Specification in Embryonic Stem Cells Through Activation and Repression

Article

Polycomb Regulates Mesoderm Cell Fate- Speciﬁcation in Embryonic Stem Cells through Activation and Repression Mechanisms

Graphical Abstract Authors Lluis Morey, Alexandra Santanach, Enrique Blanco, ..., Elphe` ge P. Nora, Benoit G. Bruneau, Luciano Di Croce

Correspondence [email protected] (L.M.), [email protected] (L.D.C.)

In Brief Morey et al. reveal that Mel18, a Polycomb-complex-associated protein, is an essential epigenetic regulator of cardiac differentiation. During directed differentiation of embryonic stem cells, Morey et al. ﬁnd that Mel18-PRC1 complexes exchange subunits in a stage- speciﬁc manner and instruct sequential gene activation and repression programs to specify mesoderm fate, prevent alternate lineage commitment, and promote cardiac differentiation.

Highlights Accession Numbers d Mel18 is required for PRC1 stability and maintenance of gene GSE67868 repression in ESCs d Mel18 is essential for ESC differentiation into early cardiac- mesoderm precursors d Different Mel18-PRC1 complexes are assembled during cardiac differentiation d Distinctive PRC1 complexes bind to highly active and repressed genes in MES cells

Morey et al., 2015, Cell Stem Cell 17, 300–315 September 3, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.stem.2015.08.009 Cell Stem Cell Article

Polycomb Regulates Mesoderm Cell Fate-Speciﬁcation in Embryonic Stem Cells through Activation and Repression Mechanisms

Lluis Morey,1,2,6,* Alexandra Santanach,1,2 Enrique Blanco,1,2 Luigi Aloia,1,2 Elphe` ge P. Nora,3,4 Benoit G. Bruneau,3,4 and Luciano Di Croce1,2,5,* 1Centre for Genomic Regulation (CRG), Doctor Aiguader 88, 08003 Barcelona, Spain 2Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain 3Gladstone Institute of Cardiovascular Disease and Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA 94158, USA 4Department of Pediatrics, Cardiovascular Research Institute, University of California at San Francisco, San Francisco, CA 94158, USA 5Institucio´ Catalana de Recerca i Estudis Avanc¸ ats, Passeig Lluis Companys 23, 08010 Barcelona, Spain 6Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA *Correspondence: [email protected] (L.M.), [email protected] (L.D.C.) http://dx.doi.org/10.1016/j.stem.2015.08.009

SUMMARY further distinguished based on their subunit composition as canonical PRC1 (cPRC1), which contains Cbx proteins, or non- Polycomb complexes (PRC1 and PRC2) are essential canonical PRC1 (ncPRC1), which lacks Cbx proteins. The high regulators of epigenetic gene silencing in embryonic level of heterogeneity in the architecture of different PRC1 com- and adult stem cells. Emerging evidence suggests plexes suggests that their specific gene targets, recruitment that the core subunit composition regulates distinct mechanisms, and enzymatic activities are context specific. biological processes, yet little is known about Consistently, we and others have shown that cPRC1 complexes the mechanistic underpinnings of how differently interchange Cbx7 in pluripotent embryonic stem cells (ESCs) for Cbx2 and Cbx4 in differentiating ESCs (Morey et al., 2012; composed Polycomb complexes instruct and main- O’Loghlen et al., 2012). tain cell fate. Here we find that Mel18, also known Although Polycomb activity is mainly associated with gene as Pcgf2 and one of six Pcgf paralogs, uniquely reg- repression, Polycomb complexes can also bind to active pro- ulates PRC1 to specify mesoderm cell fate in embry- moters, where they regulate gene expression positively (Creppe onic stem cells. Mechanistically, Mel18 functions as et al., 2014; Frangini et al., 2013; Schaaf et al., 2013; Suzuki et al., a classical Polycomb protein during early cardiac 2002). Further, in MEF cells, depleting the non-coding RNA mesoderm differentiation by repressing pluripo- (ncRNA) lincRNA-p21 upregulated a subset of PRC2 targets, tency, lineage specification, late cardiac differentia- although H3K27me3 remained unaffected in these promoters tion, and negative regulators of the BMP pathway. (and the presence of Cbx was not investigated) (Dimitrova However, Mel18 also positively regulates expression et al., 2014). It thus seems plausible that certain genes contain- of key mesoderm transcription factors, revealing an ing Cbx proteins and H3K27me3 are expressed in specific cellular processes. unexpected function of Mel18 in gene activation PRC1 heterogeneity is further defined by the six Polycomb- during cardiac differentiation. Taken together, our group RING finger protein (Pcgf) paralogs (Gao et al., 2012). findings reveal that Mel18 is required to specify Pcgf2/Mel18 (referred to herein as Mel18) and Pcgf4/Bmi1 PRC1 function in both a context- and stage-specific enhance Ring1B-mediated enzymatic activity in vitro through a manner. direct interaction of their RING domains (Buchwald et al., 2006; Elderkin et al., 2007; Li et al., 2006). Pcgf4/Bmi1 expression is strongly upregulated during neuronal differentiation and is INTRODUCTION essential for neuroprecursor (NPC) maintenance and hematopoietic stem cell (HSC) self-renewal (Bruggeman et al., 2005; The Polycomb group of complexes (PcG) are implicated in Oguro et al., 2010; Smith et al., 2011; Zencak et al., 2005). development, cancer, and stem cell fate decisions (Di Croce Pcgf1/NSPc1, together with the transcription factor Runx1, and Helin, 2013). They are classified as two main complexes, also regulate HSC differentiation and self-renewal (Ross et al., the Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) 2012). Although it is well established that Polycomb complexes (Morey and Helin, 2010). PRC2 comprises three core subunits: are essential regulators of ESC differentiation in vitro (Laugesen Eed, Suz12, and the histone methyltransferases Ezh1/2, which and Helin, 2014), very little is known about the functions of deposit di- and tri-methylation on lysine 27 of the histone H3 Pcgf proteins in this context. Mel18, Pcgf1, and Pcgf6 are highly (H3K27me2/3) (Cao et al., 2002). PRC1 contains the E3 ligases expressed core subunits of different PRC1 complexes in Ring1A/B, which monoubiquitinate lysine 119 at histone H2A ESCs, suggesting that they, similar to the Cbx proteins, can (H2AK119ub) (Wang et al., 2004). PRC1 complexes can be functionally define PRC1 activities. Indeed, Pcgf6 was recently

