PRC2-Independent Chromatin Compaction and Transcriptional Repression in Cancer

ORIGINAL ARTICLE PRC2-independent chromatin compaction and transcriptional repression in cancer

C Vallot1,2,5,AHe´ rault1,2,5, S Boyle3, WA Bickmore3,4,6 and F Radvanyi1,2,6

The silencing of large chromosomal regions by epigenetic mechanisms has been reported to occur frequently in cancer. Epigenetic marks, such as histone methylation and acetylation, are altered at these loci. However, the mechanisms of formation of such aberrant gene clusters remain largely unknown. Here, we show that, in cancer cells, the epigenetic remodeling of chromatin into hypoacetylated domains covered with histone H3K27 trimethylation is paralleled by changes in higher-order chromatin structures. Using fluorescence in situ hybridization, we demonstrate that regional epigenetic silencing corresponds to the establishment of compact chromatin domains. We show that gene repression is tightly correlated to the state of chromatin compaction and not to the levels of H3K27me3—its removal through the knockdown of EZH2 does not induce significant gene expression nor chromatin decompaction. Moreover, transcription can occur with intact high-H3K27me3 levels; treatment with histone deacetylase inhibitors can relieve chromatin compaction and gene repression, without altering H3K27me3 levels. Our findings imply that compaction and subsequent repression of large chromatin domains are not direct consequences of PRC2 deregulation in cancer cells. By challenging the role of EZH2 in aberrant gene silencing in cancer, these findings have therapeutical implications, notably for the choice of epigenetic drugs for tumors with multiple regional epigenetic alterations.

Oncogene (2015) 34, 741–751; doi:10.1038/onc.2013.604; published online 27 January 2014 Keywords: cancer; chromatin compaction; gene cluster; histone deacetylase inhibitors; polycomb

INTRODUCTION aberrant DNA methylation, can affect several neighboring genes Transcription of individual genes can be altered in cancer by in cancer and correspond to the formation of clusters of genetic but also epigenetic lesions. Focal promoter DNA corepressed genes. Recently, we and others showed that methylation can, for example, modify gene expression. However, regional epigenetic silencing is frequent in bladder and prostate 14,15 studying the regulation of genes in isolation without taking into cancer. However, little is known about the mechanisms account their genomic context limits the understanding of underlying this novel epigenetic alteration. regulatory networks in cancer. Many clusters of co-regulated We previously reported in bladder cancer that regional genes have been reported in mammalian genomes,1 such as the epigenetic silencing occurs in a particularly aggressive group of HOX clusters or the classical major histocompatibility complex. tumors, the tumors of the carcinoma in situ pathway. This The regulation of these genomic regions is complex and can silencing affects simultaneously several regions defining a new result from the interaction of multiple factors including epi- epigenetic phenotype that we called multiple regional epigenetic 15 genetic modifications,2,3 chromatin conformation and nuclear silencing (MRES). Whereas superficial bladder tumors were localization,4 non coding RNAs5 or polycomb complexes.6 PRC2 already molecularly well defined, in particular by FGFR3 16 and PRC1 polycomb complexes take part in the delineation and mutation, MRES was one of the first molecular events regulation of gene clusters, such as the HOX or Kcnoqt1 clusters,7 characterizing tumors of the carcinoma in situ pathway. Such a notably through the deposition of H3K27me3 and chromatin phenotype suggests that there might be a common mechanism compaction mechanisms.8,9 for the formation of the repressed gene clusters. We had Several studies have addressed the existence of co-regulated previously shown that, among others chromatin marks, gene clusters specific to cancer cells. Regions of expression bias, H3K27me3, a hallmark of the PRC2 enzyme EZH2, was where genes are coordinately deregulated in tumors compared characteristic of these alterations.15 Although it has been shown with normal tissue, have been reported in various tumor types that EZH2 overexpression in cancer could be key in altering using large-scale expression data sets. Many of these regions were expression profiles through aberrant silencing mechanisms,17,18 attributable to known DNA copy-number alterations,10 however, a recent data suggest that EZH2 oncogenic properties might not be significant portion of them are independent of such genetic necessarily linked to gene silencing and polycomb activities.19 alterations.11 Moreover, it has been reported that epigenetic Here, we study the involvement of EZH2, and its foot- alterations, such as histone modifications with12,13 or without11 print H3K27me3, in the deregulation of the cancer epigenome

