H2A.Z-Dependent Crosstalk Between Enhancer and Promoter Regulates Cyclin D1 Expression

ORIGINAL ARTICLE H2A.Z-dependent crosstalk between enhancer and promoter regulates Cyclin D1 expression

M 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 (2013) 32, 4243–4251; doi:10.1038/onc.2012.442; published online 29 October 2012 Keywords: H2A.Z; chromatin; acetylation; enhancer; intragenic loop; cyclin D1

INTRODUCTION cycle-independent in ERa À MDA-MB231 cells9,19 (unpublished Estrogens are intimately linked to hormone-dependent breast observations). The CCND1 ORF spans 14 kb and is flanked by 16 cancer progression by promoting G1–S transition through 50 and 30 enhancers identified by Eeckhoute et al. as DNAse I regulation of key cell cycle regulatory factors including induction hypersensitive site, to which transcription factors are recruited in þ 16,19–21 of cyclin D1, MYC, HER2 and c-SRC and repression of cyclin G2, estrogen responsive ERa cells. The 30 enhancer (enh2) caspase 9 and p21.1,2 17b-estradiol (E2) functions are mediated by defined as a major site of CCND1 regulation in these cells, was À specific estrogen receptors (ERs), which are transcription factors thought to be dysfunctional in ERa MDA-MB231 cells. It is belonging to the nuclear receptor family.3 Two ERs, ERa and ERb, plausible that the lack of enh2 function relates to poor CCND1 each expressed as several isoforms, have been characterized.4–6 transcription. To date the mechanisms responsible for the loss A majority of breast tumors (about 80%) express both receptors of hormonal regulation of gene expression, and subsequently and are hormone-dependent for growth. These tumors are less of proliferation and differentiation processes, are not fully invasive and generally not metastatic.7 In contrast, ERa-negative understood. In addition to aberrant DNA methylation, an breast tumors (ERa À ) are hormone-independent for proliferation, unidentified epigenetic mark imposes formation of a chromatin more invasive and prone to metastasize.8 In ERa À cell lines, both state that forms a barrier to ERa binding in hormone-independent ESR1 and ESR2 encoding ERa and ERb respectively, are silenced cell lines.22 Epigenetic or chromatin marks are thought to mediate together with many ER-target genes including pS2/TFF1 and the a functional dialog with DNA-binding factors to coordinate the progesterone receptor gene (PGR). On the other hand, another activity of cis-regulatory elements in space and time. Chromatin group of ER target genes including cathepsin D (CTSD), VEGF properties can be regulated by DNA methylation and histone or cyclin D1 (CCND1) escape transcriptional regulation.9,10 post-translational modifications, but also by replacement of Cyclin D1, encoded by the CCND1 gene, is a D-type cyclin histones with variants.23–29 Incorporation of histone H2A.Z into whose growth factor-induced expression and accumulation are the chromatin of inactive gene promoters and its removal tightly controlled.11–13 Cyclin D1 regulates G1–S progression and according to physiological signals participates in regulating gene gene expression via chromatin modifications and a plethora of expression.30–33 H2A.Z is principally found at the promoters of cellular functions (for a review see14). Its relative abundance has poised genes and released during gene activation in both yeast major effects on cell growth and metabolism via cell cycle regu- and human cells.31,34–36 In euchromatin, a sharp peak of H2A.Z is lated dosage of its expression.15 CCND1 transcription is regulated seen at the 50 end of numerous genes and at enhancers.37 A by an estrogen-regulated cell-type-specific enhancer and is cell recent genome-wide analysis38 showed that acetylated H2A.Z is cycle regulated in ER-positive breast cancer cells (ERa þ , MCF-7 generally found near the transcription start site (TSS) of active cells).15,16 Downregulation of Cyclin D1 has nevertheless been genes in prostate cancer cells. shown to increase migratory capacity and is associated with poor Here, we investigate the role of the histone variant H2A.Z in the prognosis.17,18 CCND1 expression is low, and hormone- and cell transcriptional regulation of the ER-target gene in ERa À breast