300 Cell Stem Cell 17, 300–315, September 3, 2015 ª2015 Elsevier Inc. demonstrated to be functionally essential for ESC self-renewal (Junco et al., 2013) and with Ring1B through their RING domains (Zdzieblo et al., 2014). PRC1 activities and functions have been (Li et al., 2006). However, as Pcgf proteins do not directly interact extensively studied mainly in the context of Ring1A/B depletion, with Cbx proteins, the reduced levels of Cbx7 in Mel18-depleted but PRC1 heterogeneity—in both complex composition and ac- cells suggested that Mel18 is required for cPRC1 complex stabil- tivity—now suggests that specific PRC1 complexes have unique ity (Figure 1D). Moreover, endogenous co-immunoprecipitation functions during development and stem cell differentiation. (coIP) experiments using a Ring1B antibody indicated that To date, no PRC1 members have been described to be impor- cPRC1 was strongly destabilized, as Phc1 and Cbx7 interactions tant for in vivo or in vitro cardiac development and differentiation, were significantly reduced when Mel18 was depleted (Figure 1D). yet the PRC2 core subunit Ezh2 has been described to be essen- As expected, the Rybp-Ring1B interaction was not affected. tial for cardiomyocyte (CM) proliferation, survival, and postnatal We conclude that Mel18 depletion specifically disrupted the cardiac homeostasis (Delgado-Olguıń et al., 2012), and Eed and cPRC1 complex in ESCs, while the ncPRC1 complex remained Jarid2 are required for heart formation (Laugesen and Helin, unaffected. 2014). Mel18 is not essential during embryogenesis, but mutant We then asked whether Ring1B, Cbx7, and PRC2 binding to mice have growth deficiencies, homeotic transformations, and chromatin was affected in Mel18 KD cells. ChIP-qPCR experi- hematopoietic defects and die a few days or weeks after birth ments indicated that Cbx7 and Ring1B recruitment was strongly (Akasaka et al., 2001). Skeletal and hematopoietic tissues derive reduced in shMel18 cells (Figure 1E), concomitant with a slight from the mesoderm lineages, suggesting that Mel18 could reduction of H2AK119ub levels (Figure S1F, left panel) and specialize during development. Suz12 occupancy (Figure S1F, right panel). As Cbx7 is required Here, we have characterized the function of Mel18 in ESC plu- for efficient binding to chromatin of Ring1B, Mel18, and PRC2 ripotency and differentiation. We show that Mel18 binds to (Morey et al., 2012), we cannot exclude the possibility that the cPRC1 target genes in ESCs and that it is essential for stabilizing reduced occupancy of Ring1B in Mel18 KD cells stemmed from cPRC1 and for cPRC1-mediated gene repression. Using direct reduced Cbx7 protein levels. Although we observed a 25% differentiation assays of ESCs to CMs, cartilage, and NPCs, reduction of H2AK119ub levels and Suz12 binding in Mel18- we elucidate a specific function of Mel18 during the early stages depleted ESCs (Figure S1F, left panel), global H2AK119ub and of mesoderm differentiation. Mechanistically, we found that, dur- H3K27me3 levels remained unaffected (Figure S1G). ing cardiac differentiation, the Mel18-PRC1 complex not only re- We observed that reduced occupancy of cPRC1 resulted in presses pluripotency genes, BMP-negative regulators, and cell gene de-repression (Figure S1H). RNA-seq experiments (Table fate identity genes, but also facilitates transcription of transcrip- S1) revealed that in shMel18 ESCs, 868 genes were deregulated tion factors that are essential for differentiation of early cardiac- (with 720 upregulated and 148 downregulated) (Figure 1F, left mesoderm precursor cells (MES cells). panel). As anticipated, Ring1B mutant cells have a major impact on gene deregulation (Figure 1F, right panel). Importantly, almost RESULTS 50% of the upregulated genes in Mel18 KD cells are Mel18 target genes, and from those, 95% are co-bound by Ring1B (Fig- Mel18 Binds to cPRC1 Target Genes, Regulates ure 1G). This result is consistent with our Mel18 ChIP-seq Transcription, and Stabilizes the cPRC1 Complex Only analysis, in which all Mel18 target genes were also Ring1B tar- in ESCs gets, and also denotes that Mel18 is important for maintaining Mel18 is extensively considered to be an integral subunit of gene repression. Gene ontology (GO) analysis of upregulated cPRC1 in ESCs (Luis et al., 2012), yet no genome-wide studies Mel18 target genes scored them as being strongly associated of Mel18 binding to chromatin have been reported to date. We to mesoderm genes, and to a lesser extent, as ectoderm genes, thus performed Mel18 chromatin immunoprecipitation (ChIP) suggesting a particular role for Mel18 in mesoderm cell fate experiments with massive-parallel sequencing (ChIP-seq). We (Figure 1H). identified 6,593 peaks, resulting in 4,162 target genes (Figure 1A; Table S1). Heat maps of Mel18 ChIP-seq signals revealed that all Mel18 Is Necessary for Proper Cardiac Differentiation of Mel18 targets are also co-occupied by Ring1B and Cbx7 and are Embryoid Bodies decorated with H2AK119ub (Figure 1A). Further analyses of We next asked whether Mel18 is essential for ESC differentia- Mel18, Ring1B, Cbx7, and H2AK119ub ChIP-seq signals at the tion. We first generated embryoid bodies (EBs) from control 4,162 Mel18 target genes corroborated that Mel18 targets are and Mel18 KD ESCs. EBs mimic early development of the em- PRC1 target genes (Figures 1B and 1C). These results further bryo and are often used as a differentiation assay to test ESC highlight that Mel18 occupancy is always within the context of pluripotency (Evans and Kaufman, 1981). AP staining of EBs at the PRC1 complex. day 7 showed that more than double the amount of colonies To determine whether Mel18 is necessary for both ESC self- appeared to be AP+ in the shMel18 ESCs as compared to the renewal and cPRC1 stability, we depleted Mel18 using two spe- control ESCs (Figure S2A), suggesting that a lack of Mel18 cific shRNAs (Figure S1A). Analysis of the pluripotency markers impairs proper differentiation. We then generated 12-day-old and alkaline phosphatase (AP) staining of control and Mel18 EBs, which have cells from all three germ layers. From days knockdown (KD) cells suggested that Mel18 is not required for 2–10, shMel18 EBs were macroscopically very similar to control ESC self-renewal (Figures S1B–S1E). Western blots of control EBs; however, at day 12, they exhibited an irregular internal and Mel18 KD extracts suggested that Mel18 is required for sta- bulk and did not present any sign of beating, which is usually bility of Phc1 and Cbx7 proteins (Figure 1D). Pcgf proteins observed from day 10 in normal EBs (Figure 2A and data directly interact with Phc proteins through their RAWUL domains not shown). Although pluripotency genes were efficiently

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Figure 1. Mel18 Only Occupies Canonical PRC1 Sites and Is Essential for cPRC1 Stability and Gene Repression Maintenance (A) ChIP-seq heat maps of Mel18, Ring1B, Cbx7, H2AK119ub, and IgG. Target genes were deﬁned by the presence of one or more Mel18 peaks within ±2.5 Kb of a given transcriptional start site (TSS). (B) Normalized signal relative to all TSSs of Mel18, Ring1B, Cbx7, H2AK119ub, and IgG ChIP-seq. (C) Genome-browser screenshots of the proﬁles of all cPRC1 components in the HOXA cluster. (D) Endogenous coIP, using Ring1B and IgG antibodies, in control and Mel18-depleted ESCs.

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Figure 2. Embryoid Bodies Derived from Mel18-Depleted ESCs Cannot Differentiate Toward CMs (A) Embryoid bodies (EBs) derived from Mel18-depleted ESCs show abnormal differentiation at day 12. Images were taken at 203 magnification at days 2, 4, 6, 10, and 12 and also at 503 at day 12. (B) Real-time qPCR of selected genes in ESCs and at day 12 of EB differentiation. Genes related to CM differentiation (Tbx5, Tbx20, Nkx2-5, Myl7, Gata4, and Mixl1) were strongly downregulated in Mel18-depleted derived EBs. Expression was normalized to the housekeeping gene RPO. Data represent the average of three experiments. (C) Semiquantitative assay to analyze beating EBs. At day 5, the control and Mel18-depleted EBs were attached to the plate, and beating clusters that appeared were counted from day 8 until day 12. Data represent the average of three experiments. (D) Real-time qPCR of CM-specific genes in ESCs and in attached EBs at day 12 of differentiation. Expression was normalized to the housekeeping gene RPO. Data represent the average of three experiments. See also Figure S2. downregulated in both control and shMel18 12-day-old EBs, we to allow quantification of beating EBs (Figure 2C). At day 11, all found striking differences in the expression of mesoderm/car- control EBs were beating, while only 20%–30% of shMel18 diac markers (Figure 2B). EBs contained beating clusters (Figure 2C). Analysis of car- To have a better readout of the cardiac differentiation potential diac-specific genes confirmed that shMel18 EBs do not properly of the shMel18 ESCs, we modified the EB differentiation protocol differentiate into beating CMs (Figure 2D).