1CNRS, UMR 144 - Cell Biology Department, Institut Curie, Paris, France; 2Institut Curie, Centre de Recherche, Paris, France; 3Chromosome and Gene Expression Section, MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine at the University of Edinburgh, Scotland, UK and 4Breakthrough Breast Cancer Research Unit, University of Edinburgh, Scotland, UK. Correspondence, Current address: Dr C Vallot, UMR7216, Universite´ Paris Diderot, 35 rue He´le`ne Brion, Paris 75205, France or Dr F Radvanyi, CNRS, UMR 144, Institut Curie, Paris 75248, France. E-mail: [email protected] or [email protected] 5These authors contributed equally to this work. 6The last two authors contributed equally to this work. Received 9 July 2013; revised 10 December 2013; accepted 20 December 2013; published online 27 January 2014 PRC2-independent silencing in cancer C Vallot et al 742 through the remodeling of large chromatin domains in bladder CL1207 and in the MRES À cancer cell line RT112 as well as in NHU cancer cells. We specifically assess the link between H3K27me3, cells. As a control, we chose a fosmid probe pair at a 1q21 locus chromatin compaction and gene repression. We show that gene that was rarely affected by DNA alterations or expression changes repression is tightly correlated to the state of chromatin in our bladder cancer data set (data not shown). We measured the compaction but not with H3K27me3 levels in cancer cells. distance (d) between the hybridization signals from these probe pairs in nuclei of the different cell lines. At probe separation of o2 Mb, there is a linear relationship between the mean-square RESULTS interphase distances between two loci (d2) and their separation in Regional epigenetic silencing events correspond to higher-order kilobases.24 Such analysis has been used to show that different chromatin alterations parts of the human genome display various levels of chromatin 25 We had previously identified seven gene clusters recurrently compaction, and that the same genomic region can change its 4,26 epigenetically repressed in bladder cancer; these chromosomal compaction state in differentiation, development or when the 8 regions are concomitantly silenced in a subgroup of aggressive activity of chromatin modifiers is ablated. In the latter cases, this tumors, defining a multiple regional epigenetic phenotype included analyses of the HoxD locus, the murine homolog of one (Figure 1a).15 Here, we studied more extensively the chromatin of the repressed gene clusters (2q31) studied here in human landscape and conformation at these clusters to get insight into cancer cells. the regulation of such epigenetic alterations. We first used At the 3p22 cluster (Figure 1c, left panel), the interphase chromatin immunoprecipitation (ChIP) coupled with quantitative separation of the probe signals (127 kb apart) in the nuclei of 2 2 PCR (qPCR) to analyze the levels of H3K27me3 and global H3 CL1207 cells (mean d ¼ 0.17 mm ) is significantly smaller than in 2 2 2 2 À 4 acetylation (H3ac), not only on promoters but also within and RT112 (d ¼ 0.33 mm , P ¼ 0.005) or NHU (d ¼ 0.53 mm , Po10 ) between the genes across the entire loci. We analyzed two of the cells. This indicates that this genomic region is in a more compact seven clusters: 2q31 (HOXD8, HOXD4, HOXD3 and HOXD1) and chromatin state in CL1207 than in RT112 and NHU cells, whereas 3p22 (VILL, PLCD1, DLEC1 and ACAA1) in normal human urothelial the chromatin compaction does not differ significantly between (NHU) cells in culture20 as well as in two bladder cancer cell lines, RT112 and NHU cells (P ¼ 0.2). A similarly more compact CL1207 and RT112. CL1207 is derived from a high-grade tumor chromatin state in CL1207 cells was seen for probes spanning 2 2 with the MRES phenotype, RT112 is from a well-differentiated 104 kb at the HOXD cluster at 2q31 (d ¼ 0.20 mm ) in comparison 2 2 tumor without the MRES phenotype.15 In CL1207 cells, but not in with RT112 cancer cells (d ¼ 0.27 mm , P ¼ 0.01) or normal NHU 2 2 RT112, genes belonging to both clusters of interest are cells (d ¼ 0.27 mm , P ¼ 0.005) (Figure 1c, central panel). This epigenetically repressed relative to normal bladder cells.15 High compaction is not a general feature of the chromatin structure levels of the polycomb-mediated histone modification H3K27me3 throughout the genome in CL1207 cells, as shown by the similar are not restricted to gene promoters in CL1207 cells (Figure 1b): all level of chromatin compaction between all three cell lines at the positions assayed in the clusters display high H3K27me3 levels in control 1q21 locus (Figure 1c, right panel). comparison with the ubiquitously expressed GAPDH in the same We also confirmed the link between clustered gene repression cells, and compared with NHU cells and RT112 cells. Conversely, and chromatin compaction in two other bladder cancer cell lines H3 was hypoacetylated across the two gene clusters in CL1207 for 2q31 (Supplementary Figure 1). In the MRES þ TCCSUP cancer cells relative to levels in NHU and RT112 cells (Figure 1b) and cell line, as in CL1207, genes of the 2q31 locus are repressed, 15 compared with GAPDH. Therefore, the repressed clusters in MRES whereas they are not in the MRES- MGHU3 cancer cell line. 2 2 cancer cells correspond to the formation of relatively homo- Compared with NHU cells (d ¼ 0.27 mm ) the chromatin at 2q31 is 2 2 geneous chromatin domains characterized by high levels of more condensed in TCCSUP (d ¼ 0.14 mm , P ¼ 0.01), but not in 2 2 H3K27me3 and low levels of H3ac. MGHU3 (d ¼ 0.32 mm , P ¼ 0.93). Histone acetylation has been shown to directly affect secondary chromatin structures: it can neutralize charges in the histone N-terminal tails and thereby nucleosome-DNA or nucleosome– EZH2 is overexpressed in tumors with multiple regional epigenetic nucleosome interactions.21,22 Moreover, the polycomb repressive alterations complexes, especially PRC1, have also been shown to be involved As we found that the regions of epigenetic alterations are covered in chromatin compaction in vitro23 and in vivo.8 Given the with abnormal H3K27me3 in cancer cells (Figure 1), we chose to observed histone modification profiles in CL1207 cells, we investigate the role of histone methyltransferases capable of examined whether there was a corresponding chromatin catalyzing the addition of a trimethyl group on H3K27: EZH127,28 compaction at the two MRES loci in bladder cancer cells. We and EZH2.29 Although a potential role of EZH1 in cancer has not used fluorescence in situ hybridation (FISH) with fosmid probe yet been addressed, the deregulated expression and activity of pairs spanning the 2q31 and 3p22 clusters to assess the state of EZH2 has been described. EZH2 is highly expressed in various chromatin compaction of these loci in the MRES þ cancer cell line tumor types,30 including breast,31 prostate,32 bladder33 or liver

Figure 1. Repressive chromatin domains form in cancer cells. (a) Location of the seven recurrent regional epigenetic alterations characteristic of the MRES phenotype. (b) Study of the 3p22 and 2q31 clusters: ChIP assays were performed for all promoters, for one position in the middle of every gene and between every pair of neighboring genes with an antibody against H3K27me3 (upper panel) and H3ac (lower panel). The graph shows the amount of immunoprecipitated target DNA expressed as a ratio of total input DNA measured by qPCR. Error bars indicate the variation between the means of two independent experiments. The map (not to scale) under each graph indicates the position of the primer pairs used for each point on the graph. The amount of immunoprecipitated DNA for the GAPDH promoter is shown as a reference. Data from the MRES bladder cancer cell line CL1207 is plotted in green; the non-MRES bladder cancer cell line RT112 in red; and normal human urothelial (NHU) cells in blue. (c) Two-dimensional FISH with a probe pair spanning the 3p22 cluster (left panel), the 2q31 cluster (middle panel) and a control locus in 1q21 (right panel). Above images, the map (to scale) shows the probe pair position (in bp) in the UCSC human genome browser (March 2006 Assembly, hg18). For the images, scale bar ¼ 5 mm. Below images, box plots showing the distribution of interprobe distances2 (d2) of each set of data, obtained from 50 nuclei. The horizontal line in the middle box represents the median, the two hinges of the middle box represent the interquartile range of the data and the empty circles indicate outliers. The whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range from the box. The statistical signiﬁcance of differences was examined by Mann–Whitney U-tests (Materials and methods).