1Laboratoire de Biologie Molećulaire Eucaryote (LBME), University of Toulouse, Toulouse, France and 2UMR 5099 CNRS; LBME, Toulouse, France. Correspondence: Professor K Bystricky, Laboratoire de Biologie Molećulaire 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; published online 29 October 2012 H2A.Z-dependent intragenic looping M Dalvai et al 4244 cancer cells. Our results demonstrate that stimulation of CCND1 CCND1 TSS slightly increased. In contrast, decreased H2A.Z transcription requires release of H2A.Z from both the TSS and binding to the PGR promoter did not allow polymerase II downstream enhancer (enh2) sequences of CCND1. Acetylation of recruitment to this silent gene (Supplementary Figure S1E). Thus, H2A.Z and Tip60 acetyltransferase regulate TSS-enhancer crosstalk H2A.Z eviction is sufficient to stimulate CCND1 transcription in via chromatin looping. ERa À breast cancer cells. Yet, its depletion alone cannot restore the 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 the RESULTS downstream enhancer enh2, previously shown to be inactive in Depletion of H2A.Z activates CCND1 transcription these cells.16 In H2A.Z siRNA transfected cells, H2A.Z binding to We first investigated whether H2A.Z was present at ER-target gene enh2 was significantly diminished (Figure 1c, right panel). In loci in ER À cells. Chromatin immunoprecipitation (ChIP) experi- addition, CCND1 activation in siH2A.Z-transfected MDA-MB231 ments revealed that H2A.Z bound to sequences flanking the cells was accompanied by a strong recruitment of pol II to enh2 CCND1 ORF, to the TSS and the downstream enhancer defined by (Figure 1c, right panel), as described previously for E2 stimulated Eeckhoute et al.16 but not to distant upstream sites in two distinct MCF-7 cells.16 We conclude that enh2 has an active role in cell lines, MDA-MB231 (Figure 1a) and MDA-MB436 (Supplemen- ER-independent CCND1 activation. H2A.Z seemed to interfere with tary Figure S1A). H2A.Z was also present at the promoters of the stimulation of transcription when bound to TSS and enhancer silent ER-target genes, PGR, TFF1 or ESR1 (Supplementary Figure sequences of CCND1. We investigates how modulating S1B). ChIP-on-chip experiments confirmed that H2A.Z associated incorporation of this histone variant regulates gene activity. exclusively with flanking regions of all tested genes and, in particular, the enh2 sites of CCND1 (J Eeckhoute, data not shown). The presence of H2A.Z at these regulatory elements in ERa À H2A.Z acetylation correlates with activation of CCND1 MDA-MB231 cells, prompted us to assess its potential role in transcription transcriptional repression of these genes. To this end, we Recently, Valdes-Mora et al.38 demonstrated in a genome-wide selectively depleted H2A.Z by small interference RNA (siRNA) study that H2A.Z acetylation marks chromatin at the TSS of active (Figure 1b, Supplementary Figures S1C, D). Seventy-two hours genes, which are frequently deregulated in prostate cancer cells. following siRNA transfection, CCND1 mRNA levels increased four- Thus, we examined whether acetylation of H2A.Z was involved in fold compared with mock-transfected cells (Figure 1b), whereas CCND1 function in breast cancer cells. A variety of inhibitors allow PGR, TFF1 and ESR1 remained silent (Figure 1b). Under these modulation of histone post-translational modifications with the conditions, a potential role of the ER in stimulating CCND1 aim to alter gene expression patterns. Exposure of mammary expression could thus be excluded (Figure 1b). ChIP confirmed tumor cell lines to trichostatin A (TSA), a pan HDACi of class 2 that H2A.Z siRNA treatment lead to markedly reduced binding of HDACs, reduces their growth rate39 and induces changes in gene H2A.Z to the CCND1 TSS (Figure 1c left panel). Concomitant to expression patterns.40–44 We tested whether TSA would stimulate increased mRNA levels, polymerase II (pol II) recruitment to the CCND1 expression. MDA-MB231 cells were cultivated in normal media and exposed to 50 ng/ml TSA for 24 h before mRNA quantification. CCND1 mRNA increased four-fold in treated compared with untreated cells (Figure 2a). We also noticed that TSS TSA treatment negatively affected H2AFZ mRNA levels, which were reduced B2-fold (Figure 2a). The ESR1 gene remained silent -2000 enh2 as previously shown22 excluding the possibility that stimulation of -3 -1 À x10 CCND1 x10 CCND1 was due to binding of ERa in these TSA treated ERa cells. 1.8 HA 1.8 These results prompted us to further investigate the mechanism 1.5 H2A.Z 1.5 by which transcription of CCND1 was stimulated. 1.2 1.2 As expected, concomitant to increased transcription in the 0.9 0.9 presence of TSA, acetylation of histone H3 at the CCND1 TSS

