figure 5d) but the presence of acetylated H2A.Z wasmarkedly reduced. We conclude from these ChIP experiments thatTip60 has a central role in CCND1 activation independently of E2and is the likely candidate for acetylating H2A.Z at both TSS andenh2 sites.

Acetylation of H2A.Z regulates promoter/enhancer crosstalk vialoop formation

To investigate whether the CCND1 TSS interacts with enh2, weused a chromatin conformation capture (3C) assay,51,52 which

detects physical proximity between distal DNA sites by ligation ofcross-linked restricted DNA fragments (Figure 6a). Ligationproducts between enh2 and promoter sequences, and between

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Figure 5. Tip60 promotes CCND1 promoter and enhancer chromatinacetylation during gene activation. (a) The presence of Tip60 on theCCND1 promoter (upper panel) and 30 enhancer (lower panel) wasanalyzed by ChIP following depletion of H2A.Z by a SMARTpool siRNA.(b) MDA-MB231 cells were transfected with an empty vector(pcDNA3.1) or with a vector expressing Tip60. CCND1, gene expressionwas analyzed after 48h by q-PCR. (c) MDA-MB231 cells were transfectedwith scrambled control siRNA (ÿ ), with H2A.Z SMARTpool siRNA (þ ),Tip60 siRNA (þ ) or both siRNAs. CCND1 gene expression was analyzedafter 72h by q-PCR. (d) MDA-MB231 cells were transfected with ascrambled control siRNA (ÿ ) or with Tip60 siRNA (þ ). The presence ofH2A.Z, Ac-H2A.Z and Pol II on the CCND1 promoter (upper panel) and30 enhancer (lower panel) was analyzed by ChIP.

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enh2 and a control fragment inside the CCND1 ORF wereamplified and normalized to the amplified enh2 PCR productused in ChIP experiments (see Materials and methods; Figure 6b).We measured significant interaction frequencies between enh2and promoter sequences in MDA-MB231 transfected withscramble siRNA (control) while in cells transfected with siH2A.Z,this interaction was reduced more than five-fold (Figure 6b). Incontrast, depletion of Tip60 alone or in combination with siH2A.Zdid not affect contact frequencies between these sites (Figure 6b).No amplification was observed between enh2 and the internal

control fragment. We did not detect amplification of any of thesefragments in undigested samples. Hence, an intragenic loopmediated by specific promoter–enhancer interactions is presentwhen CCND1 expression is low (Figure 5c). Upon transcriptionactivation, gene lopping is markedly reduced. In conclusion, wepropose a model (Figure 7) in which looping of the CCND1 50 and30 ends is mediated by histone H2A.Z and exerts repressive effects.Acetylation of H2A.Z by Tip60 reduces interactions between TSSand enh2 sites, creating a chromatin environment favorable fortranscriptional activation.

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Figure 6. H2A.Z regulates promoter–enhancer looping. (a) Schematic representation of the CCND1 locus (http://genome.ucsc.edu/cgi-bin/hgGateway). DNAseI hypersensitive sites,16 Csp6I restriction sites and ligation events tested are indicated. (b) q-PCR amplification following 3C:cross-linked DNA from cells was digested with Csp6I or incubated without restriction enzyme and ligated. Nested PCR reactions wereperformed using primers designed for each of the four possible ligation events (only one is shown). (b) MDA-MB231 cells were transfectedwith scrambled control siRNA (Ctr), H2A.Z siRNA, Tip60 siRNA or both.

Figure 7. Model of the role of H2A.Z and Tip60 in the control of loop formation between enhancer and promoter sequences implicated in thetranscriptional regulation of CCND1.

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DISCUSSION

We demonstrate that binding and acetylation of the histonevariant H2A.Z regulates CCND1 expression via specific enhancer/promoter interaction in ERaÿ breast cancer cell lines. In particular,H2A.Z was present at the CCND1 locus when the gene displayedlittle transcriptional activity. Recently, Valdes-Mora et al.38 reportedin a genome-wide study of normal and prostate cancer cells, thatacetylation of H2AZ at the TSS correlated with activation ofoncogenes and that reduced H2A.Z acetylation marked silencedtumor suppressor genes.38 Here we extend these findings tobreast cancer cells. In addition, we propose a mechanism by whichH2A.Z acetylation modulates transcription associated withderegulated gene activity in cancer cells.Reducing the pool of H2A.Z resulted in release of H2A.Z from

promoter and enhancer sequences and increased CCND1 expres-sion. This is consistent with the previously proposed model thatnucleosomes containing H2A.Z are more labile. Eviction of H2A.Zthus promotes chromatin accessibility.53 It is tempting tospeculate that modulation of the 3D intragenic organizationmay be key for creating a window of opportunity for secondaryfactors to regulate transcription. Hence, not the action of specifictranscription factors and downstream events resulting from theirassociation once favorable conditions are encountered,54 butstructure would guide activity. Concomitantly to chromatinreorganization, acetylation of the remaining H2A.Z, but not ofother histones, was a characteristic feature of chromatin in the30 enhancer region, suggesting that this modification specificallyregulates 3D organization of genetic loci.Enhancers are thought to allow transcription activators to bind

inactive genomic loci in order to facilitate the onset oftranscription. This usually involves chromatin modifying com-plexes55 such as CBP/p300 in mammalian cells.56,57 Here we showthat Tip60, a histone acetyltransferase that has been shown toacetylate histones H3, H4, H2A,58,59 and H2AX60 in vivo, is recruitedto a 30 enhancer of the CCND1 gene upon activation. Tip60 is anessential cofactor for numerous transcription factors andimportant for cell proliferation. Binding to promoter andenhancer sequences of the stimulated CCND1 gene coincidedwith an increased ratio of acetylated H2A.Z. Thus, for the first time,we provide evidence for a role of Tip60 in transcriptionalactivation of the CCND1 oncogene in ERaÿ breast cancer cellspotentially by acetylation of the histone variant H2A.Z.Replacement of H2A by H2A.Z alters nucleosome conformationcreating docking sites for distinct cofactors and chromatinremodelers.61,62 Tip60 could therefore be recruited to H2A.Zcontaining loci as has been demonstrated for the NuA4 complexin yeast.48 We hypothesize that Tip60 is capable of regulatingCCND1 gene expression by acetylating H2A.Z at the downstreamenh2, which then disturbs repressive promoter enh2 crosstalk.Here, ERa was not required for Tip60 recruitment to these CCND1regulatory elements unlike the mechanism recently described inERþ cells.62 Our data thus illustrate that the expression levels oftissue or cell-specific genes are largely modulated by the ability oftransacting factors to associate with regulatory elements.Long-range interactions between distant enhancers and pro-

moter regions via chromatin looping has been shown to allowactivation of single genes or gene clusters.63–66 This loopinggenerally occurs in the 50 part of genes and over tens or hundredsof kb. The role of such loops is thought to isolate chromatin fromsurrounding repressive environment via insulator elements.67

Long-range intragenic interactions implicate transcriptionactivators, histone variants, including H2A.Z and macro H2A.1,and chromatin remodelers bound to promoter and enhancerregions, but direct experimental evidence for intragenic loopingis still lacking.19,30,68,69 As shown by results of large-scale chromo-some conformation (4C) and ChiA-PET experiments, suchinteractions are well correlated with the existence of chromatin

domains of distinct properties around highly regulated genes,70–72

via interchromosomal but also intrachromosomal loops, but theirfunctional significance remains to be established. However, dy-namic chromosome folding within distinct domains accompaniestranscription activation73,74 (Kocanova et al., unpublished data). Itis thus plausible that local decondensation of chromatin is, at leastin part, the result of releasing looping.Finally, H2A.Z associates with a subset of ER-target genes that are

differentially regulated in different breast cancer cell lines. Hormonetreatment can lead to recruitment of H2A.Z to the TSS of the TFF1gene30 or its eviction at the PGR or CCND1 promoters (unpublishedobservations) in MCF-7 cells. These data suggest that there is nogeneral mechanism linking H2A.Z binding to transcription regulation.Indeed, H2A.Z was also associated with silenced or repressed genes inERÿ breast cancer cells. Similarly to prostate cancer cells, it is theacetylation of H2A.Z that has a key role in transcription regulation ofgenes in breast cancer cells. The ratio of acetylated relative to bulkH2A.Z bound to TSS and 30 enhancer sequences controls genefunction. Modification of this ratio allowed reversing uncontrolledgene expression in the more aggressive ERÿ breast cancer cells forwhich targeted treatments are critically lacking. Interfering with theratio of bound acetylated H2A.Z to promoter and enhancersequences in ERÿ cells mimics the effect of the estradiol bound ER.This mechanism may increase our understanding of tumorigenesisand offer new perspectives for re-activating control gene pathways.

MATERIALS AND METHODS

Cell lines, transfection and western blotting.