Oncogene (2015) 741 – 751 & 2015 Macmillan Publishers Limited PRC2-independent silencing in cancer C Vallot et al 743 cancer.34 Moreover, gain of function mutations of EZH2 have been analysis.15 As the MRES phenotype is found in invasive bladder reported in lymphomas.35,36 To understand if deregelulation of tumors, we limited our analysis to invasive tumors, so that EZH2 expression might be involved in the appearance of the MRES differences in EZH2 expression levels between MRES þ and phenotype, we compared EZH2 expression levels using Affymetrix MRES À tumors would not only be due to the heterogeneity of arrays in a wide tumor set (n ¼ 150) between invasive tumors with tumor stages between each group. EZH2 is signiﬁcantly more (n ¼ 74) and without (n ¼ 29) MRES phenotype and normal highly expressed in invasive tumors (T2–T4) with MRES phenotype urothelium samples (n ¼ 4). Tumors with the MRES phenotype than in invasive tumors without the MRES phenotype (P ¼ 0.018, were identiﬁed using Affymetrix expression data and cluster t-test) and in normal samples (Figure 2a). This was validated by

* *

1 2 3 456 7 8 9 10 11 12 1314 15 1617 1819 20 21 22

Anti-H3K27me3 Anti-H3K27me3 0.5 0.6 0.4 0.5

0.3 0.4 0.3 0.2 0.2 Input +/- SD 0.1 Ratio Bound over Ratio 0.1 0 0

NHU VILL PLCD1DLEC1 ACAA1 HOXD8 HOXD4 HOXD3 HOXD1 GAPDH

RT112 GAPDH

Anti-H3ac CL1207 Anti -H3ac 0.3 0.4

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VILL PLCD1 DLEC1 ACAA1 HOXD8 HOXD4HOXD3 HOXD1 GAPDH GAPDH

3p22 cluster 2q31 cluster Control locus 1q21

G248P80945A9 - G248P82821D6 (127 kb) G248P89002G8 - G248P82958B3 (104 kb) G248P80671B3 - G248P87792E10 (221 kb)

38000000 38100000 38200000 176700000 176800000 149900000 150000000 150100000 CL1207 CL1207 EVX2 HOXD9 PLCD1 OAZ3 THEM4 HOXD13 HOXD8 CTDSPL ACAA1 SNX27 TDRKH THEM5 HOXD12 HOXD4 DLEC1 OXSR1 CELF3 MRPL9 C2CD4D HOXD11 HOXD3 VILL MYD88 TUFT1 RIIAD1 RORC HOXD10 HOXD1 MTX2

NHU RT112 CL1207 NHU RT112 CL1207 NHU RT112 CL1207 )

2 3 3 -4 p=0.37 m p<10 μ 1.5 p=0.005 ( 2

2 2 1.0 1 1 0.5

Distance 0 0.0 0 p=0.2 p=0.005 p=0.84 p=0.01 p=0.87 p=0.44

& 2015 Macmillan Publishers Limited Oncogene (2015) 741 – 751 PRC2-independent silencing in cancer C Vallot et al 744

2 r=0.89 p<10-14 2 0 2 0 Log2 -2 Log2 Expression Affymetrix signal

-4 levels relative to 1 p=0.018 RT-qPCR levels -2 level in NHU cells

N -4 -2 0 2 MRES – MRES + Log2 Affymetrix signal T2-T4 T2-T4 MRES – MRES + Figure 2. EZH2 is speciﬁcally overexpressed in MRES tumors. (a) EZH2 log2 mRNA expression level according to Affymetrix U133plus2 arrays in normal samples (n ¼ 4), invasive tumors without the MRES phenotype (n ¼ 29) and invasive tumors with the MRES phenotype (n ¼ 74). P-value obtained from a two-tailed t-test between tumors with and without the MRES phenotype is indicated. (b) Validation of Affymetrix expression data by RT–qPCR analysis using a TLDA format for 40 tumor samples. (c) EZH2 mRNA expression levels in cell lines with (n ¼ 4) and without (n ¼ 3) the MRES phenotype relative to its expression level in NHU, indicated by the dashed line.

