1. Volume 17, Issue 3, Pages 300-315 (September 2015)
Polycomb Regulates Mesoderm Cell Fate-Specification in Embryonic Stem Cells through Activation and Repression Mechanisms 
Lluis Morey, Alexandra Santanach, Enrique Blanco, Luigi Aloia, Elphège P. Nora, Benoit G. Bruneau, Luciano Di Croce 
Cell Stem Cell 
Volume 17, Issue 3, Pages (September 2015)
DOI: /j.stem
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2. Cell Stem Cell 2015 17, 300-315DOI: (10.1016/j.stem.2015.08.009)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. 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.
(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 as a 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  , DOI: ( /j.stem )
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4. 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 20× magnification at days 2, 4, 6, 10, and 12 and also at 50× 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.
Cell Stem Cell  , DOI: ( /j.stem )
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5. 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 20× magnification.
(C) FACS analysis using FLK1 and Pdgfα 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.
Cell Stem Cell  , DOI: ( /j.stem )
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6. 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.
(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.
Cell Stem Cell  , DOI: ( /j.stem )
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7. 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+/Pdgfα+ cells were sorted.
(I) ChIP-qPCR of Mel18/Ring1B target genes in sorted FLK1+/Pdgfα+ 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  , DOI: ( /j.stem )
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8. 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 × magnification.
Cell Stem Cell  , DOI: ( /j.stem )
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9. 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 20× magnification.
(C) Western blot of α-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 20× 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 variations of Polycomb complexes are assembled, thereby allowing a higher level of fine-tuning of gene expression.
Cell Stem Cell  , DOI: ( /j.stem )
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