MDA-MB231 and MDA-MB436 cells were purchased from ATCC (Rockville,MD, USA, used up to 15 passages). MDA-MB231 cells were maintained inDulbecco’s modified Eagle’s medium (DMEM) and MDA-MB 436 in DMEM/F12 medium, both with Glutamax containing 50mg/ml gentamicin, 1mM

sodium pyruvate and 10% heat-inactivated fetal calf serum (Invitrogen,Carlsbad, CA, USA). MDA-MB231 cells were treated with 50 ng/ml TSA(Sigma-Aldrich, Inc., St Louis, MO, USA) for the indicated times. In all,4� 106 MDA-MB231 cells where mock-transfected (pcDNA3.1) or trans-fected with 2 mg of pcDNA3.1/Tip60 (gifts from Dr Didier Trouche) withAmaxa Cell line Nucleofactor Kit V program X-013 according to themanufacturer’s protocol. Breast cancer cells were mock-transfected ortransfected with H2A.Z siRNA ON-TARGET plus SMARTpool or scrambled(scr) siRNA (Dharmacon Thermo Scientific, Waltham, MA, USA) withInterferine (Ozyme, Bedford, MA, USA) according to the manufac-turer’s protocol. H2A.Z depletion was also performed with another siRNA(H2AZ-1), kindly provided by Dr Didier Trouche (Supplementary FigureS1D).75 Tip60 siRNA76 was purchased from Eurogentec, and transfectedwith Interferine (Ozyme) according to the manufacturer’s protocol. A totalof 3� 105 MDA-MB231 cells were seeded in six-well plates. Seventy-twohours following siRNA transfection, total cell extracts were isolated andprotein level of H2A.Z and Tip60 was analyzed by immunoblotting on gelSDS–PAGE 15% with anti-H2A.Z (Abcam, Cambridge, UK, ab4174), anti-Tip6076 and with anti-GAPDH (Millipore, Bedford, MA, USA, mab374) oranti-Tubulin (Sigma, DM1A) antibodies as loading control.

RNA analysis.

Total RNA was extracted using an RNeasy mini-kit (Qiagen, Valencia, CA,USA) and eluted with 35ml of RNAase-free water. First strand cDNA wasgenerated using 2 mg of total RNA in a reaction containing randomoligonucleotides as primers with the ThermoScript RT–PCR system(Invitrogen). Real-time PCR was performed on a Mastercycler ep realplex4

(Eppendorf) using the platinum SYBR Green q-PCR SuperMix (Invitrogen)according to the manufacturer’s instructions. Amplification conditions:1min at 50 1C, 3 min at 95 1C followed by 40 cycles (20 s at 95 1C, 20 s at60 1C, 20s at 72 1C). q-PCR for RPLP0 mRNA was used as an internal control.The primers used in q-PCR: ESR1: 50-CATGGTCATAACAGCCTCCTG-30 and50-TAGAATGGGCAGGAGAAA GG-30 , H2AFZ: 50-CCTTTTCTCTGCCTTGCTTG-30

and 50-CGGTGAGGTACTCCAGGATG-30 , CCND1: 50-GCGTCCATGCGGAAGATC-30 and 50-ATGGCCAGCGGGAAGAC-30 , RPLP0: 50-TGGCAGCATCTACAACCCTGAA-30 and 50-CACTGGCAACATTGCGGACA-30 , PGR: 50-CTTAATCAACTAGGCGAGAG-30 and 50-AAGCTCATCCAAGAATACTG-30 , TFF1: 50-CAGATCCCTGCAGAAGTGTCT-30 and 50-CCCCTGGTGCTTCTATCCTAAT-30 .

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ChIP assays

ChIP analyses were performed as described previously77 from MDA-MB231cells. Samples were sonicated to generate DNA fragments o500 bp.Chromatin fragments were immunoprecipitated using antibodies againstH2A.Z (ab4174, Abcam), Acetyl H2A.Z (ab18262, Abcam), RNA pol II (N20X,Santa Cruz biotechnology, Santa Cruz, CA, USA), Acetyl H3K9 (06-942,Upstate, Bedford, MA, USA), RNA pol II serine 5 phosphorylated (MPY-127R,H14, Covance, Vienna, VA, USA; IF), pan Acetyl H4 (39 243, active motif), H3(ab1791, Abcam), Tip60 78 or an irrelevant HA antibody (H6908, Sigma) ascontrol. The precipitated DNA was amplified by real-time PCR, with primersets designed to amplify the promoter region of the PGR, ESR1, TFF1 andCCND1 genes, as well the enh2 enhancer and ÿ 2000 region of CCND1. Theprimers used in q-PCR are listed here: CCND1 (TSS): 50-CGGGCTTTGATCTTTGCTTA-30 and 50-ACTCTGCTGCTCGCTGCTAC-30 , distal CCND1enhancer (enh2): 50-CAGTTTGTCTTCCCGGGTTA-30 and 50-CATCCAGAGCAAACAGCAG-30 , ÿ 2000 region: 50-GCTCTTTACGCTCGCTAACC-30 and 50-GCAAGACAGAGGAAACTGGAA-30 , PGR: 50-TCGGGGTAAGCCTTGTTGTA-30 and 50-GCCTCGGGTTGTAGATTTCA-30 , ESR1: 50-CATGGTCATAACAGCCTCCTC-30 and50-TAGAATGGGCAGGAGAAAGG-30 , TFF1: 50-ATGGGAGTCTCCTCCAACCT-30

and 50-TTCCGGCCATCTCTCACTAT-30 .All ChIP data are shown as percent input. For the ratio of acetylated

H2A.Z, the amount of Ac-H2A.Z in percent of input was divided by thepercent input of H2A.Z in order to have a percentage of global Ac-H2A.Zon a promoter.

3C assays

Chromatin conformation capture assays were performed essentially asdescribed,52 with only minor modifications. MDA-MB231 cells weretransfected with a scrambled control siRNA (ÿ ), with H2A.Z SMARTpoolsiRNA (þ ) (Dharmacon Thermo Scientific), Tip60 siRNA (þ ) or both siRNAsand cultured in phenol red-free DMEM containing 10% FBS-T for 72h beforecross-linking. The culture medium was removed, and cells were fixed with1.5% formaldehyde for 10min at room temperature. Cells were then washedtwice with cold phosphate-buffered saline solution, and resuspended in ice-cold lysis buffer (10mm Tris–HCl, pH 8.0, 10mm NaCl, 0.2% nonidet P-40, andprotease inhibitor mixture). Nuclei were resuspended in 1ml of buffer B 1.2Xbuffer (MBI Fermentas, Thermo Fisher Scientific, Rockford, IL, USA)supplemented with SDS 0.3%. Triton X-100 1.8% was added to sequesterthe SDS and incubated for 1h at 37 1C. The cross-linked DNA was digestedovernight with 400 units of restriction enzyme Csp6I (MBI Fermentas). Therestriction enzyme was inactivated by incubation at 65 1C for 20min Thereactions were diluted with ligase buffer (50mm Tris–HCl, pH 7.5, 10mmMgCl2, 10mm dithiothreitol, 1mm ATP and 25mg/ml bovine serum albumin),supplemented with Triton X-100 (1% final concentration). The DNA was ligatedusing T4 DNA ligase (New England Biolabs, Ipswich, MA, USA) overnight at16 1C and an additional 100 units for 2h at 37 1C. RNase was added for 30minat 37 1C, and samples were incubated with SDS overnight at 70 1C to reversethe crosslink. The following day, samples were incubated for 2h at 45 1C withproteinase K, and the DNA was purified by phenol–chloroform extractions andethanol precipitation. Interaction between chromatin domains was assessedby PCR amplification carried out using similar conditions as for real-time PCRamplification but with four nested primer pairs for each predicted ligationevent (four possibilities) as performed by Deschenes et al.79 with minormodifications. The second PCR reaction was performed and analyzed byq-PCR. Primers were designed on the digested BAC fragments, directly aroundthe putative site of ligation for the four possibilities. BAC clones RP11-300ID(BACPAC Resources Center at Childrens Hospital Oakland Research Institute,Oakland, CA, USA) containing the CCND1 gene and downstream 160-kb regionwere used. Forty microgram of BAC was digested by Csp6I overnight andligated. This product was purified by phenol–chloroform and precipited inorder to generate 3C control templates. PCR primer efficiency was measuredby amplifying 0.01–50ng of digested BAC product and also tested on a fixedamount (50ng) of digested genomic DNA. All primers have an annealingtemperature between 65 to 70 1C and a product size around 150–300bp. Allprimer combinations showed PCR efficiency between 90 and 100%. chromatinconformation capture assay results are presented as the average from threeindependent preparations of 3C DNA, followed by q-PCR analysis in triplicate.q-PCR for enh2 (PCR primers designed inside the Csp6I restriction fragmentincluding enh2) was used as an internal control to verify ligation events. Non-digested samples and ligation between a control fragment with enh2 arerepresented. List of primer sequences see Supplementary Figure S3.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

We would like to thank Dr Didier Trouche for the Tip60 expressing vector and

insightful discussions, J Eeckhoute, Lille for sharing unpublished work, and David

Laperriere, IRIC, Montreal, for expert advice on genome database use. This work was

supported by the Ligue Nationale Contre le Cancer (fellowship to LB), the Institut

National du Cancer (INCa grant no. 34696) and the Fondation pour la Recherche

Medicale (FRM, fellowship to MD).