(E) ChIP-qPCR of PcG target genes in control and in two different Mel18 KD ESCs, performed with the antibodies indicated below. Results are presented asa percentage of input immunoprecipitated. All ChIP experiments represent the average of at least three experiments. (F) Genes deregulated by at least 2-fold and with a FPKM value > 0.1, following Mel18 depletion in ESCs or in Ring1B KO ESCs. (G) For Mel18 target genes deregulated after Mel18 depletion, 95.4% of upregulated genes are also Ring1B targets. (H) GO analysis of Mel18 target genes upregulated after Mel18 depletion. See also Figure S1.

Cell Stem Cell 17, 300–315, September 3, 2015 ª2015 Elsevier Inc. 303 As Phc1 protein stability is greatly impaired in shMel18 ESCs, early mesoderm differentiation (Figure S3C). Similar gene cate- we next tested whether the observed differentiation defects gories were also observed when changes in gene expression were due to Phc1 depletion. CoIP experiments using a Ring1B upon Mel18 depletion were analyzed using a 5-fold cut-off antibody indicated that Ring1B and Mel18 strongly interacted, (data not shown). regardless of Phc1, but that Mel18 levels were slightly decreased Comparing the expression of the upregulated genes in ESCs and the Cbx7 interaction was strongly reduced in Phc1 KD cells from control MES cells with the deregulated genes in shMel18 (Figure S2B). shPhc1 ESCs also de-repressed Polycomb target MES cells revealed that almost 50% of the upregulated genes genes (Figure S2C), and importantly, depletion of Phc1 did not in control MES cells (199 out of 489 genes) were downregulated affect the cardiac differentiation of ESCs (Figure S2D). Thus, by at least 2-fold in shMel18 MES cells (Figure 3D). These genes we conclude that the CM differentiation defect observed in are mainly classified as mesoderm genes and, more specifically, Mel18 KD cells was not Phc1 dependent. are involved in cardiovascular system development (Figure 3E). Almost 10% (150 of 1,311) of the genes downregulated during Mel18 Is Essential for ESCs to Differentiate into Early MES differentiation were aberrantly upregulated in Mel18- MES Cells depleted MES cells; interestingly, these genes were mainly Because EB formation stimulates a chaotic ESC differentiation, it involved in ESC pluripotency (Figure 3E). Real-time qPCR anal- was difficult to estimate whether the observed differentiation de- ysis confirmed the reduced expression of MES-specific genes fects were specific for the mesoderm lineage or rather were due in Mel18-depleted MES cells (Figure 3F). T/Brachyury expression to secondary differentiation defects. To overcome this, we used was not affected in shMel18 MES cells, while Mesp1 expression a protocol for differentiating ESCs into a highly homogenous (which is downstream of T/Brachyury during mesoderm develop- population of beating CMs (Kattman et al., 2011). Further, ment; David et al., 2011; Herrmann et al., 1990; Saga et al., 1999) because early steps of cardiac differentiation, such as formation was strongly downregulated. Thus, Mel18 is not essential for the of early MES cells and cardiac precursor (CP) cells, can be initial mesoderm cell determination but is an essential regulator of studied in this protocol, we asked whether Mel18 is required the MES-specific gene signature, as its depletion resulted in a for direct CM differentiation and, if so, at which stage of cardiac block of differentiation of early MES cells. differentiation Mel18 function is required (Figure 3A). Mel18-depleted ESCs formed EB-containing MES cells (as PRC1 Complex Composition and Genome-wide Binding determined by the co-expression of the surface markers Flk1 in MES Cells [Kdr] and the Pdgfa receptor [Kattman et al., 2011]); however, We next analyzed the composition and the genome-wide distri- these were not as round and smooth as those formed from con- bution of PRC1 in MES cells. We first determined that Mel18 in- trol MES EBs (Figure 3B). In our experiments, 0.1 ng/ml and teracts with Ring1B in MES cells (Figure 4A) and measured the 0.2 ng/ml of BMP4 generated around 60% and 70%–80% of expression levels of all cPRC1 and ncPRC1 subunits (Figure 4B). Flk1+ and Pdgfa+ cells, respectively (Figure 3C). Remarkably, Cbx7 and Phc1 were strongly downregulated in MES cells, with FACS analysis for these surface markers revealed that only Cbx2 and Phc2 the main Cbx and Phc family members ex- 10% of Mel18-depleted cells are Flk1+/Pdgfa+. Importantly, we pressed in these cells. Mel18 and Pcgf6 were the strongest did not observed accumulation of an Flk1+/Pdgfa– (hematopoiet- Pcgf proteins expressed in MES cells (Figure 4B). Strikingly, ic subpopulation) or a Flk1–/Pdgfa+ population in shMel18 MES while global H2AK119ub levels were constant in ESCs as cells, suggesting that Mel18 is essential specifically for gener- compared to MES cells, H3K27me3 levels were reduced, ating MES cells and committing them to the CM lineage. concomitant with an accumulation of H3K27ac levels (Figure 4C). To explore the molecular mechanisms behind the Mel18- Concordant with the effects of Mel18 depletion in ESCs, the dependent generation of MES cells, we performed global gene levels of H2AK119ub and H3K27me3 also remained unaltered expression experiments in control and Mel18-depleted MES in shMel18 MES cells (Figure S4A). Western blot analysis of cells (Table S1). To control for the differentiation method, we first several PRC1 subunits confirmed that Cbx2 and Phc2 were analyzed genes that were up- or downregulated by at least strongly upregulated and that Mel18 was marginally downregu- 5-fold from control ESCs and MES cells (with FPKM R 1in lated in MES cells (Figure 4D). Upon Mel18 depletion, Cbx2 pro- ESCs and MES cells). We found 489 genes upregulated and tein levels were strongly impaired, yet Rybp, Ring1B, and Cbx6 1,311 genes downregulated in MES cells (Figure 3D). As expected, remained unaffected (Figure 4D). These data suggested that a upregulated genes were mainly associated with mesoderm differ- new cPRC1 complex containing Cbx2 and Phc2 was assembled entiation and genes related to pathways essential for MES differ- in MES cells and that Mel18 was required to stabilize cPRC1, but entiation (e.g., BMP, FGF/Brachyury, and Wnt). In contrast, down- not (as in ESCs) ncPRC1. regulated genes were mainly classified as ESC genes (Figure S3A). To study the architecture of the PRC1 complex in ESCs and We then analyzed the gene expression changes, using a 2-fold MES cells, we generated an ESC line that expressed endoge- cut-off, in Mel18-depleted MES cells as compared to control nous levels of a Flag-Avi-tagged Ring1B (Figure 4E). Mass spec- MES cells. We found 421 genes to be aberrantly upregulated trometry (MS) analysis confirmed that Ring1B was associated in Mel18-depleted MES cells. These genes are involved in ecto- with at least three different known complexes in ESCs: the derm differentiation and ESC pluripotency, suggesting that cPRC1, ncPRC1, and E2F6-PRC1 complexes (Morey et al., Mel18 KD MES cells have lost their cell identity and are not 2012; O’Loghlen et al., 2012; Qin et al., 2012; Tavares et al., able to fully differentiate (Figure S3B). In contrast, the 445 down- 2012; Wu et al., 2013; Farcas et al., 2012; He et al., 2013)(Fig- regulated genes were involved in mesoderm CM differentiation, ure S4B). In MES cells, Ring1B strongly associated with Mel18, with a strong impairment of the gene networks associated with Pcgf6, Rybp, Cbx2, and Phc2. Interestingly, we only found