% Input 0.6 0.6

expression expression increased (Figure 2b). In these cells, H3 was also acetylated at

0.3 mRNA Relative 0.3 0 0 enh2 suggesting that this enhancer participates in CCND1 -2000 TSS enh2 - + - + - + - + - + Si H2A.Z activation (Figure 2b). We show that H2A.Z was associated with the CCND1 TSS and that a fraction of this histone variant was evicted from the TSS in response to TSA treatment of MDA-MB231

-3 -3 cells (Figure 2c). Interestingly, upon eviction from the TSS, H2A.Z x10 TSS x10 enh2 was also lost from enh2 (Figure 2d). Under the same conditions, 1.2 1.6 1.0 we monitored binding of acetylated H2A.Z (Ac-H2A.Z) to the 1.2 0.8 CCND1 TSS and enh2 using an antibody that speciﬁcally 0.6 recognizes H2A.Z acetylated at K4, K7 and K14 (see Materials 0.8 % Input % Input 0.4 and methods). The amount of Ac-H2A.Z present at the CCND1 TSS 0.4 0.2 or enh2 did not vary following TSA treatment. However, the ratio 0 0 - + - + - + Si H2A.Z - + - + - + Si H2A.Z of Ac-H2A.Z was greatly augmented relative to the total, sharply HA H2A.Z pol II HA H2A.Z pol II reduced amount of H2A.Z detected at these sequences (Figures 2c and d right panel). Figure 1. Depletion of H2A.Z correlates with CCND1 transcriptional Based on the fact that TSA negatively affected H2AFZ mRNA activation. (a) The recruitment of H2A.Z to the CCND1 gene at levels (Figure 2a) we wondered whether stimulation of CCND1 was À 2000, TSS and enh2 was analyzed by ChIP. (b) Expression levels of the result of increased histone acetylation or of a reduction of H2AFZ, CCND1, ESR1, PGR and TFF1 in ERa À MDA-MB231 cells transfected with SMARTpool H2A.Z siRNA ( þ ) or scramble siRNA H2A.Z abundance. We then analyzed if depletion of the cellular ( À ) were analyzed by q-PCR. mRNA expression levels were pool of H2A.Z also altered Ac-H2A.Z binding at the stimulated normalized against expression levels of the RPLP0 ribosomal gene. CCND1 locus. We found nearly three-fold greater amounts of (c) The recruitment of H2A.Z and RNA pol II was analyzed by ChIP at acetylated H2A.Z at the TSS (Figure 3a, left panel) and six-fold the CCND1 promoter (left panel) or enh2 enhancer (right panel). greater amounts (Figure 3b, left panel) at the enh2 in

Oncogene (2013) 4243 – 4251 & 2013 Macmillan Publishers Limited H2A.Z-dependent intragenic looping M Dalvai et al 4245

x10-1 x10-2 TSSx10-3 enh2 1.2 2.5 1.8 1.0 2.0 1.5 0.8 1.2 1.5 0.6 0.9 1.0 % Input 0.4 % Input 0.6 0.2 0.5 0.3 0 0 0 Relative mRNA expression Relative -+ -+ -+ TSA -+ - + TSA -+ -+ TSA HA Ac-H3K9 HA Ac-H3K9

-2 -2 x10 TSS x10 TSS TSS 1.4 0.7 1.8 1.2 0.6 1.5 1.0 0.5 1.2 0.8 0.4 0.9 0.6 0.3 % Input % Input 0.6 0.4 0.2 0.2 0.1 0.3 Ac- H2A.Z / 0 0 0 - + - + TSA - + - + TSA - + TSA HA H2A.Z HA Ac-H2A.Z

x10-3 x10-4 enh2 enh2 enh2 3.0 4.0 4.8 2.5 3.5 4.0 3.0 2.0 2.5 3.2 1.5 2.0 2.4

% Input 1.5 % Input 1.0 1.6 1.0 0.5 0.5 0.8 Ac- H2A.Z / 0 0 0 - + - + TSA - + - + TSA - + TSA HA H2A.Z HA Ac-H2A.Z Figure 2. TSA modulates gene expression and increases H2A.Z acetylation at the CCND1 promoter and enhancer. MDA-MB231 cells were cultivated in DMEM medium and treated ( þ ) or not ( À ) with 50 ng/ml TSA for 24 h. (a) Gene expression was quantiﬁed by qRT–-PCR and normalized 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 was analyzed by ChIP and shown as percent input.