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9

& 2012 Macmillan Publishers Limited Oncogene (2012), 1 – 9

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TIP48/reptin and H2A.Z requirement for initiating chromatin

remodeling in estrogen activated transcription

Mathieu Dalvai 1,2, Laurence Fleury 1,2, Luca Bellucci 1,2, Silvia Kocanova 1,2 and

Kerstin Bystricky # 1,2

Affiliations:

1 - Université de Toulouse; Laboratoire de Biologie Moléculaire Eucaryote (LBME);

F-31062 Toulouse, France

2 - CNRS; LBME; F-31062 Toulouse, France

# Corresponding author

E-mail: [email protected] Phone: +33.5.61.33.58.00 Fax: +33.5.61.33.58.86

Running title: TIP48/reptin regulation of cyclin D1

Keywords: TIP48/reptin; H2A.Z; acetylation; estrogen receptor; TIP60; chromatin

remodeling; breast cancer; gene loop; transcription activation; cyclin D1;

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Abstract

Histone variants, including histone H2A.Z, are incorporated into specific

genomic sites and participate in transcription regulation. The role of H2A.Z at these

sites remains poorly characterized.

Our study investigates changes in the chromatin environment at the Cyclin D1

gene (CCND1) during transcriptional initiation in response to estradiol in estrogen

receptor positive mammary tumour cells. We show that H2A.Z is present at the

transcription start-site and downstream enhancer sequences of CCND1 when the

gene is poorly transcribed. Stimulation of CCND1 expression required release of

H2A.Z concomitantly from both these DNA elements. The AAA+ family members

TIP48/reptin and the histone variant H2A.Z are required to remodel the chromatin

environment at CCND1 as a prerequisite for binding of the estrogen receptor (ERα)

in the presence of hormone. TIP48 promotes acetylation and exchange of H2A.Z,

which triggers a dissociation of the CCND1 3’enhancer from the promoter, thereby

releasing a repressive intragenic loop. This release then enables the estrogen

receptor to bind to the CCND1 promoter. Our findings provide new insight into the

priming of chromatin required for transcription factor access to their target sequence.

Dynamic release of gene loops could be a rapid means to remodel chromatin and to

stimulate transcription in response to hormones.

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Author summary

Our study investigates changes in the chromatin environment at the Cyclin D1

gene that are a prerequisite for transcriptional initiation in response to estradiol.

Gene expression is under control of chromatin structure. Histone variants, including

histone H2A.Z, are incorporated into specific genomic sites and participate in

transcription regulation. We show that H2A.Z is present at the transcription start-site

and downstream enhancer sequences of CCND1 when the gene is poorly

transcribed. Stimulation of CCND1 expression required release of H2A.Z

concomitantly from both these DNA elements. The TIP48/reptin protein which is part

of several chromatin remodeling complexes also associated with the CCND1

regulatory elements. Here, TIP48 promotes exchange of H2A.Z, which triggers a

dissociation of the CCND1 enhancer from the promoter, thereby releasing a

repressive intragenic loop. This release then enables estrogen receptor to bind the

CCND1 promoter. Acetylation of H2A.Z is required for these processes. Our findings

provide new insight into the priming of chromatin required for transcription factor

access to their target sequence. Hence, we propose a new model for early events in

transcription activation that were not shown before. Specifically, release of looping

could be a rapid means to activate transcription efficiently in response to stimuli, in

particular estrogen.

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Introduction

Transcription activation relies on a careful choreography of local chromatin

remodeling events that include posttranslational histone modifications and

replacement of canonical histones by variants [1-4]. In ER�-positive breast cancer

cells gene activation can be advantageously studied by addition of hormone.

Numerous chromatin modifying and remodeling complexes are dynamically

exchanged during ER� target gene activation in a gene specific manner [5,6].

Chromatin immunoprecipitation (ChIP) studies have provided extensive information

on the recruitment of these complexes by the hormone bound estrogen receptor [7].

The first complex to occupy promoter sequences is the ATP-dependent SWI/SNF

chromatin remodeling complex and its catalytic subunit Brg1. Its activity enables

subsequent binding of a plethora of histone and protein modifying assemblies which

lead to transcription initiation by polymerase II [5,6,8-10]. In contrast, it is less clear

how local chromatin structure prepares for rapid and massive recruitment of the

estrogen receptor itself in the presence of estrogen.

Incorporation of histone variants constitutes a means to alter nucleosome

properties and positioning at specific genomic loci. The histone H2A variant H2A.Z is

frequently found within nucleosomes at regulatory sequences [11-14]. In particular,

H2A.Z occupancy characterizes inducible and constitutive DNAseI hypersensitive

sites to which nuclear receptors bind [15]. This variant is believed to induce a

chromatin conformation that poises genes for transcription in human cells [16]. In

yeast, H2A.Z exchange is mediated by the SWR1 complex [17,18]. However, in

mammalian cells, the mechanisms of H2A.Z deposition are still poorly characterized

and may require several distinct protein complexes depending on the cellular context

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(for a review see [19]). Among ATP-dependent chromatin remodeling complexes the

TIP48/TIP49 containing SWR1/SRCAP [20] or TIP60/p400 [21,22] complexes have

been shown to play a role in H2A.Z deposition [17,18,23]. p400 was also reported to

be required for H2A.Z incorporation into the TFF1/pS2 gene concomitant to estrogen

receptor binding [24]. In an in vitro study Choi et al. demonstrated that the AAA+

family (ATPases Associated with various cellular Activities) members TIP48/TIP49

participate in the replacement of H2A by H2A.Z [25]. This H2A.Z exchange was

facilitated by TIP60-mediated H2A acetylation. TIP48/TIP49 proteins (also known as

TIP49b and TIP49a, Rvb2 and Rvb1, reptin and pontin) are important for assembly

and activity of the histone TIP60 acetyltransferase complex [26].

To gain a better understanding of the early steps required in estrogen receptor

mediated transcription activation and the coordination between remodeling

complexes and chromatin structure, we analyzed transcription of the cyclin D1 gene

(CCND1) in ER�-positive MCF-7 breast cancer cells. This oncogene is frequently

overexpressed in human breast tumors [27]. Its down-regulation increases migratory

capacity and is linked to unfavorable prognosis [28,29]. Cyclin D1 is a mitogenic

sensor that modulates cell cycle progression. CCND1 transcription is stimulated by

17�-estradiol (E2), inhibited by antiestrogens and cell cycle regulated in ER�-positive

breast cancer cells [30,31].

Here we show that TIP48 and H2A.Z associate with CCND1 promoter and

enhancer sequences. TIP48 is required for chromatin reorganization which is initiated

by release of H2A.Z and opening of a repressive promoter-enhancer gene loop

enabling TIP60 and the E2 bound estrogen receptor to be loaded to stimulate

CCND1 transcription.

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Results

TIP48 and H2A.Z association with CCND1 regulatory sites is required for

activation by estradiol.

In ER-positive MCF-7 cells grown in steroid stripped media only basal

transcription levels of the Cyclin D1 gene (CCND1) were measured. Addition of E2

initiated CCND1 transcription activation lead to a 2.5-fold increase in mRNA levels in

cells treated 6h with 100nM E2 (Fig. 1A). H2A.Z has been reported to act in concert

with ER to regulate the TFF1 gene [24] prompting us to examine H2A.Z association

with CCND1 regulatory elements. Analysis of ChIP-on-chip data revealed that H2A.Z

was highly enriched at sequences 5’ and 3’ flanking the CCND1 gene, and largely

absent from the open reading frame (Fig. 1B) [32]. By conventional ChIP, we found

that the amount of H2A.Z present at the CCND1 promoter was reduced by 50% at

the TSS (Fig. 1C). Eeckhoute et al. identified an enhancer (enh2) at the 3’end of the

CCND1 gene which acts as the primary site for ER� and cofactor binding during

CCND1 transcriptional regulation [9]. We thus analyzed the chromatin organization of

enh2. Similar to the promoter, H2A.Z present at enh2 was removed during

transcription activation (Fig 1C). Reduced binding was not due to a decrease in

H2AFZ expression in the presence of E2 (Fig. 1A). Chromatin modifications at the

CCND1 promoter and enhancer appear to be coordinated. Removal of H2A.Z from

promoter sequences upon transcription activation correlates with observations in

yeast and several mammalian cells and points to a mechanism of regulation distinct

from the one of the ER� target gene TFF1 [33] [24,34]. Replacement of nucleosomal

H2A with H2A.Z has been shown to be catalyzed by the TIP48/49 complex in vitro

[25]. The TIP48/49 complex was thus a good candidate for regulating H2A.Z

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dynamics at CCND1 regulatory sequences. TIP48 and TIP49 are ubiquitously

expressed and are often part of the same complex. In most cell types, and in

particular in epithelial cancer cells such as MCF-7 cells, silencing one of the partners

by interference RNA lead to degradation of the other partner [35]. Better antibody

specificity and efficiency prompted us to investigate TIP48. TIP48 was associated

with the promoter and enh2 of CCND1 in non-induced ER�-positive MCF-7 cells (Fig.