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Figure 3. Mel18-Depleted ESCs Do Not Properly Differentiate into Early MES Cells (A) Schematic representation of the protocol used to differentiate ESCs to CMs (Kattman et al., 2011). (B) Representative pictures of control and Mel18-depleted MES cells. Images were taken at 203 magniﬁcation. (C) FACS analysis using FLK1 and Pdgfa antibodies in control and Mel18-depleted MES cells induced by two different concentrations of BMP4. (D) RNA-seq heat map of up- and downregulated genes in control or Mel18-depleted MES cells as compared to control ESCs. Only genes up- or downregulated at least 5-fold from ESCs are shown. (E) GO analysis of genes deregulated by at least 2-fold in Mel18-depleted MES cells as compared to control MES cells. (F) Real-time qPCR of speciﬁc MES genes in control or Mel18-depleted MES cells. Expression was normalized to the housekeeping gene RPO. Data represent the average of six experiments. See also Figure S3.

Pcgf6 as a core subunit of the E2F6-PRC1 complex, suggesting MES cells, Ezh2 was strongly downregulated during ESC differ- that this complex might not be involved in ESC differentiation into entiation (Figure S4C). We did not observe changes in Ezh1 MES cells (Figure 4F). We next analyzed a time course of ESC-to- expression at these differentiation stages (Table S1). MES cell differentiation to determine the time frame in which the We next performed ChIP-seq experiments of Ring1B, Mel18, switch of the cPRC1 complex composition occurs. Changes in Rybp, and Cbx2 in MES cells (Table S1). As expected, Mel18, protein levels of PRC1 subunits seemed to be cytokine depen- Ring1B, and Rybp occupied genes not previously occupied dent, since major protein changes occurred 24 hr post-induction by PRC1 in ESCs and were displaced from genes previously (Figure S4C). In agreement with reduced levels of H3K27me3 in occupied in ESCs, indicating a partial re-localization of PRC1

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Figure 4. PRC1 Composition, Genome-wide Occupancy, and Expression of Polycomb Target Genes in MES Cells (A) Endogenous coIP, using a Ring1B and IgG antibodies from MES cells. (B) RNA-seq expression of PRC1 subunits in MES cells represented by FPKM values. (legend continued on next page) 306 Cell Stem Cell 17, 300–315, September 3, 2015 ª2015 Elsevier Inc. genome binding in differentiating cells (Figure 4G). Surprisingly, co-activators. Interestingly, we also observed that about one-third de novo Ring1B target genes in MES cells were mainly associ- of class 1 genes also contained H3K36me3 (Figure 4L; Table S1). ated with metabolic processes (Figure S4D). Cbx7 binding GO analysis revealed that these genes were mainly associated is substituted for Cbx2 in 1,589 genes previously bound by with cell-cell adhesion and heart development (Figure 4M). Genes Cbx7 in ESCs (Morey et al., 2012), and Cbx2 binds to 1,382 within this category are strongly related to the BMP pathway and MES-specific genes (Figure 4G and Figures S4E and S4F). the transcription factors indispensable for early heart develop- Cbx7 ESC genes and common Cbx7/Cbx2 target genes in ment (Table S1). Genes containing H3K36me3 from the six clas- ESC/MES cells were related to development, yet Cbx2 MES ses have different biological functions (Figure S4J), suggesting cell targets were also strongly associated with metabolic pro- that genes containing different combinations of PRC1 members cesses (Figure S4E). and histone modifications regulate distinct cellular pathways Detailed analyses of Ring1B, Mel18, Rybp, and Cbx2 target and processes. Overall, these data demonstrate that a new genes disclosed at least six different types of genes robustly cPRC1 complex was assembled in MES cells and that cPRC1 bound by different combinations of PRC1 subunits. A genome and ncPRC1 complexes bind to more than 2,000 genes in these browser screenshot of a large region of chromosome 1 shows cells, yet different gene classes can contain distinct combinations genes co-bound by all four PRC1 subunits (#1), by Ring1B either of Polycomb subunits. Strikingly, binding of Polycomb proteins alone (#2) or with Rybp (#3), or by Mel18 and Rybp, but not Cbx2 was not always associated with gene repression. (#4) (Figure 4H). We also found Rybp bound to 1,058 genes that contained no traces of any PRC1 subunit (Morey et al., 2013) and Mel18 Directly Regulates Expression of MES-Specific genes with Mel18, Ring1B, and Cbx2 without Rybp (Figure 4I). Genes The 2,186 genes co-bound by Ring1B, Mel18, Cbx2, and We hypothesized that the Mel18-mediated phenotype in MES Rybp (Figure 4I) were classified as developmental genes and, cells might be connected with the direct regulation of genes en- more specifically, as ectoderm and mesoderm genes (Figure 4J). coding transcription factors directing cardiac differentiation. We also found examples of genes that were co-bound by all four Since the set of 647 class 1 active genes contained Mel18 (see PRC1 members involved in cardiac differentiation (Bruneau, Figure 4L), we investigated whether Mel18 is also involved in 2013), endoderm differentiation, and pluripotency/ectoderm gene transcription. (Figures S4G and S4H). Compared to their wild-type counterparts, Mel18-depleted Hypothesizing that genes bound by different combinations of MES cells aberrantly deregulated a very similar number of genes, PRC1 subunits might be expressed differently, we intersected with an altered expression of more than 300 Mel18 targets (Fig- our RNA-seq of control MES cells with the different ChIP-seq ure 5A). A higher number of Mel18 targets were downregulated datasets and with an H3K27me3 dataset (Wamstad et al., (201) than upregulated (116) (Figure 5A). The downregulated 2012). Indeed, Ring1B and Rybp target genes that do not contain 201 genes were expressed and contained H3K36me3 in wild- Mel18, Cbx2, and/or H3K27me3 were expressed at higher levels type cells, suggesting again that Mel18 might be required for than genes containing all PRC1 members and PRC2 (Figure 4K). proper gene transcription of these genes. To confirm that Moreover, genes containing Cbx2, but not Rybp (class 5 versus Mel18 was bound to H3K36me3-containing genes, we per- class 6), were also more repressed than the genes containing formed a sequential Mel18/H3K36me3 ChIP assay (Figure 5B). Rybp, but not Cbx2 (Morey et al., 2013)(Figure 4K). GO analysis GO analysis revealed that the Mel18 targets downregulated of the six gene classes indicated that different genes containing upon its depletion were mainly related to mesoderm specifica- particular combinations of PRC1 subunits have specific biolog- tion, whereas the upregulated genes were associated with regu- ical functions (Table S1). lation of the other cell linages and stem cells (Figure 5C). To substantiate the observation that Polycomb-containing To strengthen the observation that Mel18 might control genes are transcriptionally active, we looked for H3K36me3 his- expression of active genes in MES cells, we selected the tone modifications (Wamstad et al., 2012). Almost all genes from highest-induced genes from ESC-to-MES differentiation, which the classes 2, 3, and 4 contained H3K36me3, thus confirming were upregulated by at least 25-fold (FC25) and had an that they are active (Figure 4L; Figure S4I) and suggesting that FPKM R 1 in MES cells (Table S1). Analysis of Mel18, Ring1B, recruitment of Ring1B or Rybp per se was not enough to repress Cbx2, and Rybp ChIP-seq signal at these 98 genes clearly transcription. We thus hypothesized that they might function as showed a positive signal over the IgG ChIP-seq (Figure 5D).