-3 siH2A.Z-transfected cells ( þ ) compared with scrambled siRNA x10 TSS TSS ( ) treated MDA-MB231 cells. As the total amount of H2A.Z was 4.0 12 À reduced three-fold at these sites upon H2A.Z depletion, the 3.2 10 8 relative ratio of Ac-H2A.Z to total H2A.Z increased significantly 2.4 6 (Figures 3a and b, right panels). % Input 1.6 4 In conclusion, these results reveal that the presence of hypo- 0.8 2 acetylated H2A.Z at the proximal promoter around the TSS and at Ac-H2A.Z /H2A.Z 0 0 0 the 3 enhancer negatively controls CCND1 expression in triple - + - + Si H2A.Z - + Si H2A.Z negative mammary tumor cells. Reducing the pool of free H2A.Z HA Ac-H2A.Z led to an increase in the amount of Ac-H2A.Z present at both regulatory sequences at the expense of unmodified H2A.Z. We -3 x10 enh2 enh2 therefore attempted to identify the factors involved in acetylation 1.0 10 of H2A.Z and chromatin modifications associated with CCND1 0.8 8 transcriptional activation. 0.6 6 0.4 4 % Input Recruitment of Tip60 and H2A.Z acetylation regulates enhancer 0.2 2

Ac-H2A.Z /H2A.Z chromatin structure 0 0 - + - + Si H2A.Z - + SiH2A.Z Histone acetylation has long been associated with active 45,46 HA Ac-H2A.Z transcription and thus the increase in acetylation of H2A.Z bound to the CCND1 locus may be part of general acetylation of Figure 3. Acetylation of H2A.Z correlates with activation of gene expression. (a, b) Recruitment of Ac-H2A.Z to CCND1 promoter chromatin in MDA-MB231 cells. Indeed, acetylation of H3K9 and (a) and enh2 enhancer (b) sites was analyzed by ChIP (left panels) H4 increased upon depletion of H2A.Z by siRNA at the proximal after depletion of H2A.Z by a SMARTpool siRNA. Data are presented promoter (Figures 4a and b, upper panel). Global acetylation was as percent input. The panels on the right show the ratios of ac- accompanied by the recruitment of elongating pol II phosphory- H2A.Z/H2A.Z calculated from these experiments. lated at serine 5 (P-Ser 5; Figure 4d, upper panel). In contrast,

& 2013 Macmillan Publishers Limited Oncogene (2013) 4243 – 4251 H2A.Z-dependent intragenic looping M Dalvai et al 4246

-3 -1 -2 -2 x10 TSSx10 TSSx10 TSSx10 TSS 0.5 2.1 1.2 0.90 1.8 0.4 1.0 0.75 1.5 1.8 0.60 0.3 1.2 0.6 0.45 0.2 0.9 % Input % Input 0.6 % Input 0.4 % Input 0.30 0.1 0.3 0.2 0.15 0 0 0 0 - + - + - + - + - + - + - + - + Si H2A.Z HA Ac-H3K9 HA Ac-H4 HA H3 HA p -Ser 5

x10-3 x10-1 x10-2 -2 enh2 enh2 enh2x10 enh2 0.5 2.5 0.8 0.30 2.0 0.25 0.4 0.6 0.3 1.5 0.20 0.4 0.15 0.2 1.0 % Input % Input % Input % Input 0.10 0.2 0.1 0.5 0.05 0 0 0 0 - + - + - + - + - + - + - + - + Si H2A.Z HA Ac-H3K9 HA Ac-H4 HA H3 HA p -Ser 5 Figure 4. H2A.Z depletion is accompanied by global histone acetylation at the proximal promoter but not at the enhancer site. (a–d) After 72 h of H2A.Z depletion by a smartpool siRNA, the presence of Ac-H3K9, pan Ac-H4, histone H3 and RNA pol II phosphorylated on Ser5 was analyzed by ChIP at CCND1 promoter (upper panel) and enh2 enhancer (lower panel) sites and shown as percent input.