1D). Binding of TIP48 to the CCND1 TSS and enh2 decreased rapidly following

addition of 100 nM estradiol (E2). Expression of the gene coding for TIP48 was

insensitive to E2 (Fig. S1A). To examine the relationship between TIP48 and H2A.Z,

we selectively depleted TIP48 by siRNA (Fig. S1A). In siTIP48 transfected cells

treated or not with E2, H2A.Z binding to CCND1 was reduced compared to control,

non–specific siRNA transfected cells. Levels of H2A.Z binding in the absence of

TIP48 were roughly equivalent to levels in E2 treated control cells (Fig. 2A).

Moreover, nucleosome density assessed by immunoprecipitating histone H3 was

unchanged near the TSS (Fig. 2B). Thus, eviction of H2A.Z upon initiation of E2

stimulated transcription was not due to general chromatin decondensation around the

CCND1 gene and its promoter region in particular. TIP48 appears to be necessary

for recruiting H2A.Z to the CCND1 gene in MCF-7 mammary tumor cells.

We thus asked whether binding of H2A.Z or its release were important for

regulating CCND1 transcription. H2A.Z mRNA expression levels were reduced ~5-

fold in cells 48h post transfection with a smartpool siRNA directed against H2A.Z

compared to control cells (Fig. S1B). Reduced levels of H2A.Z did not alter basal

CCND1 expression levels, but impeded activation by E2 (Fig. 2C). Similarly, in the

absence of TIP48, basal transcription levels were conserved, while activation of

CCND1 by E2 was compromised (Fig. 2C). H2AFZ mRNA levels were not affected

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by selective knockdown of TIP48 (Fig. S2A). Thus, Stimulation of CCND1 expression

required release of H2A.Z concomitantly from both these DNA elements.

TIP48 promotes ER� binding during E2 activated transcription.

Absence of activation was likely due to failure of ER� fixation to the CCND1

promoter. Under standard conditions, E2 stimulated ER� binding to both the

promoter and enh2 of CCND1 (Fig. 3A) [9]. Selective knock down of TIP48 hindered

ER� binding to these sites (Fig. 3A). Reduced binding could not be attributed to

altered or decreased ESR1 expression patterns in cells transfected with control or

TIP48 siRNAs (Fig. 3B). Therefore, TIP48 appears to be necessary to remodel

CCND1 chromatin structure for productive ER� binding in the presence of hormone.

TIP48 promotes recruitment of TIP60 to CCND1

TIP48 and TIP60 have been found as part of the same complex [20,25]. TIP60

also cooperates with ER� and other chromatin-remodeling enzymes during estrogen-

induced transcription [36,37]. We tested whether TIP48 and TIP60 binding to the

CCND1 promoter was coordinated. In the presence of E2, TIP60 was recruited to the

CCND1 promoter (Fig. 4A). Upon depletion of TIP48, TIP60 no longer associated

with the CCND1 TSS (Fig. 4A). Cooperation between TIP48, ER� and TIP60 binding

was likely to be necessary for transcription activation. To unravel a functional link, we

first over-expressed TIP60 in MCF-7 cells (Fig. S2B). TIP60 overexpression

stimulated E2 activated CCND1 transcription nearly 5-fold compared to control

untreated cells, without affecting neither basal, non-induced mRNA levels (Fig. 4B)

nor the expression pattern of the H2AFZ gene (Fig. S2C). In siTIP48 transfected

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cells, overexpression of TIP60 was no longer able to stimulate CCND1 transcription

upon E2 stimulation (Fig. 4C), suggesting that TIP48 is required for TIP60 function.

TIP60 is found in protein complexes able to acetylate histones, with a

preference for lysine 5 of H2A [38]. Core histones are generally acetylated in the

promoter region of transcribed genes. Acetylation of the histone variant H2A.Z was

shown to characterize active genes in yeast and recently also in prostate cancer cells

[13,39]. Using an antibody that specifically recognizes H2A.Z acetylated at 3 N-

terminal lysines, we determined that a large fraction of H2A.Z bound to the CCND1

promoter and the 3’ enh2, was highly acetylated (Fig. 5A). Acetylation levels of

H2A.Z did not vary following E2 induced CCND1 gene activation in control samples

(Fig. 5A). However, because H2A.Z was released during transcription activation, the

ratio of acetylated H2A.Z/total H2A.Z increased nearly 2-fold at these sites (Fig. 5B).

In siTIP48 transfected cells, we observed a decrease in acetylated H2A.Z present at

the TSS and the enh2 (Fig 5A). The increased ratio of acetylated H2A.Z associated

with the CCND1 gene following E2 was abolished in cells transfected with siTIP48

(Fig. 5B). The reduced ratio of H2A.Z acetylation thus correlated with impeded

transcription activation in siTIP48 transfected MCF-7 cells (Figs. 5B and Fig. 2C).

In conclusion, failure of TIP60 to associate with CCND1 in the absence of

TIP48 correlated with reduced binding of ER� (Fig. 3), reduced levels of H2A.Z

acetylation at the CCND1 gene (Fig. 5) and the inability to activate this gene by

estrogen (Fig. 2C).

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TIP48 modulates CCND1 chromatin structure by controlling gene looping

Long-range chromatin interactions between ERα recognition sequences and

enhancers have been proposed to regulate ERα-target genes in breast cancer cells

[40,41]. The main enhancer regulating CCND1 is located at the 3’ end of the gene,

14kb distant from the promoter [9]. Gene looping via promoter-enhancer crosstalk is

associated with repressed, low CCND1 expression in ERα-negative, MDA-MB231

cells [42]. Thus we asked whether this loop also existed in MCF-7 cells and more

importantly, whether looping was sensitive to hormone.

We used a chromatin conformation capture (3C) assay. The 3C method

detects physical proximity between distal DNA sites by ligation of cross-linked

restricted DNA fragments [43,44]. Ligation products between enh2 and promoter, and

between enh2 and a control fragment inside the CCND1 ORF were amplified and

normalized to an amplified enh2 PCR product (see Materials and Methods) (Figure

6A). We measured significant interaction frequencies between enh2 and promoter

sequences in MCF-7 cells grown in hormone-stripped media (-E2). Interaction

frequencies were reduced ~10-fold 45min after addition of E2 to the cell culture (Fig.

6B). No significant amplification of ligation products between enh2 and the internal

control fragment was detectable. Hence, an extragenic loop mediated by specific

promoter enhancer interactions was present when CCND1 expression is low (Figs.

6B and 2D). Upon transcription activation, gene looping is markedly reduced.

It was tempting to speculate that TIP48 plays a role in regulating looping. We

assessed the relative frequencies of interaction between enh2/promoter and

enh2/internal control fragments in MCF-7 cells transfected or not by siTIP48.

Depletion of TIP48 had no impact on enh2/promoter contacts in the absence of E2

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(Fig. 6B). This observation correlated with identical basal expression levels of

CCND1 in control and siTIP48 transfected cells (Fig. 2C).

45min after addition of E2 to the cells, the frequency of enh2/promoter

interaction was 5-fold greater in siTIP48 transfected cells compared to control cells

(Fig. 6B). Conservation of significant repressive gene looping could thus account for

impeded E2 bound ERα binding to the CCND1 promoter and compromised

transcription activation. We propose a model (Fig. 7) in which TIP48 is required at

early steps during transcription activation which is initiated by release of H2A.Z and

subsequent dissociation of the enhancer from the promoter. E2 bound estrogen

receptor can then recognize the promoter and stimulate transcription of CCND1.

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Discussion

We unraveled a role for TIP48 in initiating transcription activation of the

CCND1 oncogene. Recruitment of the histone acetyltransferase TIP60 is dependent

on TIP48 and H2A.Z binding to the promoter and 3’ enhancer of the CCND1 gene.

We propose that low levels of CCND1 expression are regulated because the

associated gene loop is transcription-dependent. This regulation is brought about by

the activity of TIP48 containing complexes which locally act upon chromatin structure

to release a disabling loop. Such a mechanism allows fine-tuning transcription

regulation of genes pivotal for the cellular equilibrium in rapidly changing

environments.

Our work describes early events implicated in E2 induction of CCND1. These

events include dynamic exchange of a series of cofactors, namely the TIP48 complex

and histone variant H2A.Z, recruitment of TIP60 and acetylation of H2A.Z enabling

the main transcription factor, the estrogen receptor, to associate with its target

sequences. TIP60 can directly interact with ERα and its acetyltransferase activity is

important during transcription initiation once ERα is bound to target gene promoters

[36].

TIP60 is a versatile enzyme that functions with a variety of partners in a gene

and cell specific manner [36,45]. Selective knock-down of TIP60 by siRNA

compromises activation of some, but not all ERα target genes in MCF-7 cells, as well

as nuclear receptor independent genes in several cell lines (unpublished

observations). CCND1 was one of the genes found to be insensitive to siTIP60 [36].

This observation denotes that TIP60 can be replaced by other histone

acetyltransferases in CCND1 transcription activation. Thus, dependency of early

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chromatin remodeling steps on TIP48 and H2A.Z may be more generally applicable

to allow cofactor recruitment for productive ERα binding in stimulated transcription.