(C) Western blots of histone modifications analyzed from acid-extracted histones. (D) Western blots of PRC1 subunits from control or shMel18 ESCs or from MES cells. (E) Western blots of Ring1B and streptavidin in control BirA ESCs or BirA ESCs that expressed Ring1B. (F) Purification protocol and MS analyses of PRC1 subunits associated to Ring1B in MES cells. (G) Venn diagram of Ring1B, Mel18, Cbx2, and Rybp target genes in MES cells. (H) Genome browser screenshots of Ring1B, Mel18, Cbx2, and RYBP ChIP-seq assays performed in MES cells. Boxes represent different classes of genes bound by specific PRC1 members. (I) Venn diagram of Ring1B, Mel18, Cbx2, and Rybp target genes in MES cells. (J) GO analysis of the 2,186 genes co-bound by Ring1B, Mel18, Cbx2, and Rybp. (K) Expression of six different classes of genes bound by Ring1B, Mel18, Cbx2, Rybp, and/or H3K27me3. (L) Number of class 1, 2, and 4 genes that contain H3K36me3. (M) GO analysis of the 647 genes co-bound by Ring1B, Mel18, Cbx2, and Rybp that contain H3K27me3 and H3K36me3 histone modifications. See also Figure S4.

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308 Cell Stem Cell 17, 300–315, September 3, 2015 ª2015 Elsevier Inc. While not all 98 genes contained a PRC1 subunit, 24 were bound 80% of FLK1+/Pdgfa+cells) (Figure 5H). We confirmed that the by all four subunits (Figure S5A). Further, Rybp was present in Gata4, Lhx1, Hand1, and Six2 promoters, but not the T or more genes than Cbx2, reinforcing the concept that genes con- Mesp1 promoters, were bound by Mel18 and Cbx2 and were taining Rybp, rather than just any Cbx protein, are more ex- decorated with H3K27me3 in FLK1+/Pdgfa+ cells (Figure 5I) and pressed (Figure S5A). Genome-browser screenshots of the that the expression of all six genes was very high in these cells Ring1B, Mel18, Rybp, and Cbx2 ChIP-seq signal at some of as compared to ESCs (Figure S5F). Second, to rule out potential the FC25 genes (Hand1, Lhx1, and Six2) and Gata4 are shown off-target shRNA effects, we generated two Mel18 knockout in Figure 5E. We then assessed whether these promoters, which (KO) ESCs lines (named 12.1 and 12.2) by CRISPR/Cas9. Similar were highly expressed yet contained PRC1 complexes, also to the shMel18 ESCs, Phc1 protein levels were also strongly contained H3K27me3. These four promoters (and in general, downregulated in both KO cell lines (Figure S5G), and ChIP- all the FC25 genes) were decorated not only with H3K4me3 qPCR assays confirmed that Mel18 was required for full Ring1B and H3K36me3, but also with H3K27me3 (Figure 5E; Fig- binding to chromatin (Figure S5H). MES-derived EBs from ure S5B). Interestingly, the H3K4me3 profiles for the Hand1, Mel18 KO cells were also affected (Figure S5I). Finally, ChIP ex- Lhx1, and Six2 loci were not restricted to the transcriptional start periments confirmed that Ring1B and Mel18 co-occupied the site (TSS), but rather, they resembled the broad H3K4me3 do- Gata4, Lhx1, Hand1, and Six2 promoters and that Ring1B was mains present in genes that define cell identity (Benayoun strongly displaced in Mel18 KO MES cells (Figure S5J). et al., 2014). ChIP-qPCR assays confirmed that Ring1B, Rybp, and Mel18 co-bound to the promoter of these four genes (Fig- Mel18 Directly Controls Negative Regulators of the BMP ures S5C and S5D). Similar to ESCs, Mel18 was required in Pathway in MES Cells MES cells for full Ring1B binding and H2AK119ub and In addition to regulating genes implicated in early MES differen- H3K27me3 deposition, while Rybp binding remained unaltered tiation (Figure 5D), Mel18 also functions as a classical Polycomb (Figure S5D, lower panel). Not all FC25 genes contained PRC1 protein (Figures 4H–4J). As the BMP pathway needs to be tightly subunits in MES cells; for instance, Mel18 and Ring1B bind at regulated during early mesoderm differentiation, Mel18 could high levels to T and Mesp1 in ESCs but were displaced from their also be involved in repressing negative regulators of the pathway promoters in MES cells (Figures S5C and S5D). to ensure its proper activation and function. Indeed, we found In ESCs, Cbx7 depletion reduces the chromatin binding for that Mel18, together with Ring1B, Rybp, and Cbx2, were bound Mel18, but not Rybp (Morey et al., 2013); similarly, in Cbx2- to the promoter of four well-known negative regulators of the depleted MES cells, Mel18 binding was partially reduced, while BMP pathway (Ramel and Hill, 2012)(Figure 6A). ChIP-qPCR ex- Rybp recruitment was either not affected or even slightly periments confirmed that Ring1B and Mel18 co-occupy these increased (Morey et al., 2012)(Figure S5E). four promoters and that Ring1B recruitment was partially depen- In addition to affecting the FC25 direct target genes, Mel18 dent on Mel18 (Figure 6B). Notably, Mel18 and Ring1B displace- depletion also downregulated FC25 genes that did not contain ment in shMel18 cells resulted in upregulation of Nbl1 and Chrd Polycomb proteins, implying a secondary effect probably medi- (Figure 6C). Mechanistically, Chrd prevents BMP binding to its ated by the deregulation of FC25 genes bound by Mel18 (Fig- receptor (Larraıń et al., 2000), subsequently blocking the ure 5F, right panel); see also examples for Gata4, Lhx1, Hand1, pathway, which in turn can be reestablished when high doses Six2, and T expression after Mel18 depletion (Figure 5G; Table of BMP4 are delivered to the cells. Administrating 1 ng/ml of S1). The expression of genes containing Rybp, but not Cbx2, BMP4 to shMel18 cells induced re-expression of Flk1 and was less affected by Mel18 depletion, suggesting that the Mesp1 (Figure 6D). Moreover, the features of shMel18 MES Mel18 function was strongly associated with the cPRC1 com- EBs closely resembled those of control MES EBs (Figure 6E). plex (Figure 5F, middle panel). To establish controls, we first assured that our results were not Mel18 Depletion Blocks CM and Cartilage affected by cell heterogeneity by FACS sorting to select the Differentiation FLK1+/Pdgfa+ cell population (as differentiation of MES cells We next determined whether shMel18 MES cells can terminally in vitro is never 100% efficient—we routinely achieved 70%– differentiate into beating CMs. In accordance with our gene