acetylation of H3K9 was low at the downstream enhancer -2 -3 x10 TSS x10 enh2 enh2 and did not vary with transcription activation (Figure 4a, 0.25 0.25 lower panel). Acetylation of H4 also remained unchanged 0.20 0.20 (Figure 4b, lower panel). In addition, H3 binding to the CCND1 0.15 0.15 0

0.10 % Input 0.10

3 enhancer but not to the TSS varied following H2A.Z eviction % Input in these ERa À cells (Figure 4c). In contrast to previous 0.05 0.05 observations in prostate cancer cells,38 nucleosome density did 0 0 -+ -+ Si H2A.Z - +-+ Si H2A.Z not vary at the promoter of the activated CCND1 gene. However, HA Tip60 HA Tip60 decreased nucleosome density at enh2 points to chromatin

decondensation usually associated with DNAse I hypersensitive -1 x10 x10-1 sites, which reinforces the notion that the enh2 site is a key CCND1 CCND1 16 0.8 1.2 element for CCND1 gene activation. P-ser 5 binding did not 0.7 1.0 0 47 0.6 vary at the 3 end of the gene as expected (Figure 4d, lower 0.5 0.8 panel). Hence, acetylation of chromatin is linked to transcription 0.4 0.6 0.3 0.4

0 expression initiation and elongation near the 5 end of CCND1 while 0.2 expression

Relative mRNA Relative 0.2

0.1 mRNA Relative acetylation of H2A.Z characterized the active state at both the 0 0 TSS and the 30 enh2. Mock Tip60 - + - + Si Tip60 Several studies in yeast have shown that the NuA4 (an ortholog Over-expression - - + + Si H2A.Z

of the human Tip60/p400 complex) and the SAGA histone -2 -3 x10 TSS x10 enh2 acetyltransferase complexes acetylate multiple lysine residues of 0.6 48–50 0.30 H2A.Z. Tip60 could then be one of the HATs capable of 0.5 0.25 acetylating H2A.Z in mammalian cells. Indeed, we found that 0.4 0.20 Tip60 was recruited to both the TSS and enh2 of the activated 0.3 0.15

% Input 0.2 % Input 0.10 CCND1 gene in MDA-MB231 cells transfected with siH2A.Z 0.1 0.05 (Figure 5a). Upon overexpression of Tip60 in MDA-MB231 cells 0 0 (Supplementary Figure S2A) CCND1 expression increased nearly - + -++ - + - Si Tip60 - + -++ - + - Si Tip60 HA H2A.ZAc- pol II HA H2A.ZAc- pol II two-fold (Figure 5b), whereas transcription levels of ESR1 and H2A.Z H2A.Z H2AFZ remained unchanged (Supplementary Figure S2B). The selective knockdown of Tip60 did not affect the basal, uninduced Figure 5. Tip60 promotes CCND1 promoter and enhancer chromatin acetylation during gene activation. (a) The presence of Tip60 on the expression level of CCND1 (Figure 5c, Supplementary Figure S2C), 0 but prevented gene activation upon H2A.Z depletion (Figure 5c, CCND1 promoter (upper panel) and 3 enhancer (lower panel) was analyzed by ChIP following depletion of H2A.Z by a SMARTpool siRNA. Supplementary Figure S2D). In the absence of Tip60, the amount (b) MDA-MB231 cells were transfected with an empty vector of H2A.Z and pol II bound to the TSS and enh2 of CCND1 did not (pcDNA3.1) or with a vector expressing Tip60. CCND1, gene expression vary (Figure 5d) but the presence of acetylated H2A.Z was was analyzed after 48h by q-PCR. (c) MDA-MB231 cells were transfected markedly reduced. We conclude from these ChIP experiments that with scrambled control siRNA ( À ), with H2A.Z SMARTpool siRNA ( þ ), Tip60 has a central role in CCND1 activation independently of E2 Tip60 siRNA ( þ )orbothsiRNAs.CCND1 gene expression was analyzed and is the likely candidate for acetylating H2A.Z at both TSS and after 72 h by q-PCR. (d) MDA-MB231 cells were transfected with a enh2 sites. scrambled control siRNA ( À )orwithTip60siRNA(þ ).Thepresenceof H2A.Z, Ac-H2A.Z and Pol II on the CCND1 promoter (upper panel) and 30 enhancer (lower panel) was analyzed by ChIP. Acetylation of H2A.Z regulates promoter/enhancer crosstalk via loop formation detects physical proximity between distal DNA sites by ligation of To investigate whether the CCND1 TSS interacts with enh2, we cross-linked restricted DNA fragments (Figure 6a). Ligation used a chromatin conformation capture (3C) assay,51,52 which products between enh2 and promoter sequences, and between