We found that H2A.Z was removed from CCND1 regulatory elements while

this variant had previously been shown to be recruited to the promoter of the TFF1

gene upon E2 treatment of MCF-7 cells [24]. Differences in promoter structure are a

plausible explanation for divergent remodeling mechanisms. It is also likely that post-

translational modifications of H2A.Z are important as shown in a recent genome wide

study by Valdes-Mora et al. who found that H2A.Z acetylation at the TSS correlates

with active transcription in prostate cancer cells [13]. Indeed, the level of acetylation

of H2A.Z near the TSS of CCND1 was equivalent at non-activated and E2 stimulated

cells. We propose that, in ER�-positive breast cancer cells, the ratio of acetylated

H2A.Z / H2A.Z rather than the total amount of H2A.Z bound to the CCND1 promoter

correlates with transcriptional activity.

Chromatin remodeling events are crucial for hormone stimulated activation of

estrogen receptor target genes. However, so far, all data available describe the

recruitment of remodeling complexes and cofactors once the estrogen receptor is

bound. The Brg1 subunit of the SWI/SNF complex is one of the first proteins to

associate with ERα and, although transcription is no longer activated in its absence,

ERα remains bound in siBrg1 transfected cells [36]. Here we demonstrate that

chromatin remodeling events prior to ERα binding are essential for initiating

transcription. These events depend on TIP48 and H2A.Z specific nucleosome

conformation. Chromatin structure impedes ERα loading via intragenic looping.

Notably, interaction between promoter and enhancer sequences forms a repressive

complex. Reduced distances between 5’ and 3’ ends of gene loci have been

attributed to greater chromatin density. In this case, looping does not require

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14

changes in chromatin compaction. Dynamic release of gene loops is consistent with

rapid chromatin remodeling and transcription activation by hormone.

Finally, addition of hormone triggers large scale chromatin remodeling. In

breast cancer cells gene response to progestin is mediated by nucleosomes[46] and

estradiol treatment leads to expansion of chromosome territories within minutes [47].

This latter phenomenon was also observed in ER negative cells (unpublished)

suggesting that chromatin decondensation is independent of the receptor and may

prepare its binding in ER positive cells. It is thus tempting to speculate that the

signaling mechanism by which hormone addition primes chromatin triggers histone

exchange and remodeling prior to ER binding.

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Materials and Methods

Cell lines, transfection and western blotting.

MCF-7 cells were purchased from ATCC and were maintained in DMEM/F12

without phenol red with Glutamax containing 50 mg/ml gentamicin, 1 mM sodium

pyruvate and 10% heat-inactivated and steroid free fetal calf serum (FCS)

(Invitrogen). MCF-7 cells were treated with 10-7 M estrogen E2 (Sigma) for the

indicated times. 5x106 MCF-7 cells were transfected with 20 nM of H2A.Z siRNA ON-

TARGET plus SMARTpool, TIP48 siRNA ON-TARGET plus SMARTpool or

scrambled (scr) siRNA (Dharmacon Thermo Scientific) using Interferine (Ozyme).

Cells were mock-transfected (pcDNA3.1) or transfected with 1 µg of pcDNA3.1/TIP60

(gift from Dr. Didier Trouche) using the Amaxa® Cell line Nucleofactor Kit V program

P-020 according to the manufacturer’s protocol. TIP60 siRNA [45] was purchased

from Eurogentec, and transfected using Interferine (Ozyme). 5x105 MCF-7 cells were

seeded in 6 well plates. 72 h following siRNA transfection, total cell extracts were

isolated and protein levels of H2A.Z, TIP48 and TIP60 analyzed by immunoblotting

on gel SDS-page 15% using antibodies against H2A.Z (ABCAM, ab4174), TIP48 (gift

of Dr. Mikhaïl Grigoriev) TIP60 [45] or GAPDH (Millipore, mab374).

RNA analysis.

Total RNA was extracted using an RNeasy mini-kit (Qiagen) and eluted with

35 µl of RNAase-free water. First strand cDNA was generated using 2 µg of total

RNA in a reaction containing random oligonucleotides as primers with the

ThermoScript RT-PCR system (Invitrogen). Real-time PCR was performed on a

Mastercycler® ep realplex 4 (Eppendorf) using the platinum SYBR Green q-PCR

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SuperMix (Invitrogen) according to the manufacturer’s instructions. Amplification

conditions: 1 min at 50 °C, 3 min at 95 °C followed by 40 cycles (20s at 95 °C, 20s at

60 °C, 20s at 72 °C). mRNA expression were normaliz ed against expression levels of

the RPLP0 ribosomal gene used as an internal control. qRT-PCR primers: H2AFZ:

5’-CCTTTTCTCTGCCTTGCTTG-3’ and 5’-CGGTGAGGTACTCCAGGATG-3’,

CCND1: 5’-GCGTCCATGCGGAAGATC-3’ and 5’-ATGGCCAGCGGGAAGAC-3’,

RPLP0: 5’-TGGCAGCATCTACAACCCTGAA-3’ and 5’- CACTGGCAACATTG

CGGACA-3’, TIP48: 5’-TGAAGAGCACTACGAAGACGC-3’ and 5’-

CCTTACTACCCAGCTC CTGA- 3’

ChIP assays

ChIP analyses were performed as described previously [48]. Samples were

sonicated to generate DNA fragments <500 bp. Chromatin fragments were

immunoprecipitated using antibodies against H2A.Z (ab4174, ABCAM), Acetyl H2A.Z

(ab18262, ABCAM), TIP48 (gift of Dr. Mikhaïl Grigoriev), ER� (sc-543, Santa Cruz),

H3 (ab1791, ABCAM), TIP60 [49] or an irrelevant HA antibody (H6908, Sigma). The

precipitated DNA was amplified by real-time PCR, with primer sets designed to

amplify the promoter (TSS) and enh2 enhancer regions of the CCND1 gene (Fig.

1B). qRT-PCR primers: CCND1 (TSS): 5’-CGGGCTTTGATCTTTGCTTA-3’ and 5’-

ACTCTGCTGCTCGCTGCTAC-3’, distal CCND1 enhancer (enh2): 5’-

CAGTTTGTCTTCCCGGGTTA-3’ and 5’- CATCCAGAGCAAACAGCAG-3’. All ChIP

data are shown as percent input.

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3C Assays

3C assays were performed essentially as described [50,51], with minor

modifications. MCF-7 cells were treated with E2 10-7 M for 45 mn or transfected with

a scrambled control siRNA, with TIP48 SMARTpool siRNA (Dharmacon Thermo

Scientific), and cultured in phenol red-free DMEM containing 10% FBS-T for 72h

before cross-linking. The culture medium was removed, and cells were fixed with

1.5% formaldehyde for 10 min at room temperature. Cells were then washed twice

with cold phosphate-buffered saline solution, and resuspended in ice-cold lysis buffer

(10 mm Tris-HCl, pH 8.0, 10 mm NaCl, 0.2% Nonidet P-40, and protease inhibitor

mixture). Nuclei were resuspended in 1 ml of Buffer B 1.2X buffer (MBI Fermentas)

supplemented with SDS 0,3%. Triton X-100 1,8% was added to sequester the SDS

and incubated for 1 h at 37 °C. The cross-linked DN A was digested overnight with

400 units of restriction enzyme Csp6I (MBI Fermentas). The restriction enzyme was

inactivated by incubation at 65 °C for 20 min. The reactions were diluted with ligase

buffer (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 10 mm dithiothreitol, 1 mm ATP, and

25 �g/ml bovine serum albumin), supplemented with Triton X-100 (1% final

concentration). The DNA was ligated using T4 DNA ligase (New England Biolabs,

Ipswich, MA) overnight at 16°C and an additional 10 0 units for 2 h at 37°C. RNase

was added for 30 min at 37°C, and samples were incu bated with SDS overnight at

70°C to reverse the crosslink. The following day, s amples were incubated for 2 h at

45°C with proteinase K, and the DNA was purified by phenol-chloroform extractions

and ethanol precipitation. Interaction between chromatin domains was assessed by

real-time-PCR amplification for each predicted ligation event [50,52]. Primers have

been designed on the digested BAC fragments, directly around the putative site of

ligation for the four possibilities. BAC clones RP11-300ID (BACPAC Resources

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Center at Childrens Hospital Oakland Research Institute, Oakland, CA) containing

the CCND1 gene and downstream 160-kb region were used. 40 ug of BAC was

digested by Csp6I overnight and ligated. This product was purified by phenol

chloroform and precipitated in order to generate 3C control templates. PCR primer

efficiency was measured by amplifying 0.01 to 50 ng of digested BAC product and

also tested on a fixed amount (50 ng) of digested genomic DNA. All primers have an

annealing temperature between 65 to 70° C and a pro duct size around 150-300 bp.

All primer combinations showed PCR efficiency between 90 and 100%. 3C assay

results are presented as the average from three independent preparations of 3C

DNA, followed by qPCR analysis in triplicate. qPCR for enh2 (PCR primers design

inside the Csp6I restriction fragment enh2) was used as an internal control to verify

ligation events. Non-digested sample and ligation between a control fragment and

enh2 were also performed (data not shown). Primers used for one of the four ligation

event tested: Enh2/Prom: 5’-CTGGGAGAGATGGAGCTGAG-3’ and 5’-

GGTTTTGTTGGGGGTGTAGA-3’, Enh2/ctrl: 5’-AAGCTCTCCCACAACCCATT-3’

and 5’-GTCAGCCCCACTGTTGACTC-3’. Other primers available upon request.