Figure 5. PRC1 Complexes Bind to Highly Active, Early Cardiac-Mesoderm-Speciﬁc Genes (A) Number of deregulated genes in shMel18 MES cells crossed with Mel18 target genes. (B) Re-ChIP-qPCR performed using a Mel18 antibody or IgG. Eluted material was then used for re-ChIP using an H3K36me3 or IgG antibody. Results of the sequential-ChIP were analyzed by qPCR and are represented as a percentage of input immunoprecipitated from the Mel18 ChIP. Re-ChIP experiments represent the average of two experiments. Sox2 and T loci were used as negative controls for the Mel18-H3K36me3 re-ChIP. (C) GO analysis of Mel18 target genes downregulated or upregulated upon Mel18 depletion in MES cells. (D) Normalized Ring1B, Mel18, Cbx2, and Rybp ChIP-seq signals at genes upregulated at least by 25-fold (FC25) from ESCs to MES cells. (E) Genome-browser screenshots of Ring1B, Mel18, Cbx2, Rybp, H3K27me3, H3K36me3, and H3K4me3 occupancy at three FC25 gene loci and Gata4. (F) Expression of FC25 genes in ESCs and control and Mel18-depleted MES cells divided in genes containing Ring1B, Mel18, Cbx2, and Rybp (all PRC1); Ring1B, Mel18, and Rybp (with no Cbx2); and FC25 genes with no PRC1 subunits. (G) RNA-seq FPKM values of genes analyzed in (F), and T, in control and Mel18-depleted MES. (H) FACS-sorting proﬁle example in which FLK1+/Pdgfa+ cells were sorted. (I) ChIP-qPCR of Mel18/Ring1B target genes in sorted FLK1+/Pdgfa+ cells. T and Mesp1 promoters were used as negative control genes, and IgG antibody as a negative control, for the Mel18, Cbx2, and H3K27me3 antibodies. All ChIP experiments represent an average of two experiments. See also Figure S5.

Cell Stem Cell 17, 300–315, September 3, 2015 ª2015 Elsevier Inc. 309 A

B CD

Figure 6. A Mel18-Mediated Differentiation Block of Cardiac-Mesoderm Cells Was Partially Rescued by a High BMP4 Dose (A) Genome-browser screenshots of ChIP-seq profiles of Ring1B, Mel18, Cbx2, and Rybp in MES cells at negative regulators of the BMP pathway. (B) ChIP-qPCR of negative regulators of the BMP pathway in control and Mel18-depleted MES. All ChIP experiments represent the average of three experiments. (C) Real-time qPCR of the same genes from (A) and (B) in control and Mel18-depleted MES cells. (D) Real-time qPCR of MES-specific genes in control and Mel18-depleted MES cells induced with two BMP4 concentrations. Expression was normalized to the housekeeping gene RPO. Data represent the average of three experiments. (E) Representative pictures of control MES cells and Mel18-depleted MES cells induced to differentiate with a higher BMP4 dose. Images were taken at 20 3 magnification. expression profiles, T protein levels were unaffected in Mel18- contrast to shMel18 MES cells, more genes were upregulated depleted cells during the course of ESC differentiation into in shMel18 CMs using both cut-offs (Figures 5A and 7D). Impor- MES cells, while Mesp1 protein levels were reduced, but not tantly, downregulated genes were mainly involved in heart devel- completely abolished (Figure 7A). Mechanistically, both T and opment and function, and these were also Polycomb target Mesp1 promoters showed reduced levels of Ring1B and Mel18 genes in the heart (Figure 7E; Figure S6B). Upregulated genes after induction of MES differentiation (Figure S6A). To generate were strongly related to ESC genes and specifically, to different CM precursors and beating CMs, the EBs containing the MES cardiomyopathies (Figure 7F; Figure S6C). GO analysis of the de- cells needed to be disaggregated and plated in monolayer (Fig- regulated genes using a 5-fold cut-off revealed the same gene ure 3A). Disaggregated shMel18 MES cells attached to the categories as observed when a 2-fold cut-off was applied plate as the wild-type and could be maintained in cardiac (data not shown). media; however, they did not grow in monolayers but rather To better understand the function of the deregulated genes in formed cell clumps (Figure 7B). Strikingly, very few shMel18 shMel18 CMs, we first analyzed the ESC, MES, and CM gene cells were capable of beating (Movie S1 and Movie S2). We signatures. We defined the characteristic gene signature of observed that two contraction-associated proteins, cTnT (car- each cell type (ESC, MES, and CM) as the set of genes ex- diac troponin T) and a-actinin, were strongly reduced in Mel18- pressed (FPKM R 1) that are at least 5-fold upregulated as depleted CMs (Figure 7C). To evaluate whether these cells compared to the other cell types (Figure 7G). Analysis of genes remained undifferentiated or if Mel18 depletion resulted in cell deregulated by at least 2-fold in Mel18-depleted CMs compared fate changes, we performed RNA-seq experiments. Around to CM-specific genes revealed that 218 genes were down- 1,500 genes were deregulated by at least 2-fold, and around regulated, while 171 ESC-specific genes were upregulated (Fig- 700 genes were deregulated by at least 3-fold, in Mel18- ure 7G). In agreement with the previous analysis, downregulated depleted CMs as compared to control CMs (Figure 7D). In genes were associated with ESCs (Figure S6D), and upregulated