RT–qPCR on 40 tumors of our tumor set r ¼ 0.89 (P ¼ 10 À 14, lines, compared with the negative control GAPDH. When we Pearson’s correlation test, Figure 2b). In seven bladder cancer cell reduced H3K27me3 levels with the EZH2 inhibitor GSK343, lines (all derived from invasive bladder tumors), we also found RING1B binding was not abolished. These results suggest that higher EZH2 expression in cell lines with MRES phenotype than the aberrant levels of chromatin compaction observed in CL1207 those without, which displayed an expression level closer to the in 3p22 are not a consequence of high levels of H3K27me3 and one of NHU cells (Figure 2c). Altogether, EZH2 expression level is that PRC1 can bind independently of H3K27me3 at these loci in significantly higher in cells harboring multiple regional epigenetic cancer cells. alterations. EZH2-mediated histone methylation is secondary to gene silencing and hypoacetylation Erasing H3K27me3 does not lead to transcriptional activation or relieve chromatin compaction and RING1B binding To better decipher the links between H3K27me3 and gene repression in the MRES bladder cancer gene clusters, we started To get insight into the putative role of EZH2 in the establishment by erasing aberrant marks from the chromatin at 2q31 and 3p22 of large chromatin repressive blocks in cancer cells, we used two in CL1207 cells and setting the levels of histone modification back siRNA to efficiently knock down EZH2 at the mRNA and protein to ones more similar to those found in normal NHU cells, that is, levels (Figure 3a, siEZH2 A and B). The full knockdown of EZH2 promoters with higher histone acetylation and low H3K27me3. To using siEZH2 A led to a significant decrease of H3K27me3 levels at do so, we used a treatment with 5-aza-20-deoxycytidine combined gene promoters in 3p22 as well as at HOXD in 2q31 to a lesser with TSA. 5-aza-20-deoxycytidine is not only known to inhibit DNA extent, close to the levels found in NHU cells (Figure 3b).15 methylation but also to affect repressive histone modifications in a Incomplete knockdown of EZH2, using siEZH2 B, led to a moderate non-specific manner;37 this combination was the most effective of decrease of H3K27me3 in 3p22 and at the HOXD9 promoter in those we tested at removing H3K27me3 and restoring higher 2q31. However, efficient EZH2 knockdown by siEZH2 A did not acetylation levels (data not shown). activate (42 fold) gene expression in the MRES clusters We measured gene expression (Figure 5a), H3K27me3 (Figure 3c), compared with non-transfected cells (UN), or (Figure 5b) and H3K9ac (Figure 5c) for genes of the two MRES compared with normal bladder samples. In contrast, treatment clusters before and after treatment, as well as 24 and 48 h after of cancer cells with the histone deacetylase inhibitor (HDACi), removal of the drugs. As expected, we observed that transcription Trichostatin A (TSA), leads to gene re-expression compared with of the two clusters is induced just after treatment. However 24 h normal bladder samples: from 10 to 100% of normal gene after removal of the drugs, the mRNA levels decreased again for all expression is restored (Figure 3c). These results were confirmed genes except VILL. The dynamics of histone acetylation levels of using the EZH2 inhibitor GSK343, which led to a significant repressed genes (HOXD9 to HOXD1 in 2q31 and VILL, PLCD1 and decrease of H3K27me3, yet unchanged gene expression in CL1207 DLEC1 in 3p22) were similar: treatment transiently increased the cells, as well as in other MRES þ cancer cells TCCSUP level of H3K9ac at promoters, however, only 24 h after removal of (Supplementary Figure 2). These data suggest that decreasing the drugs acetylation levels decreased. In contrast, H3K27me3 H3K27me3 levels at MRES clusters is not sufficient for transcription levels for these genes were decreased immediately on treatment activation. and remained low up to 48 h after removal of the drugs, except for H3K27me3 has been shown to be a docking site for the PRC1 DLEC1 for which H3K27me3 levels were not significantly complex, which in turn compacts the targeted chromatin.8 We decreased by 5-aza-20-deoxycytidine þ TSA treatment. These therefore investigated whether removing H3K27me3 in CL1207 results show that, after treatment with epigenetic drugs, histone could decrease chromatin compaction and/or PRC1 binding at the modifications follow different kinetics: the decrease of histone studied MRES loci. We first analyzed the level of compaction in the H3K9 acetylation seems simultaneous to the loss of transcription, 3p22 locus, where the decrease of H3K27me3 was the most whereas H3K27me3 levels follow a more complex kinetics; in most marked after siRNA transfection using the most effective siRNA cases it decreases after treatment, but does not recover its initial targeting EZH2 (siEZH2 A). We observed no significant (P ¼ 0.53) levels even 48 h after removal of the drugs. Our findings suggest variation in chromatin compaction in CL1207 after EZH2 that H3K27me3 recapitulation is not necessary for gene knockdown compared with untreated cells (Figure 3d). As it is repression. PRC1, rather than PRC2, that is responsible for chromatin compaction,at least in stem cells,8 we analyzed the enrichment of RING1B, a member of the PRC1 complex, at MRES loci. In H3K27me3 removal is not necessary for transcriptional activation untreated cancer cells, we observe that RING1B binds to the and chromatin decondensation repressed genes of the HOXD cluster in CL1207 and TCCSUP cells To further investigate the link between histone acetylation, (Figures 4a and b). In the 3p22 cluster, we observe moderate H3K27me3, gene repression and chromatin compaction, we enrichment of RING1B only on the promoter of DLEC1, in both cell repeated the TSA treatment using different batches of TSA

Oncogene (2015) 741 – 751 & 2015 Macmillan Publishers Limited PRC2-independent silencing in cancer C Vallot et al 745 Anti-H3K27me3 0.3

0.2 UN 1.5 si ctrl 0.1 si EZH2 A

1 siEZH2 A siEZH2 B UN si ctrl siEZH2 B 0.0 100kDa VILL PLCD1 DLEC1 ACAA1 GAPDH 80kDa CL1207 0.5 EZH2 0.8 60kDa 50kDa α-tubulin

to untransfected 0.6 0

Fold-change compared 0.4 0.2 Ratio Bound over Input +/- SD 0.0 HOXD9 HOXD8 HOXD4 HOXD3 HOXD1 MTX2

2.5 1.6 UN 1.4 siEZH2 ctrl siEZH2 A 2 1.2 siEZH2 B 1 1.5 TSA 0.8 0.6 1 Fold-change Fold-change 0.4 0.5 compared to normal compared to normal 0.2 bladder samples +/- SD bladder samples +/- SD 0 0 VILL PLCD1 DLEC1 ACAA1 HOXD9 HOXD8 HOXD4 HOXD3 HOXD1 MTX2