Acknowledgements

We would like to thank Mikhaïl Grigoriev for the generous gift of the TIP48

antibody, Didier Trouche for kindly providing the TIP60 expression vector and

antibody.

Conflict of interest

The authors declare no competing financial interests.

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Figure legends

Figure 1: TIP48 and H2A.Z are required for CCND1 activation by estradiol. A)

MCF-7 cells were cultivated 3 days in steroid stripped (white) medium and then

induced by E2 10-7 M for 6 h. CCND1, H2AFZ mRNA was quantified by real-time RT-

PCR. The mean and SD from three independent experiments are shown. (*)

indicates a p value < 0.05 (Student t-test). B) MCF-7 H2A.Z ChIP on chip signal

detected around the CCND1 gene on human chromosome 11 loaded from [32] on

UCSC. C, D) H2A.Z (C) and TIP48 (D) occupancy at CCND1 TSS and enh2 before

and after 30 min and 60 min of E2 10-7 M treatment analysed by ChIP. The values of

ChIP efficiencies are given in % of input with s.e.m indicated (n=3). Positions of

DNAseI hypersensitive sites and primers used for ChIP are indicated in C).

Figure 2: TIP48 regulates estrogen-activated transcription via H2A.Z release. A,

B) MCF-7 cells were cultivated 3 days in steroid free medium, transfected with,

scramble (ctrl) or TIP48 siRNA for 72 h and induced by E2 10-7 M for 30 min.

Association of H2A.Z (A) and histone H3 (B) with TSS and enh2 sites was analyzed

by ChIP and shown as % input (n=2). C) MCF-7 cells were cultivated 3 days in

steroid free medium and transfected at day 0 with a Scramble (Ctrl), a H2A.Z siRNA

or a TIP48 siRNA for 72 h and then induced by E2 10-7 M for 6 h. CCND1 mRNA

levels were determined by qRT-PCR. Control (Ctrl) without E2 treatment

was set to 1. The mean and SD from three independent experiments are shown. (*)

indicates a p value < 0.05.

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Figure 3: TIP48 is required for ER� binding during E2 activated transcription.

MCF-7 cells were cultivated 3 days in steroid free medium, transfected with scramble

(ctrl) or TIP48 siRNA for 72 h and induced by E2 10-7 M for 30 min. A) ChIP analysis

of ER� occupancy at the CCND1 TSS and enh2 sites. Results are shown as % input

(n = 2). B) qRT-PCR quantification of ESR1 mRNA expression relative to RPLO. The

mean and SD from three independent experiments are shown. (**) indicates a p

value < 0.01.

Figure 4: TIP48 promotes TIP60 binding and CCND1 transcription activation.

MCF-7 cells were cultivated 3 days in steroid free medium and induced by E2 10-7 M

for 6 h. A) Cells were transfected with scramble (-) or TIP48 siRNA for 72 h and

induced by E2 10-7 M for 30 min. TIP60 binding to the CCND1 promoter was

analyzed by ChIP and shown as percent of input (n=2). B) After 24 h in steroid free

medium, MCF-7 cells were transfected with a mock vector or a vector expressing

TIP60 for 48 h. CCND1 mRNA expression was analyzed by qRT-PCR. The mean

and SD from three independent experiments are shown. (*) indicates a p value <

0.05. C) MCF-7 cells were first transfected with scramble (-) or TIP48 siRNA as in

(A). After 24 h, same cells were transfected with a mock vector or a vector

expressing TIP60 for 48 h as in (B). CCND1 gene expression was analyzed by qRT-

PCR after 30 min of E2 10-7 M induction.

Figure 5: TIP48 promotes acetylation of H2A.Z. MCF-7 cells were cultivated and

transfected as in Fig 2. A) Association of Acetyl H2A.Z with CCND1 TSS and enh2

sequences analyzed by ChIP and shown as % input (n=2). B) Ratio of acetylated

versus total H2A.Z in %.

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Figure 6: Release of CCND1 gene looping requires TIP48. A) Schematic

representation of the CCND1 locus on h.s. chromosome 11 (adapted from the USCS

genome browser http://genome.ucsc.edu/cgi-bin/hgGateway). DNAseI hypersensitive

sites [9], Csp6I restriction sites and fragments assayed by 3C are represented. B)

Quantitative PCR amplification of ligation events in a 3C assay in MCF-7 cells treated

or not with E2 10-7 M for 45 min and transfected 72 h before with scramble (ctrl) or

TIP48 siRNA. Crosslinked DNA was digested with the Csp6I restriction enzyme and

ligated. qRT-PCR was performed with primers designed for the 4 possible ligation

events (only one is shown).

Figure 7: Model for the role of TIP48, H2A.Z and TIP60 in initiation of transcription

activation of CCND1 via promoter enhancer crosstalk.

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Supplemental Figure legends

Figure S1: Depletion of TIP48 and H2A.Z by siRNA. MCF-7 cells were cultivated 3

days in steroid free medium and then induced by E2 10-7 M for 6 h. A) TIP48 mRNA

expression levels analyzed by qRT-PCR and TIP48 protein analyzed by

immunoblotting in siTIP48 transfected compared to control cells. B) H2AFZ mRNA

expression levels analyzed by qRT-PCR and H2A.Z protein analyzed by

immunoblotting in siH2A.Z transfected compared to control cells.

Figure S2: TIP60 overexpression and H2AFZ gene expression. MCF-7 cells were

cultivated 3 days in steroid free medium and then induced by E2 10-7 M for 6 h. A)

TIP60 gene expression analyzed by qRT-PCR. TIP60 protein expression analyzed

by western-blotting. B) H2AFZ gene expression levels were analyzed by qR-PCR in

siTIP48 transfected compared to control cells. C) H2AFZ mRNA expression levels

analyzed by qRT-PCR in siTIP48 transfected compared to control cells.

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Dalvai et al. Fig. 1 R

ela

tive m

RN

A e

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n

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2.0

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NT

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ChIP H2A.Z

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B

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0-

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Dalvai et al. Fig. 2

si ctrl si TIP48 si ctrl si TIP48

% Input

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+ - + -

* *

E2 treatment

E2 treatment

E2 treatment

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Dalvai et al. Fig. 3

% Input

x 10-3

0

0.2

0.4

0.6

0.8

1.0

A

TSS enh2 x 10-3

0

0.2

0.4

0.6

0.8

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0

x 10-3

0.3

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0.9

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C

Dalvai et al. Fig. 4

*

E2 treatment

E2 treatment

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120

0

60

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ChIP H2A.Z acetylation/H2A.Z B

0

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Dalvai et al. Fig. 5

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0.15

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0.25

0.05

0.4

0

0.2

0.3

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% Ac-H2A.Z / H2A.Z

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B

Dalvai et al. Fig. 6

6kb

TSS Exon1 Exon2,3 Exon 4 Exon 5

3’ UTR CCND1 RefSeq:NM_053056.2

DNAse I hypersensitive site enhancer2 promoter

ChIP primers

Csp6I restriction site

3C experiment Control Amplification

Loop Enh2/Prom

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Dalvai et al. Fig. 7

Enh2 TSS

ac

Opening of chromatin

and remodeling

ac ac ac

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blocks ER fixation

TIP

48

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48

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Z Z

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Tip60 and ER recruitment leads to

CCND1 Expression

ER ER

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Dalvai et al. Fig. S1

0

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Dalvai et al. Fig. S2

TIP60 mRNA

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� ���

IX. Supplementary data.

During the last years, aspects of cellular biology became important to understand

biological mechanisms and regulation. The spatial and temporal behavior of the genome

is now recognized as intimately linked to control genome function. Further studies about

the contribution of nuclear architecture and its impact on gene expression will be required

to better understand physiological processes such as differentiation, development and

diseases at the cellular level (Misteli 2005).

It is now clear that chromatin organization in the vertebrate nucleus is non-random.

Chromosomes adopt preferential position with regard to the center or edge of the nucleus

and genes adopt preferential positions with regard to their own chromosome territory

(Fraser and Bickmore 2007). In particular, the relative position of a gene with regard to its

chromosome territory may be related to transcriptional gene regulation (Chambeyron and

Bickmore 2004); (Kocanova, Kerr et al. 2010).

Each chromosome’s neighbor in the nucleus varies between cell types, and it has

consequences for the chromosome’s ability to interact in trans with other parts of genome,

as revealed by the frequency of specific chromosome translocations (Bickmore and

Teague 2002; Parada, McQueen et al. 2004). The positional organizations of

chromosomes within the nucleus and of genes within chromosome territories impinge on

other aspects of genome function and, in particular, on the regulation of gene expression

(Xu, Tsai et al. 2006), (Bacher, Guggiari et al. 2006)).