310 Cell Stem Cell 17, 300–315, September 3, 2015 ª2015 Elsevier Inc. genes, to muscle system, heart development, and cardiac-asso- DISCUSSION ciated diseases (Figure S6E). Real-time qPCR analysis of CM-specific proteins and tran- Here we propose Mel18 as a new epigenetic factor that scription factors confirmed that cells lacking Mel18 did not specifically controls mesoderm differentiation by at least two properly differentiate into functional CMs (Figure 7H). We also opposite mechanisms. While Mel18 functions as a classical performed the full cardiac differentiation assay using wild-type Polycomb gene in pluripotent cells, it is associated with a and Mel18 KO ESCs as a control. Indeed, Mel18 KO cells also PRC1 complex that functions as both a repressor and an failed to express several CM genes (Figure S6F), and FACS anal- activator during early cardiac mesoderm cell differentiation ysis using a cTnT antibody confirmed that the population of (Figure 7L). We propose that Mel18 directly controls the beating CMs was greatly reduced in these cells (Figure S6G). activity of genes encoding transcription factors essential for In Mel18-depleted CMs, ESC markers were strongly upregu- early cardiac mesoderm cell specification yet also directly lated, and cardiac markers, downregulated, while chondrocyte, controls the expression of the negative regulators of the neuroectoderm, endoderm, and hematopoietic genes were not BMP pathway and genes involved in late cardiac differentia- expressed, confirming that shMel18 CM cells did not change tion, pluripotency, and ectoderm and endoderm cell fate to their cell fate and are ultimately non-differentiated cells with a ensure their transcriptional repression in the early steps of ESC-like gene expression profile (Figure S6H; Table S1). Finally, cardiac differentiation. we asked whether PRC1 subunits were differentially expressed Mutant Mel18 mice have an apparently normal heart (yet die in control CMs as compared to ESCs and MES cells. Cbx4, shortly after birth, so it is not clear how long this heart is func- Phc3, and Pcgf5 were upregulated in CM cells, suggesting that tional). How can we explain this apparent inconsistency with alternative PRC1 complexes might exist in these cells (Figures our observation that Mel18 is an essential epigenetic factor S6I and S6J). implicated in CM differentiation in vitro? Inherent differences be- Because Mel18 KO mice do not display evident defects in heart tween in vitro and in vivo environments, such as local fluxes development, and as Pcgf5 is upregulated in CM cells, we hypoth- in vivo that could compensate for differentiation challenges, esized that Pcgf5 might be also involved in CM differentiation. We could be partially responsible. For instance, we observed depleted Pcgf5 from ESCs (Figure S7A) and then submitted these in vitro that increasing BMP4 rescued the differentiation defects ESCs to EB differentiation (as for Figure 2C). shPcgf5 ESCs ex- of the Mel18 mutant cells, and local variations in BMP signaling pressed normal levels of pluripotent markers and could be main- occur in vivo, during, for example, gastrulation. Additionally, tained in culture (Figure S7B). We observed that shPcgf5 EBs had another Pcgf protein could compensate for the lack of Mel18 a delay in their beating capacity, yet the effect was not as strong as during heart development in vivo. One candidate could be that observed for shMel18 ESCs (Figures S7C–S7D). These data Pcgf5, which we found to be strongly upregulated during late suggest that Pcgf5 is also involved in cardiac differentiation CM differentiation in vitro, and the depletion of which delayed in vitro. We then generated ESCs depleted for Mel18 and Pcgf5 CM differentiation. Further studies using mice with a conditional to assess whether a Pcgf5 KD would have an additive effect on Mel18 mutant in the heart or with a double Mel18/Pcgf5 mutant cells depleted for Mel18. Mel18/Pcgf5 KD ESCs can also be will shed light about the in vivo contributions of Mel18 and cultured and express normal pluripotency genes (Figures S7E– other Pcgf proteins during heart development and homeostasis S7G), and levels of PRC1 members are similar to the shMel18- of the tissue. Finally, deregulated genes in Mel18-depleted ESCs (Figure S7H). Real-time qPCR analysis of control, shMel18, CMs are related to cardiomyopathies. None of the genes previ- double KD (dKD) control cells, and Mel18/Pcgf5 dKD 12-day-old ously reported to be mutated in cardiomyopathies were deregu- EBs indicated that Pcgf5 cooperates with Mel18 during cardiac lated in Mel18-depleted CMs, suggesting that either Mel18 is a differentiation (Figures S7I and S7J). gene newly associated with heart abnormalities, or that Mel18 Finally, we examined whether the Mel18 function during differ- controls molecular pathways deregulated in heart diseases. entiation is restricted to cardiac cells or whether it is also neces- Further analyses at both the genetic and molecular levels are sary for differentiation of ESCs into other mesoderm-derived required to determine the possible role of PRC1 proteins in heart cells or cells derived from other cell lineages in vitro. We differen- diseases. tiated control and Mel18-depleted ESCs into cartilage cells Our functional and genome-wide studies suggest that Mel18 (Craft et al., 2013)(Figure 7I) and NPCs. Mel18-depleted cells directly controls several pathways important for CM differentia- were not capable of fully differentiating into cartilage cells (Fig- tion in vitro. Mel18 positively regulates expression of essential ures 7J and 7K), but efficiently differentiated into NPCs (Figures transcription factors involved in early cardiac differentiation, S7K–S7M). RNA-seq experiments in control and shMel18- suggesting a function of a Polycomb protein as a co-activator. derived NPCs indicated that around 200 and 70 genes were One possibility could be that that expression of highly active up- or downregulated by at least 2- or 3-fold, respectively, in genes containing cPRC1, ncPRC1, and PRC2 complexes is Mel18-depleted cells. Analyzing the ESC and NPC gene signa- regulated by ncRNAs (such as enhancer RNAs) that cooperate tures, we found that only 57 genes were downregulated, while with positive transcription regulators. Another characteristic of 70 ESC-specific genes were upregulated (Figure S7O). GO anal- the MES-specific highly active genes containing Polycomb ysis of these sets of genes did not show significant association to complexes was the presence of a specific breadth of the any biological process (Figure S7O). We therefore conclude that H3K4me3 domain and a particular deposition pattern of Mel18 specifically controls ESC differentiation toward meso- H3K36me3. Interestingly, the breadth of broad H3K4me3 do- derm-derived cells, but not toward other cell linages, such as mains has been recently linked to transcriptional consistency neuroectoderm precursors. at genes essential for cell identity (Benayoun et al., 2014).

Cell Stem Cell 17, 300–315, September 3, 2015 ª2015 Elsevier Inc. 311 AB C D 900 2 fold change 800 3 fold change CM shCTR CM shMel18 #72 shCTR shMel18 #72 700

shCTR shMel18 #72shCTR shMel18 #72shCTR shMel18 #72 α-actinin 600 500 T cTnT 400 300 Mesp1 Mel18 200 ESC 24h 40h (MES) GAPDH 100

Deregulated genes shMel18 Deregulated 0 CM Up Down

EFGO genes > 2 fold downregulated in CM-shMel18 GO genes > 2 fold upregulated in CM-shMel18 G Mouse Gene Atlas Mouse Gene Atlas Heart Embryonic stem cells ESC MES CM CM-shMel18 Skeletal muscle Intestine large Mammary gland Intestine small Lung 171 genes UP CM-shMel18 vs CM-shCTR Umbilical cord Placenta 7 genes DOWN -40 -30 -20 -10 0 -20 -15 -10 -5 0 CM-shMel18 vs CM-shCTR -log2 (p-value) -log2 (p-value) 845 genes Biological process Cross Species Phenotype Regulation blood circulation Muscle filament sliding Dilated cardiomyopathy Muscle system process Decreased ventricle contraction 137 genes Regulation system process Atrial fibrillation Muscle contraction Heart contraction Decreased size cardiac ventricle 22 genes UP Abnormal heart contraction CM-shMel18 vs CM-shCTR Striated muscle contraction Regulation of muscle -12 -9 -6 -3 0 218 genes DOWN -log2 (p-value) CM-shMel18 vs CM-shCTR

Actin-myosin sliding 1,066 genes

Regulation muscle contraction H Mel18 Nkx2-5 cTnT Tbx20 Tbx5 Myl2 Myl7 1.2 2500 600 300 1200 1000 1500

2000 500 800 1200 shCTR 0.9 900 400 200 shMel18 #72 1500 600 900 0.6 300 600 1000 400 600 200 100 0.3 300 200 500 100 300 Relative expression Relative 0.0 0 0 0 0 0 0 ESC CM ESC CM ESC CM ESC CM ESC CM ESC CM ESC CM

I J K Sox5 Sox9 shCTR shMel18 #72 800 14000 Embryoid bodies Monolayer Activin A 600 shCTR 10500 Wnt3a ROCKi BMP4 shMel18 no serum EBsVEGF P.SFGFb CM FGFb Cartilage ESC 400 7000 48h 40h 5 days 14 days cells 200 3500 Fold Induction Fold

0 0 L M ESC CM day 14 ESC CM day 14 PRC1 types Aggrecan Col2a1 ESC MES CM 400 800

PRC1 300 600

Late cardiac genes (Tbx5, Tbx20...) 200 400 off Lineage specific genes (Sox17...) Pluripotency genes (Sox2...) Cbx Pcgf Rybp off BMP pathway regulators (Chrd, Nog...) Mel18 Lineage genes Mel18 Induction Fold 100 200 on ESC-cPRC1 MES-cPRC1 Cardiac-MES TF (Gata4, Lhx1, Six2...) Cell differentiation 0 0 ESC CM day 14 ESC CM day 14 ESC MES ESC-like off Cardiac genes Cbx-PRC1 variants on (i.e Cbx7-PRC1 in ESCs vs Pluripotency genes Cbx2/4/8-PRC1 differentiating ESCs) Mesp1, Flk1, Pcgfa Ring1B off Cbx7 Cardiac-MES TF (Gata4, Lhx1, Six2...) Pcgf-PRC1 variants Late cardiac genes (Tbx5, Tbx20...) off/on (i.e Cbx7-Mel18-PRC1 in ESCs vs Mel18 Lineage genes Lineage specific genes (Sox17...) Cbx2-Mel18-PRC1 MES cells) Phc1 on Pluripotency genes (Sox2...) BMP pathway regulators (Chrd, Nog...) Rybp-PRC1 variants ?