3p22 cluster Control locus 1q21 siEZH2 A siEZH2 A

1.5 p=0.53 1.5 p=0.94 ) ) 2 2 m m

μ 1.0 μ 1.0 ( ( 2 2

0.5 0.5 Distance Distance

0.0 0.0 UN si ctrl siEZH2 A UN si ctrl siEZH2 A Figure 3. Removing H3K27me3 is not sufﬁcient for transcriptional activation and chromatin decompaction. (a) Validation of siRNA efﬁciency using qRT–PCR analysis to study EZH2 mRNA levels and western blot to analyze EZH2 protein levels in transfected (siEZH2 A and B), control (si ctrl) and untransfected cells (UN). (b) ChIP analysis for gene promoters in 3p22 (upper panel) and 2q31 (lower panel) with an antibody against H3K27me3 for CL1207 cells transfected or non-transfected cells (UN) with siRNAs targeting EZH2 or GAPDH. The bar chart shows the amount of immunoprecipitated target DNA expressed as a ratio of total input DNA, measured in duplicate by qPCR. The error bars indicate the variation between the means of two independent experiments. (c) We measured the expression of the genes in 3p22 and 2q31 in CL1207 transfected or non-transfected cells with siRNAs targeting GAPDH or EZH2, and in CL1207 cells treated with TSA. Results are expressed as a ratio of the average expression levels in three normal bladder samples. For each condition, RT–qPCR analyses were performed on two independent experiments, with each qPCR performed in duplicate. The error bars indicate the variation between the means of the two independent experiments. (d) Three-dimensional FISH with a probe pair spanning the 3p22 cluster, below images, box plots showing the distribution of interprobe distances2 (d2) for non-transfected CL1207 cells (UN) and cells transfected with a control siRNA or an siRNA targeting EZH2. For the images, scale bar ¼ 5 mm.

(n ¼ 3) and also used a different HDACi: sodium butyrate (NaBu). EZH2 for 72 h and NaBu treatment within the last 24 h of All TSA treatments led to equivalent gene re-expression, but only transfection (Supplementary Figure 3). These results suggest rare decrease of H3K27me3 levels. Treatment of CL1207 cells with that HDAC and EZH2 do not seem to act in synergy to repress NaBu led to the re-expression of genes in the 3p22 and 2q31 gene expression. clusters (Figure 6a). However, NaBu did not induce any decrease in HDACi such as TSA are known to be able to relieve chromatin H3K27me3 levels (Figure 6b). These experiments suggest that compaction,38,39 including at polycomb repressed Hox loci in removing H3K27me3 from gene promoters is not necessary for mouse embryonic stem (ES) cells, and without the removal of genes in MRES domains to be re-expressed. Moreover, genes in H3K27me3.40 We therefore used FISH to determine whether the 3p22 and 2q31 are expressed at similar levels after treatment chromatin compaction at repressed gene clusters in CL1207 with NaBu alone for 24 h or after siRNA transfection targeting cancer cells could also be relieved by TSA treatment. We observed

& 2015 Macmillan Publishers Limited Oncogene (2015) 741 – 751 PRC2-independent silencing in cancer C Vallot et al 746 Anti-RING1B Anti-RING1B 0.012 0.012 CL1207 NT 0.01 0.01 CL1207 GSK343

0.008 0.008

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Anti-RING1B Anti-RING1B 0.025 0.025 TCCSUP NT TCCSUP GSK343 0.02 0.02

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0.005 0.005 Ratio Boundover Input +/- SD Ratio Bound over Input +/- SD 0 0 HOXD4 HOXD3 HOXD1 MTX2 VILL PLCD1 DLEC1 ACAA1 GAPDH Figure 4. RING1B binding at 3p22 and 2q31 is independent of H3K27me3 levels. (a) ChIP analysis for genes promoters in 2q31 and 3p22 in CL1207 cells with and without GSK343 treatment. The bar chart shows the amount of immunoprecipitated target DNA with anti-RING1B antibody expressed as a ratio of total input DNA, measured in duplicate by qPCR. The error bars indicate the variation between the means of two independent experiments. (b) Same analysis for the TCCSUP cell line.

a specific decompaction of chromatin at the 3p22 and 2q31 domain-like distribution, they are generally restricted to promoter clusters in TSA-treated CL1207 cells (CL1207 TSA, d2 ¼ 0.60 mm2 proximal regions.42,43 The repressed clusters we have studied in and d2 ¼ 0.33 mm2, respectively) in comparison with non-treated cancer cells resemble normal repressed gene clusters in that cells (CL1207 NT, d2 ¼ 0.17 mm2 (Po10 À 9) and d2 ¼ 0.20 mm2 transcription is lost from broad domains with high levels of (P ¼ 0.02), respectively) (Figure 7a left panel and Figure 7b). There H3K27me3. They are also characterized by broadly distributed was no change in compaction at the control 1q21 locus (P ¼ 0.18) histone hypoacetylation, which has been shown to be a (Figure 7a right panel). These results show that the aberrant characteristic of repressed gene clusters in prostate cancer.14 chromatin condensation observed in the MRES þ CL1207 cells at One unresolved issue is to understand why the same regions the 3p22 and 2q31 regions are independent of H3K27me3 and are recurrently repressed and targeted by aberrant epigenetic may instead depend on the hypoacetylated state of the under- modifications in tumors. We have shown in bladder cancer that lying chromatin. aggressive tumors display multiple regional epigenetic alterations at the same loci, including parts of the HOXD and HOXA clusters.15 Here, we have demonstrated that, when part of HOXD is repressed DISCUSSION in cancer cells, there are high levels of H3K27me3 and the In this paper, we have shown that repression of gene clusters in chromatin is condensed. This is the reverse of what has been cancer cells, whether they are groups of genes known to be co- observed in mouse embryogenesis, where H3K27me3 is regulated in normal cells (2q31) or not (3p22), is accompanied by progressively lost and chromatin decompacts as Hox loci are marked changes in histone modification profiles over the entire activated.8,26,44,45 This suggests that the MRES phenotype may cluster, and a consequent remodeling of the higher-order result from the reimposition of a gene-silencing pathway used in chromatin structure of these domains to a compact state. In normal development. It has recently been shown in breast cancer cancer cells harboring multiple repressed gene clusters (the MRES that the DNA methylation pattern might reflect developmental phenotype), not only is the epigenome remodeled compared with processes and the cell lineage of tumors more than the tumor normal and other cancer cells, but the higher-order chromatin progression itself.46 Bladder stem cells have not been isolated, and structure is also modified and aberrantly condensed at specific so it is not possible at present to access the epigenome of this cell loci. By modulating the levels, or activity, of histone-modifying type and further assess this hypothesis. enzymes, we have shown that removal of H3K27 methylation is The loose link we observed between H3K27me3 and gene neither necessary nor sufficient to activate transcription and to repression challenges previous findings about the role of EZH2 in relieve chromatin compaction at these loci. In contrast, gene cancer cells. Several studies have demonstrated that EZH2 is transcription is highly correlated to the state of chromatin directly involved in the transcriptional repression in cancer, and compaction and acetylation levels. that knocking down EZH2 restores gene expression.47,48 However, Repressive histone marks have been shown to cluster in large here we show that knocking down EZH2 and removing H3K27me3 domains in normal mammalian cells, and not only around the is neither necessary nor sufficient for gene expression and well-known HOX clusters. Transcriptionally silent domains display- chromatin relaxation at repressed clusters in bladder cancer ing high levels of H3K27me3 have been identified in MEFs2 and cells. Instead we found that gene transcription was tightly linked human ES cells.3 Differentiated cells can also display to the state of chromatin compaction and acetylation levels, both homogeneous chromatin domains, termed LOCKs for large factors being independent of H3K27me3 levels in this context. organized chromatin K9 modifications, in comparison with the Moreover, we demonstrate that RING1B, member of PRC1, binds originating stem cells.41 In contrast, active chromatin marks, to these repressed clusters independently of H3K27me3 levels. such as histone acetyl or H3K4me3, do not usually display such Altogether, such observations are in accordance with previous