To study the organization of the genome in the nucleus, there are, essentially, two main

techniques: fluorescent in situ hybridization (FISH) and chromosome conformation

capture (3C).

In 3C, chromatin fragments in close proximity in the nucleus are captured by fixation,

restriction-enzyme digestion and intramolecular ligation. The interaction between two

designated loci is tested by PCR with specific primers for the loci under investigation.

In FISH, the relative nuclear position of genes, genomic region or even whole

chromosomes are analyzed by the hybridization of fluorescent probes to nuclei fixed on

glass slides. The hybridization signals are then visualized with fluorescent microscopy.

With this technique in our laboratory, we performed several studies. In particular, we were

interested in gene movements within nucleus and with regard to their chromosome

territory, during gene activation.

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� ���

Using estrogen receptor target genes as a model, we can study and analyze in 3D the

genes displacements and “interactions” (Kocanova, Kerr et al. 2010), clarifying the

existence of a link between nuclear gene position and its transcriptional state.

During my doctoral project, I tested the 3D nuclear organization preserving FISH

technique on other genes,in particular, on non-estrogen receptor target genes. I analyzed

p21 gene position relative to chromosome territories in ER�-positive, p53-positive MCF-7

cells, and asked whether its position changed after estradiol treatments.

First, I performed 2D FISH on metaphase cells to know how many chromosomes 6 and

p21 gene copies are present in MCF-7 cells (Figure 1).

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So,�I performed FISH experiments on interphase cells, using DNA from BAC clones as a

fluorescent probe to visualize p21 gene and its chromosomal territory (chromosome 6). I

treated or not the cells with estradiol (10nM) during 3h and I observed p21 gene behavior

on fixed cells (Figure 2).

CT6

p21

DAPI

ESR1

10µm

+E2-E2

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The first thing that we see is that the chromosomal territory occupancy is very precise and

discrete in each nucleus. Each chromosome 6 territory includes a p21 copy, the p21

localization is not fixed within territory and seems to be independent of transcriptional

state. No relevant and statistical difference has been observed between treated and non-

treated cells. These types of experiments were thus inconclusive.

The only relevant and interesting observation was the presence of some “doublets”

(shown in figure 2B), indicating a decondensation of the locus, which may be in relation

with an active transcription.

The localization experiments on p21 were abandoned, but not the strategy. In the

laboratory, investigation about gene movements and localization during activation and in

different cellular contexts is actively pursued. Now, we search to visualize, using shorter

fluorescent probes based on fosmids, chromatin behavior during gene activation. In

particular, using the CCND1 gene as a model, we try to visualize the opening of the loop

between its TSS and 3’ enhancer region (see results and discussion).

Moreover, we try to perform the same observations but in living cells. In this manner, we

will be able to follow all the steps of gene activation and the eventual displacements in

nucleus.

CT6

p21

DAPI

5µm

CT6

p21

DAPI

5µmA B

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� ���

A. Materials and Methods.

For 2D FISH on metaphase cells. The cells were grown in DMEM (10% FCS, 1%

Antibiotic, 1% glutamine), at 70-80% confluency, before addition of 2mM Thymidine for

24h (S-phase block). Thymidine was then removed by washing with 1xPBS; and cells

cultured in fresh DMEM for 3-4h. After cell releasing, 100ng/ml Colcemid was added for

12h (mitotic block). Cells were harvested in mitotic phase by shaking, after light

trypsinization The rest of the cells were also harvested. They were centrifuged for 10 min

at 1000 rpm. The supernatant was removed, and cell pellets resuspended. Gently add 75

mM KCl (37°C) for hypotonic treatment. Incubate for 10 min in 37°C water bath.

Centrifuge 10 min at 1000 rpm. Remove supernatant, leave about 1 ml, resuspend pellet.

Add 2 ml fixative (ethanol/acetic acid 3 : 1; 150ml = 111ml EtOH + 37ml AcA), mix well

and gently. Add fixative until 10 ml, mix meanwhile. Repeat the pellet is white. Remove

fixative until about 1ml final volume. Resuspend pellet and apply suspension on clean

slides stored at 4°C in 50% EtOH.

For 3D FISH on interphase cells. Cells were grown for 3 days on coverslips in DMEM

without phenol red, containing 5% charcoalstripped FCS, before addition of 10 nM or 100

nM E2 for the indicated times. Coverslips were washed twice with PBS, fixed in 4%

paraformaldehyde (pFA)/PBS for 10 min at room temperature and during the last minute

200 ml of 0.5% Triton X-100/PBS were added into 500 ml of 4% PFA. After fixation, the

cells were washed three-times for 3 min in 0.01% Triton X-100/PBS, incubated in 0.5%

Triton X-100/PBS for 10 min at room temperature and then incubated with 0.1 mg/ml

RNase in 2xSSC for 1 hour at 37°C. After 3 washes o f 10 min in PBS, cells were

incubated in 0.1 M HCl for 5 min at room temperature, twice in 2xSSC for 3 min and then

equilibrated overnight in 50% formamide/2xSSC (pH =7.2).

DNA from BAC clones which include p21(RZPDB737B0916) and ESR1 (RP11-450E24)

were labeled using nick translation (BioPrime DNA Labeling System, Invitrogen) by

incorporation of fluorochrome-conjugated nucleotides ChromaTide AlexaFluor 488-5-

dUTP (Molecular Probes). Chromosome paints (chromosome 6) was obtained from

Genetix Ltd., UK. We used 100 ng labeled DNA probe or 100 ng chromosome paint

together with 7 mg Cot-1 DNA and 5 mg sonicated salmon sperm DNA per slide.

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� ���

X. Discussion and outlook.

Biological complexity and variability is key to all evolutionary processes. Control of gene

expression that underlies this complexity is tissue, cell or, even, gene dependent. If on the

one side this constitutes the beauty of research, on the other side it represents a problem.

Indeed, new findings cannot be generalized to all genes in a complex multicellular

organism. It is thus necessary to select specific model genes and choose the appropriate

cellular models to define molecular mechanisms regulating gene expression and to gain

insight into functional pathways.

We focus our interest, in particular, on epigenetic mechanisms. The cellular epigenetic

pattern is like an “identity card” for the cell. Epigenetic mechanisms are involved in all

“normal” cellular processes (cell cycle, cell division, transcription etc. etc.). At the same

time, epigenetics can be involved in pathological situations, such as cancer, hyper-

proliferation, or drug resistances. Finally, epigenetics are involved, especially, in control

and regulation of gene expression. In this case, the epigenetic programs represent a sort

of “cellular memory”, a heritage that characterizes cell types, tissues, cancers etc.

A case-by-case study of the biological mechanisms as well as epigenetic involvement in

transcriptional gene regulation is important. In this manner, we respect the rudiments of

biological investigation: understand the complexity and try to address it in our favor if it is

possible. All our studies were performed keeping this idea in mind. In addition to our aim

to define generally applicable mechanisms, we keep in mind that each gene (for example)

is different and may, in a particular environment or condition, have different mechanisms

of control.

One of the most intriguing epigenetic mechanisms is to modulate chromatin structure by

exchanging canonical histones with histones variants. Several studies show the

involvement of specific histones variants in particular cellular processes. Among the

plethora of the discovered and studied histone variants, we are interested in clarifying role

of the histone variant H2A.Z in transcriptional gene regulation. We choose to use human

breast cancer cell lines as a model. In fact, studying the activation of ER� target genes,

we are able to investigate both early and late processes of transcriptional activation.

Moreover, we know that different breast cancer cell types have different epigenetic

characteristics. In particular, there are some interesting differences between ER�-positive

and negative cells. ER�-negative cells are more proliferative and aggressive due to

“special” epigenetic patterns. Therefore, in these cell lines, epigenetic programs play a

critical role for gene expression, activation and repression.

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We have shown that both the presence and the acetylation of H2A.Z are important for

gene activation. Acetylation of H2A.Z seems to be truly critical for transcriptional gene

regulation more than its presence. Treatment of ER-negative cells MDA-MB231 with TSA

activates p21 expression. Interestingly H2A.Z depletion prevented TSA activation of p21.

Looking at the H2A.Z acetylation on the p21 starting site, we show that it increases after

TSA treatments leading to p21 activation. Depleting H2A.Z and its possibility to be

acetylated, impairs p21 activation. Therefore, the critical element for p21 activation is

H2A.Z acetylation more than its presence or not.

Our findings are not the first that give due importance to H2A.Z acetylation and its

involvement in gene expression. Several studies show that acetylated H2A.Z is present on

gene promoters and represent a mark for gene activation, both in yeast and mammalian

cells (Bruce, Myers et al. 2005; Millar, Xu et al. 2006).

We still have to elucidate by which mechanisms H2A.Z interacting with the p21 promoter

can be acetylated. In general, a good candidate for H2A.Z acetylation is the

acetyltransferase Tip60 (Dalvai, Bellucci et al. 2012). However, this factor is not

responsible for modification of H2A.Z on the p21 promoter. Likely, histone-modifying

complexes are specific to various pathways and cells. In MDA-MB231, the activation

HDACi dependent of p21 involving H2A.Z acetylation, did not require Tip60, but involved a

strong recruitment of the HAT p300 to the p21 promoter accompanied p21 expression.