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312 Cell Stem Cell 17, 300–315, September 3, 2015 ª2015 Elsevier Inc. Detailed analyses of these multivalent regions warrant further were performed as previously described (Craft et al., 2013; Kattman et al., investigation. 2011) with the following modifications. In our hands, generation of a highly en- It has been recently proposed that a PRC1 variant containing riched population of MES cells was strongly influenced both by the dose and the batch of BMP4. Typically, we obtained the best differentiation results with AUST2 and a phosphorylated form of Ring1B, but no Cbx pro- 0.1–0.3 ng/ml of BMP4. We also noticed that the amount of MES cells seeded teins, binds to active genes in neurons (Gao et al., 2014). There- to obtain cardiac precursor cells was crucial to obtain a high percentage of fore, another possibility could be that the PRC1 complexes that beating CMs, with the best results obtained when 125,000–150,000 MES cells regulate gene activity in MES cells also contain a post-transcrip- were seeded in a 96-well plate. For cartilage differentiation, we also titrated the tionally modified Ring1B (or other subunit). Due to the lack of amount of BMP4 and the number of cells; we obtained the best differentiation specific antibodies against the CK2-mediated Ring1B phos- using 5 ng/ml of BMP4 and seeding 3,000 cells in a 96-well plate. phorylated form, we could not assess whether this is the case in our cellular system. ACCESSION NUMBERS Could post-transcriptional modifications of Polycomb pro- All the databases generated in this paper have been deposited in the NCBI teins be involved in tuning the enzymatic activity of the GEO under the accession number GEO: GSE67868. Polycomb complexes? We suggest that specific PRC1 complexes (Figure 7M) might cooperate with specific molecules, SUPPLEMENTAL INFORMATION in different cell types and biological process, to regulate and modulate Polycomb activities. Additionally, the involvement Supplemental Information for this article includes seven figures, Supplemental of Mel18 in CM differentiation makes it also worth investiga- Experimental Procedures, one table, and two movies and can be found with ting whether any disturbances in this mechanism underlie this article online at http://dx.doi.org/10.1016/j.stem.2015.08.009. cardiomyopathies. AUTHOR CONTRIBUTIONS EXPERIMENTAL PROCEDURES L.M. designed the study and conducted all the experiments, except the carti- Generation of Stable ESC KD Cultures lage differentiation experiments (carried out by A.S.) and the NPC experiments ESCs expressing stable pLKO-shRNA (Sigma) vectors against Mel18, Phc1, (conducted by L.A and A.S.). Mel18 KO ESCs lines were generated by E.P.N. in and Pcgf5 mRNAs were generated as previously described (Morey et al., the lab of B.G.B., who was supported by a grant from NHLBI Bench to 2013). To generate Mel18/Pcgf5 dKD cells (dKD ESCs), shMel18 ESCs were Bassinet Program U01HL098179 to B.G.B. and fellowships from European transduced with lentiviruses expressing a shPcgf5-Hygromycin vector. We Molecular Biology Organization (ALTF523-2013) and Human Frontier Science also transduced the shCTR-ESCs with a pLKO-Hygromycin-control vector Program to E.P.N. E.B. performed the bioinformatics analysis. A.S. and E.B. to generate ESCs resistant to puromycin and hygromycin (dKD control ESCs). contributed equally. L.D.C. supervised the experiments and provided intellec- tual support toward design and interpretation of the results. L.M. and L.D.C. ChIP and Re-ChIP Assays wrote the manuscript. ChIP and re-ChIP experiments in ESCs were performed as previously described (Morey et al., 2013). ChIP assays in MES cells were performed ACKNOWLEDGMENTS with the following modifications: MES cells were centrifuged 3 min at 800 rpm to discard cells that did not form EBs. EBs were trypsinized with We are indebted to V.A. Raker for help in preparing the manuscript, members TrypLE Express and then crosslinked, lysated, and sonicated as described of the Di Croce laboratory for discussions, the CRG Genomic Unit, the CRG for ESC ChIPs. Antibodies and primers used for ChIP-qPCR are given in Advanced Light Microscopy Unit, and the CRG Proteomics Unit. We also Table S1. thank John Wylie and Dr. April Craft for help with the cardiomyocyte and cartilage differentiation protocols, respectively. This work was supported by grants Differentiation Assays from the Spanish ‘‘Ministerio de Educacio´ n y Ciencia’’ (SAF2013-48926-P), ESCs were differentiated into EBs and NPCs as previously described (Aloia from AGAUR, from Fundacio´ ‘‘La Marato´ de TV3,’’ and from the European et al., 2014; Morey et al., 2012). CMs and cartilage differentiation assays Commission’s 7th Framework Program 4DCellFate (277899).

Figure 7. Generation of CMs and Cartilage Cells Was Impaired in Cells Lacking Mel18 (A) Western blot of T and Mesp1 in ESCs and during their differentiation to MES cells in control and Mel18-depleted cells. (B) Representative pictures of control CM and the resulting Mel18-depleted cells at day 10 of differentiation. Images were taken at 203 magnification. (C) Western blot of a-actinin and cTnT at day 10 of CM differentiation in control and Mel18-depleted cells. (D) Deregulated genes in Mel18-depleted CM. (E and F) GO analysis of genes downregulated (E) and upregulated (F) by at least 2-fold in shMel18-CMs. (G) Heat map of ESCs, MES, and CM-specific genes and genes deregulated in shMel18 CMs as compared to shControl CMs (see text for details). (H) Real-time qPCR of Mel18 and CM-specific genes in control and Mel18-depleted cells at day 10 of differentiation. Data represent the average of four experiments. (I) Schematic representation of the protocol used to differentiate ESCs to cartilage cells (Craft et al., 2013). (J) Representative pictures of control Mel18-depleted cells at day 14 of differentiation. Images were taken at 203 magnification. (K) Real-time qPCR of cartilage-specific genes in control and Mel18-depleted cells at day 14 of differentiation. Data represent the average of three experiments. (L) Proposed model for Mel18 function during cardiac differentiation. In ESCs, cPRC1 complexes containing Mel18 represses lineage genes. When cells are induced to differentiate into early MES cells, a new cPRC1 complex is assembled. To ensure proper differentiation, this new cPRC1 complex positively regulates expression of mesoderm transcription factors and negatively regulates pluripotency genes, lineage genes, late cardiac differentiation genes, and negative regulators of the BMP pathway. Mel18 depletion results in a disassembly of the cPRC1 complex in ESCs, which is followed by an aberrant expression of pluripotency genes, ectoderm genes, and negative BMP regulators. Reduced expression of mesoderm transcription factors leads to an impairment in late cardiac differentiation. (M) PRC1 complexes are characterized by the presence of Cbx, Pcgf, and Rybp. We propose that during differentiation and disease, different variationsof Polycomb complexes are assembled, thereby allowing a higher level of fine-tuning of gene expression.

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