Oncogene (2013) 4243 – 4251 & 2013 Macmillan Publishers Limited H2A.Z-dependent intragenic looping M Dalvai et al 4247 Chromosome 11 69165022 pb 69178423 pb CCND1 RefSeq:NM_053056.2 TSS 3’ UTR

DNAse I hypersensitive site promoter enhancer2

Csp6I restriction site

3C experiment Control Amplification

Loop Enh2/Prom

Ligation Enh2/Prom Ligation Enh2/Control 2.4 MDA-MB231 2.0

1.6

1.2

0.8 amplification 0.4 Relative ligation events Relative

10-2 +-+- +-+- +-+- +-+- Csp6I Si CtrSi Tip60 Si H2A.Z Si H2A.Z + Si Tip60 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 were performed using primers designed for each of the four possible ligation events (only one is shown). (b) MDA-MB231 cells were transfected with 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 the transcriptional regulation of CCND1.

enh2 and a control fragment inside the CCND1 ORF were control fragment. We did not detect amplification of any of these amplified and normalized to the amplified enh2 PCR product fragments in undigested samples. Hence, an intragenic loop used in ChIP experiments (see Materials and methods; Figure 6b). mediated by specific promoter–enhancer interactions is present We measured significant interaction frequencies between enh2 when CCND1 expression is low (Figure 5c). Upon transcription and promoter sequences in MDA-MB231 transfected with activation, gene lopping is markedly reduced. In conclusion, we scramble siRNA (control) while in cells transfected with siH2A.Z, propose a model (Figure 7) in which looping of the CCND1 50 and this interaction was reduced more than five-fold (Figure 6b). In 30 ends is mediated by histone H2A.Z and exerts repressive effects. contrast, depletion of Tip60 alone or in combination with siH2A.Z Acetylation of H2A.Z by Tip60 reduces interactions between TSS did not affect contact frequencies between these sites (Figure 6b). and enh2 sites, creating a chromatin environment favorable for No amplification was observed between enh2 and the internal transcriptional activation.