P300 has been found to regulate p21 via promoter acetylation in other cellular contexts

(Billon, Carlisi et al. 1999).

Based on our results, we think that H2A.Z is involved in gene activation in different

manners depending of observed gene, pathway, mechanism or cell type. We think, also,

that the complexes (such as acetyltransferases) involved depend on cellular context. It

was shown that Tip60 and p400 play an antagonistic role during p21 activation. Tyteca et

al. showed that Tip60 participates in p21 activation through the stimulation of p53 DNA

binding activity. In contrast, p400 represses p21 expression (Tyteca, Vandromme et al.

2006). So, our cellular context being p53-negative, it is not surprising if p21 activation is

Tip60 independent. At the same time, we find that p400 remains a negative factor for p21

expression. It is highly enriched on the p21 promoter. p400 binding is reduced upon p21

activation via HDACi treatments. We can thus speculate that modulation of the factors

involved in gene regulation, also for the same gene, depends on cellular context and is

the key during evolution and adaptation.

Some proof for this may come from our other work. When we changed the gene, in the

same cell type, the same complexes changed their role and behavior. We demonstrate

that in MDA-MB231 H2A.Z, its acetylated form and Tip60 are critical for CCND1 gene

expression (Dalvai, Bellucci et al. 2012). Moreover, we show that an H2A.Z-dependent

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� ��

short-range loop exists between the transcription starting site and 3’ enhancer sequence.

This loop correlates with downregulation of CCND1 expression. Unlike what was shown

for p21, the depletion of H2A.Z leads to CCND1 expression accompanied by an increase

of its acetylated form bound to the promoter. Acetylation of H2A.Z is, one more time, the

fundamental mark for gene activation. Moreover, inducing CCND1 expression by TSA, we

show that H2A.Z acetylation increases and Tip60 is the HAT involved in this process. In

fact, depletion of Tip60 compromises the TSA-mediated CCND1 activation. It is the first

time that we have evidence for a role of Tip60 in transcriptional activation of the CCND1

oncogene in ER�-negative breast cancer cells. Interestingly, H2A.Z is present both at the

transcriptional starting site and the 3’ enhancer. Increase in H2A.Z acetylation is observed

at both gene positions upon CCND1 activation by TSA.

But the most interesting finding is the presence of a short-range loop that negatively

controls CCND1 expression in MDA-MB231 cells. Long range interactions between

distant enhancers and promoters regions via chromatin looping have been shown to allow

single gene and gene cluster activation. This has been shown for example for Myb proto-

oncogene transcription (Stadhouders, Thongjuea et al. 2012) and for the beta-globin locus

(Tolhuis, Palstra et al. 2002). The looping, in these cases, occurs in the 5’ part of genes

and over tens or hundreds of kb. The role of these loops is to isolate a chromatin portion

from surrounding repressive environment (Vogelmann, Valeri et al. 2011). RNA pol II

seems to be involved in loop formation and maintenance. In fact, another role for

chromatin loops might be to “help” the RNA pol II to recycle, converting it from the

elongation form into initiation form and delivering it back to the promoter (Kadauke and

Blobel 2009). In contrast, the short-range loop observed for CCND1 gene has a

repressive role. Upon activation, the loop is open, H2A.Z is acetylated by Tip60 and the

gene can be transcribed. We think that in this case the chromatin loop has a different role.

We hypothesize that this type of loop helps chromatin compaction but at the same time,

allows the possibility to activate rapidly gene transcription. This possibility is ensured by

H2A.Z recruitment to regulatory regions including promoter and enhancer. Previous model

have shown that H2A.Z containing nucleosomes are more labile and that H2A.Z eviction

promotes chromatin accessibility (Jin, Zang et al. 2009).

Interestingly, this process seems to be fundamental to the acetylation of H2A.Z. H2A.Z

acetylation is the only histone modification that increases (acetylation of the other histones

remains low and stable) after gene activation in 3’ enhancer region, suggesting that this

modification specifically regulates 3D organization of genetic loci.

In a recent study, we use MCF-7 ER�-positive cells to investigate the mechanisms that

characterize the early steps of gene activation. In these cells, CCND1 expression is

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estradiol-dependent. The events involved in E2 induction of CCND1 include dynamic

exchange of a series of cofactors, namely the Tip48 complex and the histone variant

H2A.Z, Tip60 recruitment and H2A.Z acetylation enabling ER� to associate with its target

sequences. Tip60 can directly interact with ER� and its acetyltransferase activity is

important during transcription initiation once ER� binds to the promoters of target genes

(Jeong, Kim et al. 2011).

We show that for CCND1 activation in MCF-7, the eviction of H2A.Z performed by Tip48

(AAA+ family member) as a prerequisite for the ER� binding in presence of hormone.

Tip48 promotes acetylation and exchange of H2A.Z, which triggers the opening of the

loop between TSS and 3’ enhancer region. This release enables the estrogen receptor to

bind to the CCND1 promoter. Removal of H2A.Z from CCND1 regulatory elements is

accompanied by Tip60 recruitment and the change in the ratio of acetylated H2A.Z / total

H2A.Z. Increase of acetylated H2A.Z amount correlates with transcriptional activation in

ER�-positive breast cancer cells. Looking at the early steps that characterize hormonal

activation of CCND1 gene, we show that these events depend on Tip48 and H2A.Z

specific nucleosome conformation. In fact, H2A.Z eviction, acetylation (by Tip60) and the

loop release depend on Tip48 chromatin remodeling factor and prepare chromatin for the

estrogen receptor binding.

All our studies demonstrate that the histone variant H2A.Z has a central role in gene

regulation. In particular, its acetylation seems to play a fundamental role for gene

activation. For a long time, several studies focused exclusively on the presence or not of

this variant. We are first to uncover the importance of the acetylation of H2A.Z.

Each time that we stimulate gene transcription with HDACi, as in the case of p21 or

CCND1 in MDA-MB231 cells, or with E2, as in the case of CCND1 in MFC-7 cells, the

modification that characterizes this activation, is the increase in H2A.Z acetylation. This

evidence paves the way for different outlooks.

Our observations are very significant for several reasons. We show an alternative and

p53-independent pathway to activate p21 in ER�-negative breast cancer cells. These cells

represent a more aggressive and hyper-proliferative tumor. This tumor type is a problem

because it escapes hormonal therapies. We show that HDACi treatments induce p21

expression, leading to proliferation arrest, and this activation depends on H2A.Z

acetylation. This suggests that H2A.Z could have a critical role in tumor aggressiveness.

In the absence of the possibility to acetylate H2A.Z, we can lose the possibility to activate

p21 and to stop cell proliferation. The evidence that the increase of H2A.Z acetylation is a

mark for gene activation opens new potential strategies to arrest cell proliferation, in some

specific cases, such as breast cancer.

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It will be interesting understand how H2A.Z acetylation can recruit the complexes

necessary to start transcription. Is the acetylation of H2A.Z a specific signal to recruit RNA

pol II to gene promoters? We could address this question using in vitro studies, which

allow to create a system with a “constitutively” acetylated form of H2A.Z to understand the

impact on transcriptional gene control. The same study in vivo is nearly impossible.

Introducing a constitutively acetylated form of H2A.Z induces genome-wide alteration and

may change the normal cellular physiology.

Our studies open new avenues for understanding chromatin organization and its

impact on the control of gene expression. The existence of a short-range loop that

represses gene transcription is an exciting finding. We initiated studies to analyze the

chromatin organization by microscopy in fixed and living cells. We can use FISH

(Fluorescent In Situ Hybridization) to test loop presence in different conditions. Using two

probes to visualize the extremities of an expected loop, we are able to see the opening or

not of the loop in a transcriptional activity dependent manner. In particular, in the case of

the CCND1 gene, if we use two probes recognizing TSS and 3’ enhancer sequence, we

can visualize the presence of the loop and its opening after CCND1 activation. The same

strategy can be used for other genes too, for example, for the estrogen receptor target

genes. In the future, we would like to perform the similar approaches in living cells using a

recently implemented and patented chromosome labeling technique (WO/2012/127047).

In this manner, we could observe loop dynamic step by step during gene activation.

Moreover, we can use shorter probes. Indeed, small-range loops are difficult to visualize

using classical FISH probes. Classical probes are long sequences but the sequences

involved in loop formation are short. Using these probes some problems during spots

analysis arise due to microscopy resolution. To prevent these problems we can use

shorter probes, such as fosmide probes, to have more precise signals easier to visualize

and analyze.

The role of H2A.Z and cofactors involved in H2A.Z exchange and modification are cell-

and gene-dependent. Our findings open new research fields of investigation to

increase our understanding of breast cancer biology, but also of epigenetic

mechanisms in general. Our aim is to refine models that approach reality more and

more, keeping in mind that we will never be able to have all the answers. The “tension” to

find one answer is enough.

Page 112: THÈSE - Paul Sabatierthesesups.ups-tlse.fr/1939/1/2013TOU30011.pdfFigure 2 . Schematic representation of DNA methylation. 1. DNA Methyl Transferases There are two classes of DNMTs.

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References

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