Oncogene (2015) 741 – 751 & 2015 Macmillan Publishers Limited PRC2-independent silencing in cancer C Vallot et al 747 plate cells AZT AZT+24h AZT+48h

5aza 5aza +TSA

a 1000 40 900 NT 35 800 AZT 30 700 AZT+24h 600 AZT+48h 25 500 20 400 15 300 10 treated cells +/- SD 200 treated cells +/- SD Fold-change vs to non 100 Fold-change vs to non 5 0 0 VILL PLCD1 DLEC1 ACAA1 MTX2 HOXD9 HOXD8 HOXD4 HOXD3 HOXD1

b Anti-H3K27me3 Anti-H3K27me3 0.9 0.25 0.8 0.7 0.20 0.6 0.15 0.5 0.4 0.10 0.3 0.2 0.05 0.1 Ratio Bound over Input +/- SD Ratio Bound over Input +/- SD 0.0 0.00 VILL PLCD1 DLEC1 ACAA1 GAPDH MTX2 HOXD9 HOXD8 HOXD4 HOXD3 HOXD1

c Anti-H3K9ac Anti-H3K9ac 0.5 1

0.4 0.8

0.3 0.6

0.2 0.4

0.1 0.2 Ratio Bound over Input +/- SD 0 Ratio Bound over Input +/- SD 0 VILL PLCD1 DLEC1 ACAA1 GAPDH MTX2 HOXD9 HOXD8 HOXD4 HOXD3 HOXD1 Figure 5. Once erased with epigenetic drugs, repressive histone marks are reimposed in a multi-step process. (a) The cartoon depicts the experiments and time points at which materials were collected. We measured the expression of the genes in 2q31 (left panel) and 3p22 (right panel) in CL1207 before (NT) and after treatment with 5-aza-deoxycytidine and TSA (AZT), as well as 24 h (post AZT 24 h) and 48 h (post AZT 48 h) after having removed the drugs. Transcript values were measured by RT–qPCR and are expressed as the ratio between expressions in treated versus non-treated samples. For each condition, RT–qPCR analyses were performed on three independent experiments, with each qPCR performed in duplicate. The error bars indicate the s.d. between three independent experiments. (b) ChIP assays for samples previously studied by RT–qPCR using an antibody against H3K27me3. The bar chart shows the amount of immunoprecipitated target DNA expressed as a ratio of total input DNA, measured in duplicate by qPCR. The error bars indicate the s.d. between three independent experiments. (c) Same experiment with an antibody targeting H3K9ac.

ﬁndings in mouse ES cells that presence of hypoacetylated The discrepancies about H3K27me3 and gene repression in histones40 and PRC18 are required both for gene silencing and cancer cells between previous ﬁndings and our results could be chromatin compaction. We also show that HDACi can alter higher- explained by the existence of multiple tumor pathways, where order chromatin conformation and restore expression levels H3K27me3 might have different roles. In some tumors, H3K27me3 comparable to what is measured in a normal bladder (around could be an early event causing gene repression, which could be 50% of expression in average), without decreasing high reverted by H3K27me3 removal, whereas in others, and in our H3K27me3 levels. Such PRC2-indepedent decompaction had case, it might only be a consequence of previous alterations and previously been observed in Ring1B null mouse ES cells that still not the initiator of gene repression. Alternatively, EZH2 and had PRC2-mediated H3K27 methylation.8 Moreover, the ability of H3K27me3 could also have been necessary for the initial silencing HDACis to circumvent H3K27me3 for gene expression and of the regions at one stage in tumor progression, but might have chromatin decompaction at polycomb targets has previously induced a more permanent mark, which once present becomes been reported in normal human49 and mouse ES cells.50 independent of EZH2. Moreover, we have observed in all our

& 2015 Macmillan Publishers Limited Oncogene (2015) 741 – 751 PRC2-independent silencing in cancer C Vallot et al 748

a Anti-H3K27me3 Anti-H3K27me3 NT 1 TSA 0.4 NaBu 0.8 0.3 0.6 0.2 0.4 Input +/- SD Input +/- SD 0.1 Ratio Bound over

0.2 Ratio Bound over

0 0 HOXD8 HOXD4 HOXD3 HOXD1 MTX2 VILL PLCD1 DLEC1 ACAA1 GAPDH b NT 750 TSA 20 NaBu 450 15 150 10 100 5 Fold-change vs