& 2013 Macmillan Publishers Limited Oncogene (2013) 4243 – 4251 H2A.Z-dependent intragenic looping M Dalvai et al 4248 DISCUSSION domains of distinct properties around highly regulated genes,70–72 We demonstrate that binding and acetylation of the histone via interchromosomal but also intrachromosomal loops, but their variant H2A.Z regulates CCND1 expression via specific enhancer/ functional significance remains to be established. However, dy- promoter interaction in ERa À breast cancer cell lines. In particular, namic chromosome folding within distinct domains accompanies 73,74 H2A.Z was present at the CCND1 locus when the gene displayed transcription activation (Kocanova et al., unpublished data). It little transcriptional activity. Recently, Valdes-Mora et al.38 reported is thus plausible that local decondensation of chromatin is, at least in a genome-wide study of normal and prostate cancer cells, that in part, the result of releasing looping. acetylation of H2AZ at the TSS correlated with activation of Finally, H2A.Z associates with a subset of ER-target genes that are oncogenes and that reduced H2A.Z acetylation marked silenced differentially regulated in different breast cancer cell lines. Hormone 38 treatment can lead to recruitment of H2A.Z to the TSS of the TFF1 tumor suppressor genes. Here we extend these findings to 30 breast cancer cells. In addition, we propose a mechanism by which gene or its eviction at the PGR or CCND1 promoters (unpublished H2A.Z acetylation modulates transcription associated with observations) in MCF-7 cells. These data suggest that there is no deregulated gene activity in cancer cells. general mechanism linking H2A.Z binding to transcription regulation. Indeed, H2A.Z was also associated with silenced or repressed genes in Reducing the pool of H2A.Z resulted in release of H2A.Z from À promoter and enhancer sequences and increased CCND1 expres- ER breast cancer cells. Similarly to prostate cancer cells, it is the sion. This is consistent with the previously proposed model that acetylation of H2A.Z that has a key role in transcription regulation of nucleosomes containing H2A.Z are more labile. Eviction of H2A.Z genes in breast cancer cells. The ratio of acetylated relative to bulk 0 thus promotes chromatin accessibility.53 It is tempting to H2A.Z bound to TSS and 3 enhancer sequences controls gene function. Modification of this ratio allowed reversing uncontrolled speculate that modulation of the 3D intragenic organization À may be key for creating a window of opportunity for secondary gene expression in the more aggressive ER breast cancer cells for factors to regulate transcription. Hence, not the action of specific which targeted treatments are critically lacking. Interfering with the ratio of bound acetylated H2A.Z to promoter and enhancer transcription factors and downstream events resulting from their À association once favorable conditions are encountered,54 but sequences in ER cells mimics the effect of the estradiol bound ER. structure would guide activity. Concomitantly to chromatin This mechanism may increase our understanding of tumorigenesis reorganization, acetylation of the remaining H2A.Z, but not of and offer new perspectives for re-activating control gene pathways. other histones, was a characteristic feature of chromatin in the 30 enhancer region, suggesting that this modification specifically regulates 3D organization of genetic loci. MATERIALS AND METHODS Enhancers are thought to allow transcription activators to bind Cell lines, transfection and western blotting. inactive genomic loci in order to facilitate the onset of MDA-MB231 and MDA-MB436 cells were purchased from ATCC (Rockville, transcription. This usually involves chromatin modifying com- MD, USA, used up to 15 passages). MDA-MB231 cells were maintained in plexes55 such as CBP/p300 in mammalian cells.56,57 Here we show Dulbecco’s modified Eagle’s medium (DMEM) and MDA-MB 436 in DMEM/ F12 medium, both with Glutamax containing 50 mg/ml gentamicin, 1 mM that Tip60, a histone acetyltransferase that has been shown to 58,59 60 sodium pyruvate and 10% heat-inactivated fetal calf serum (Invitrogen, acetylate histones H3, H4, H2A, and H2AX in vivo, is recruited Carlsbad, CA, USA). MDA-MB231 cells were treated with 50 ng/ml TSA to a 30 enhancer of the CCND1 gene upon activation. Tip60 is an (Sigma-Aldrich, Inc., St Louis, MO, USA) for the indicated times. In all, essential cofactor for numerous transcription factors and 4 Â 106 MDA-MB231 cells where mock-transfected (pcDNA3.1) or trans- important for cell proliferation. Binding to promoter and fected with 2 mg of pcDNA3.1/Tip60 (gifts from Dr Didier Trouche) with enhancer sequences of the stimulated CCND1 gene coincided Amaxa Cell line Nucleofactor Kit V program X-013 according to the with an increased ratio of acetylated H2A.Z. Thus, for the first time, manufacturer’s protocol. Breast cancer cells were mock-transfected or we provide evidence for a role of Tip60 in transcriptional transfected with H2A.Z siRNA ON-TARGET plus SMARTpool or scrambled À (scr) siRNA (Dharmacon Thermo Scientific, Waltham, MA, USA) with activation of the CCND1 oncogene in ERa breast cancer cells Interferine (Ozyme, Bedford, MA, USA) according to the manufac- potentially by acetylation of the histone variant H2A.Z. turer’s protocol. H2A.Z depletion was also performed with another siRNA Replacement of H2A by H2A.Z alters nucleosome conformation (H2AZ-1), kindly provided by Dr Didier Trouche (Supplementary Figure creating docking sites for distinct cofactors and chromatin S1D).75 Tip60 siRNA76 was purchased from Eurogentec, and transfected remodelers.61,62 Tip60 could therefore be recruited to H2A.Z with Interferine (Ozyme) according to the manufacturer’s protocol. A total 5 containing loci as has been demonstrated for the NuA4 complex of 3 Â 10 MDA-MB231 cells were seeded in six-well plates. Seventy-two in yeast.48 We hypothesize that Tip60 is capable of regulating hours following siRNA transfection, total cell extracts were isolated and protein level of H2A.Z and Tip60 was analyzed by immunoblotting on gel CCND1 gene expression by acetylating H2A.Z at the downstream SDS–PAGE 15% with anti-H2A.Z (Abcam, Cambridge, UK, ab4174), anti- enh2, which then disturbs repressive promoter enh2 crosstalk. Tip6076 and with anti-GAPDH (Millipore, Bedford, MA, USA, mab374) or Here, ERa was not required for Tip60 recruitment to these CCND1 anti-Tubulin (Sigma, DM1A) antibodies as loading control. regulatory elements unlike the mechanism recently described in ER þ cells.62 Our data thus illustrate that the expression levels of RNA analysis. tissue or cell-specific genes are largely modulated by the ability of transacting factors to associate with regulatory elements. Total RNA was extracted using an RNeasy mini-kit (Qiagen, Valencia, CA, USA) and eluted with 35 ml of RNAase-free water. First strand cDNA was Long-range interactions between distant enhancers and pro- generated using 2 mg of total RNA in a reaction containing random moter regions via chromatin looping has been shown to allow 63–66 oligonucleotides as primers with the ThermoScript RT–PCR system activation of single genes or gene clusters. This looping (Invitrogen). Real-time PCR was performed on a Mastercycler ep realplex4 generally occurs in the 50 part of genes and over tens or hundreds (Eppendorf) using the platinum SYBR Green q-PCR SuperMix (Invitrogen) of kb. The role of such loops is thought to isolate chromatin from according to the manufacturer’s instructions. Amplification conditions: surrounding repressive environment via insulator elements.67 1 min at 50 1C, 3 min at 95 1C followed by 40 cycles (20 s at 95 1C, 20 s at Long-range intragenic interactions implicate transcription 60 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 and activators, histone variants, including H2A.Z and macro H2A.1, 0 0 0 0 and chromatin remodelers bound to promoter and enhancer 5 -TAGAATGGGCAGGAGAAA GG-3 , H2AFZ:5-CCTTTTCTCTGCCTTGCTTG-3 and 50-CGGTGAGGTACTCCAGGATG-30, CCND1:50-GCGTCCATGCGGAAGA regions, but direct experimental evidence for intragenic looping 0 0 0 0 19,30,68,69 TC-3 and 5 -ATGGCCAGCGGGAAGAC-3 , RPLP0:5-TGGCAGCATCTACAACC is still lacking. As shown by results of large-scale chromo- CTGAA-30 and 50-CACTGGCAACATTGCGGACA-30, PGR:50-CTTAATCAACTAG some conformation (4C) and ChiA-PET experiments, such GCGAGAG-30 and 50-AAGCTCATCCAAGAATACTG-30, TFF1:50-CAGATCCCTG interactions are well correlated with the existence of chromatin CAGAAGTGTCT-30 and 50-CCCCTGGTGCTTCTATCCTAAT-30.

Oncogene (2013) 4243 – 4251 & 2013 Macmillan Publishers Limited H2A.Z-dependent intragenic looping M Dalvai et al 4249 ChIP assays ACKNOWLEDGEMENTS ChIP analyses were performed as described previously77 from MDA-MB231 We would like to thank Dr Didier Trouche for the Tip60 expressing vector and cells. Samples were sonicated to generate DNA fragments o500 bp. insightful discussions, J Eeckhoute, Lille for sharing unpublished work, and David Chromatin fragments were immunoprecipitated using antibodies against Laperriere, IRIC, Montreal, for expert advice on genome database use. This work was H2A.Z (ab4174, Abcam), Acetyl H2A.Z (ab18262, Abcam), RNA pol II (N20X, supported by the Ligue Nationale Contre le Cancer (fellowship to LB), the Institut Santa Cruz biotechnology, Santa Cruz, CA, USA), Acetyl H3K9 (06-942, National du Cancer (INCa grant no. 34696) and the Fondation pour la Recherche Upstate, Bedford, MA, USA), RNA pol II serine 5 phosphorylated (MPY-127R, Me´dicale (FRM, fellowship to MD). 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) as control. The precipitated DNA was amplified by real-time PCR, with primer REFERENCES sets designed to amplify the promoter region of the PGR, ESR1, TFF1 and CCND1 genes, as well the enh2 enhancer and À 2000 region of CCND1. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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