50 Fold-change vs non treated cells +/- SD

0 non treated cells +/- SD 0 HOXD8 HOXD4 HOXD3 HOXD1 VILL PLCD1 DLEC1 ACAA1

0.5 Anti-H3K9ac Anti-H3K9ac c NT TSA 1.0 0.4 NaBu 0.8 0.3 0.6 0.2 0.4 Input +/- SD Input +/- SD Ratio Bound over 0.1 0.2 Ratio Bound over 0.0 0.0 HOXD8 HOXD4 HOXD3 HOXD1 VILL PLCD1 DLEC1 ACAA1 GAPDH Figure 6. K27me3 loss is not necessary for gene re-expression. (a) We measured the expression of the genes in 2q31 (left panel) and 3p22 (right panel) in CL1207 before (NT) and after treatment with TSA (TSA) or NaBu (NaBu). Results are expressed as the ratio between the expression in treated versus non-treated samples. For each condition, RT–qPCR analyses were performed on three independent experiments, with each qPCR performed in duplicate. The error bars indicate the s.d. between three independent experiments. Far right panel: comparative scope of TSA and NaBu. (b) ChIP assays for samples previously studied by RT–qPCR using an antibody against H3K27me3. The bar chart shows the amount of immunoprecipitated target DNA expressed as a ratio of total input DNA, measured in duplicate by qPCR. The error bars indicate the s.d. between three independent experiments. (c) Same as in (b) with an antibody against H3K27me3.

experiments that gene repression was best correlated to the state approach toward patient selection using molecular means, that is, of chromatin compaction than to H3K27me3; in this line, recent the MRES phenotype. evidence of PRC2-independent compaction mechanisms could account for our observations,51 in particular the existence of PRC1 variants that act independently of PRC2.52–54 MATERIALS AND METHODS Understanding how these repressed clusters form in cancer Patients and tissue samples cells and how they are regulated is a major step toward We used 150 tumors to study gene expression. These carcinomas were therapeutic perspectives. We had previously demonstrated that obtained from patients included between 1988 and 2001 in the the occurrence of MRES corresponded to a clinically and prospective database established in 1988 at the Department of Urology of Henri Mondor Hospital. Four normal urothelial samples, obtained as molecularly deﬁned tumor progression pathway of bladder 15 cancer, the carcinoma in situ pathway, and that MRES clusters previously described were also used for transcriptome analysis. Forty of contained known or potential tumor suppressor genes. Here, we the 150 tumor samples and three normal samples were analyzed by RT–qPCR with the TLDA format (Applied Biosystems, Life Technologies, have shown that interfering with HDACs, but not with EZH2, and Saint Aubin, France). All patients provided informed consent and the study thereby enhancing acetylation and relaxing chromatin compac- was approved by the ethics committees of the hospital. tion is the key to gene re-expression in these clusters. These ﬁndings clearly suggest that for patients with the MRES Cell culture phenotype, therapeutic approaches involving HDACis and not histone methyltransferase inhibitors could be of major interest. The bladder cancer cell line CL1207 was derived from a high-grade tumor.56 RT112 was obtained from DSMZ (Braunschweig, Germany). HDACis, such as Vorinostat, are currently used in clinical trials TCCSUP was obtained from the laboratory of D Chopin (Hoˆpital Henri alone or in combination for the treatment of various tumor 55 Mondor, Cre´teil, France) and MGHU3 from the laboratory of Y Fradet types, ranging from T-cell lymphoma to glioblastoma; however, (University of Laval, Que´bec, Canada). To verify the identity of the various targeting the subgroup of patients who will be responsive remains cell lines used, we analyzed the genomic alterations with comparative a priority. For bladder cancer, our results might give a pioneer genomic hybridization (CGH) arrays57 and FGFR3, TP53, HRAS and KRAS

Oncogene (2015) 741 – 751 & 2015 Macmillan Publishers Limited PRC2-independent silencing in cancer C Vallot et al 749 3p22 cluster Control locus 1q21

CL1207 NTCL1207 TSA CL1207 NT CL1207 TSA

) 3

2 3 m μ

( 2 2 2

1 1

Distance 0 0 p<10-9 p=0.18 2q31 cluster

CL1207 NT CL1207 TSA ) 2 1.5 m μ (

2 1.0 0.5

Distance 0.0

p=0.02 Figure 7. Chromatin compaction at the 2q31 and 3p22 clusters is reverted by TSA. (a) Two-dimentional FISH for nuclei of non-treated (CL1207 NT) versus TSA-treated (CL1207 TSA) cells with probes at 3p22 (left panel) and at 1q21 (right panel). (b) Same analysis at 2q31. mutations with the SNaPshot technique (for FGFR3)58 or classical Nanterre, France). The amount of immunoprecipitated target was deter- sequencing for the other genes. CGH array profiles were found to be mined by real-time PCR, in duplicate, using the ABI PRISM 7900HT sequence similar to published CGH profiles and/or consistent with previously detection system (Applied Biosystems, Life Technologies) or the LightCycler reported genomic alterations, and mutations were found to be identical ROCHE (Roche Applied Science, Meylan, France). The primer sequences used to those reported (data not shown). All bladder cancer cell lines for amplification are available on request. In Figure 1, several positions were cultured in Dulbecco’s modified Eagle’s medium Glutamax within each cluster were studied by ChIP: a position near the transcription supplemented with 10% fetal bovine serum (Life Technologies). NHU start site of every gene, a position located in the middle of each gene body cells were established as finite cell lines and cultured in a complete and positions between every gene pair. For each sample and each promoter, 20 keratinocyte serum-free medium, as described. For analyses of the effect an average CT value was obtained for the immunoprecipitated material and of TSA (Calbiochem, Fontenay-sous-Bois, France) or sodium butyrate for the input chromatin. The amount of immunoprecipitated material was (NaBu) (Sigma-Aldrich, Saint-Quentin Fallavier, France) on CL1207 cells, defined as 2ðC Tðinput DNAÞÀC TðÞÞ immunoprecipitated DNA . cells were seeded in 25 cm2 dishes at a density of 8 Â 105 cells/dish. Cultures were treated the next day with 300 nM TSA or 2 mM NaBu for 16 h. To erase repressive marks from gene clusters in CL1207 cells (Figure 4), FISH CL1207 cells were treated for 48 h with 5 mM 5-aza-deoxycytidine (Calbiochem) followed by 100 nM TSA for 10 h. For EZH2 inhibitor Two-dimensional FISH analysis with fosmid probe pairs that span the 3p22 treatment, CL1207 and TCCSUP cells were seeded in 25 cm2 dishes at a and HOXD regions, and a control locus in 1q21 was as detailed in Chambeyron et al.26 and Morey et al.44 Briefly, nuclei were isolated in density of 8 Â 105 cells/dish and treated the next day with 2 mM GSK343 (kindly provided by the Structural Genomics Consortium, Toronto, ON, hypotonic buffer (0.25% KCl, 0.5% trisodium citrate), fixed with 3:1 v/v Canada) for 24 h. methanol/acetic acid and dropped on slides. Slides were then denatured in 70% formamide/2xSSC at 70 1C for 80 s. Fosmid clones were prepared and labeled with digoxigenin-11-dUTP or with biotin-16-dUTP. Approximately Chromatin immunoprecipitation 150 ng of biotin- and digoxigenin-labeled fosmid probes were used per ChIP assays were carried out in duplicate in three 150 cm2 dishes, as slide, together with 5 mg of human Cot1 DNA (Life Technologies) and 10 mg previously described for histone marks.15 Chromatin was prepared with an salmon sperm DNA (Ambion, Life Technologies). Probes were denatured at enzymatic kit (Active Motif, Rixensart, Belgium). The complexes were 70 1C for 5 min, re-annealed with Cot1 DNA for 15 min at 37 1C and immunoprecipitated with 4 mg of antibodies against H3K27me3 (07–449, hybridized to the denatured slides overnight. Washes and detection were Upstate Biotechnology, Santa Cruz, CA, USA) or acetyl histone H3 (06–599, as described in Chambeyron et al.4 Three-dimensional FISH was performed Upstate Biotechnology). For RING1B ChIP, chromatin was sheared by for the analysis of the 3p22 locus and 1q21 locus in CL1207 transfected (or sonication,59 to obtain a 200–600-bp sample and the complex immunopre- not) with siRNAs targeting EZH2 and GAPDH. In this case, cells were grown cipitated with 4 mg of antibody against RING1B (D139-3, MBL Clinisciences, on coverslips and denatured for 30 min in 50% formamide/2xSSC.

& 2015 Macmillan Publishers Limited Oncogene (2015) 741 – 751 PRC2-independent silencing in cancer C Vallot et al 750 Image analysis and statistics banding pattern on a mouse autosomal chromosome. Genome Res 2009; 19: Slides were examined using a Zeiss Axioplan II fluorescence microscope 221–233. (Zeiss, Marly le Roi, France). Grayscale images were captured with a 3 Zhao XD, Han X, Chew JL, Liu J, Chiu KP, Choo A et al. Whole-genome mapping of Hamamatsu Orca AG CCD camera (Hamamatsu Photonics Ltd, Massy, histone H3 Lys4 and 27 trimethylations reveals distinct genomic compartments in France). Image capture and analysis were performed using written for human embryonic stem cells. Cell Stem Cell 2007; 1: 286–298. IPLab Spectrum (Spectra, Ontario, NY, USA): the mean squared distance 4 Chambeyron S, Bickmore WA. Chromatin decondensation and nuclear reorgani- (d2) between the centroids of two probes hybridization signals was as zation of the HoxB locus upon induction of transcription. Genes Dev 2004; 18: previously described.8 Statistical analysis and numerical calculations were 1119–1130. carried out with R 2.12.1 (R Foundation for Statistical Computing). Each 5 Nagano T, Mitchell JA, Sanz LA, Pauler FM, Ferguson-Smith AC, Feil R et al. The Air data set consisted of at least 50 nuclei for each cell line and for each noncoding RNA epigenetically silences transcription by targeting G9a to chro- combination of probes. The statistical significance of differences in od24 matin. Science 2008; 322: 1717–1720. was assessed using the nonparametric Mann–Whitney U-test to examine 6 Simon JA, Kingston RE. Occupying chromatin: polycomb mechanisms for getting the null hypothesis that two sets of data show the same distribution. to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell 2013; A P-valueo0.05 was considered significant. 49: 808–824. 7 Pandey RR, Mondal T, Mohammad F, Enroth S, Redrup L, Komorowski J et al. Transfection Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell 2008; 32: 232–246. CL1207 cells were transfected with siRNAs targeting EZH2 or GAPDH as a 8 Eskeland R, Leeb M, Grimes GR, Kress C, Boyle S, Sproul D et al. Ring1B compacts control (Ambion, Life Technologies) using Lipofectamine RNAiMAX chromatin structure and represses gene expression independent of histone ubi- transfection agent (Life Technologies) following the supplier’s recommen- quitination. Mol Cell 2010; 38: 452–464. dations for reverse transfection. The siRNA/transfection agent complexes 9 Margueron R, Reinberg D. Chromatin structure and the inheritance of epigenetic were prepared inside the dishes, after which cells and medium were 2 5 information. Nat Rev Genet 2010; 11: 285–296. added. Cells were seeded in 25 cm dishes at a density of 8 Â 10 cells/dish. 10 Crawley JJ, Furge KA. Identification of frequent cytogenetic aberrations in hepa- Two siRNAs targeting EZH2 and a control siRNAs targeting GAPDH were tocellular carcinoma using gene-expression microarray data. Genome Biol 2002; 3: used to monitor gene expression in the studied clusters. The efficiency of RESEARCH0075. the knockdown was checked using qRT–PCR and western blot. 11 Stransky N, Vallot C, Reyal F, Bernard-Pierrot I, de Medina SG, Segraves R et al. Regional copy number-independent deregulation of transcription in cancer. Nat Gene expression analysis by quantitative RT–PCR Genet 2006; 38: 1386–1396. RNAs were extracted from cell lines with the Qiagen RNeasy kit (Qiagen, 12 Frigola J, Song J, Stirzaker C, Hinshelwood RA, Peinado MA, Clark SJ. Epigenetic Courtaboeuf, France). One microgram of total RNA was used for reverse remodeling in colorectal cancer results in coordinate gene suppression across an transcription, with random hexamers (20 pmol) and 200 U MMLV reverse entire chromosome band. 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