Abstract:Polycomb group (PcG) proteins are major determinants of gene silencing and epigenetic memory in higher eukaryotes. Here, we systematically mapped the human PcG complexome using a robust affinity purification mass spectrometry approach. Our high-density protein interaction network uncovered a diverse range of PcG complexes. Moreover, our analysis identified PcG interactors linking them to the PcG system, thus providing insight into the molecular function of PcG complexes and mechanisms of recruitment to target … Show more
“…To address the composition of PCGF6 complexes in mouse ESCs, we stably expressed an epitope-tagged form of PCGF6 in mouse ESCs and affinity purified it from nuclear extracts, then used LC-MS/MS analysis to identify associated proteins. We observed strong association of PCGF6 with MGA, RING1B, RING1A, CBX3, CBX1, RYBP, L3MBTL2, YAF2 and TFDP1 (Figure 1A,B), indicating that the mouse ESC PCGF6 complex is similar to those purified from human cells (Gao et al, 2012; Hauri et al, 2016; Kloet et al, 2016; Ogawa et al, 2002; Trojer et al, 2011). We however did not detect considerable amounts of MAX in the PCGF6 complexes in mouse ESCs.10.7554/eLife.21064.002Figure 1.Biochemical properties of PCGF6-PRC1 and its target genes in ESCs.( A ) Affinity purification of PCGF6-containing complexes in ESCs.…”
Section: Resultsmentioning
confidence: 82%
“…Consistent with this notion, we observed considerable overlap between PCGF6-bound and MAX-bound genes, while the overlap between PCGF6-bound and MYC-bound genes was much less (Figure 4A). Interestingly, MGA, a transcription factor that also forms a heterodimer with MAX and binds the CACGTG E-box motif (Hurlin et al, 1999), is included in the PCGF6 complex determined by us (Figure 1B) and others (Gao et al, 2012; Hauri et al, 2016; Kloet et al, 2016; Ogawa et al, 2002; Qin et al, 2012; Trojer et al, 2011). Therefore, we hypothesized that MAX/MGA heterodimer could play a role in PCGF6-PRC1 recruitment.10.7554/eLife.21064.009Figure 4.The role of MAX/MGA in recruiting PCGF6-PRC1 to its target genes.( A ) Considerable overlap of genes bound by PCGF6 and MAX.…”
Section: Resultsmentioning
confidence: 83%
“…Previous proteomic approaches have repeatedly identified PCGF6 as a component of multimeric protein complexes designated as PCGF6-PRC1 that included MAX, MGA, E2F6, TFDP1, RING1B, RING1A, CBX3, RYBP, L3MBTL2, YAF2 and WDR5 in human cell lines (Gao et al, 2012; Hauri et al, 2016; Ogawa et al, 2002; Trojer et al, 2011). To address the composition of PCGF6 complexes in mouse ESCs, we stably expressed an epitope-tagged form of PCGF6 in mouse ESCs and affinity purified it from nuclear extracts, then used LC-MS/MS analysis to identify associated proteins.…”
Section: Resultsmentioning
confidence: 99%
“…This complex was annotated as PCGF6-PRC1, and was shown to form stable complexes with several other well-known epigenetic factors such as RING1A/B, PCGF6, L3MBTL2 [lethal(3)malignant brain tumor-like 2], RYBP, YAF2, G9A (also known as EHMT2: euchromatic histone-lysine N-methyltransferase 2), GLP (G9a-like protein 1, also known as EHMT1), and CBX1/3 (Gao et al, 2012; Hauri et al, 2016; Kloet et al, 2016; Ogawa et al, 2002; Qin et al, 2012; Trojer et al, 2011). Interestingly, PCGF6-PRC1 also interacts with sequence-specific DNA binding proteins such as E2F6, MAX, MGA and TFDP1 (transcription factor Dp-1) (Gao et al, 2012; Hauri et al, 2016; Kloet et al, 2016; Ogawa et al, 2002; Qin et al, 2012; Trojer et al, 2011). This suggests that such DNA binding proteins could play a role in sequence specific recruitment of PCGF6-PRC1 to target loci; however, this notion has not been experimentally validated.…”
The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.DOI:
http://dx.doi.org/10.7554/eLife.21064.001
“…To address the composition of PCGF6 complexes in mouse ESCs, we stably expressed an epitope-tagged form of PCGF6 in mouse ESCs and affinity purified it from nuclear extracts, then used LC-MS/MS analysis to identify associated proteins. We observed strong association of PCGF6 with MGA, RING1B, RING1A, CBX3, CBX1, RYBP, L3MBTL2, YAF2 and TFDP1 (Figure 1A,B), indicating that the mouse ESC PCGF6 complex is similar to those purified from human cells (Gao et al, 2012; Hauri et al, 2016; Kloet et al, 2016; Ogawa et al, 2002; Trojer et al, 2011). We however did not detect considerable amounts of MAX in the PCGF6 complexes in mouse ESCs.10.7554/eLife.21064.002Figure 1.Biochemical properties of PCGF6-PRC1 and its target genes in ESCs.( A ) Affinity purification of PCGF6-containing complexes in ESCs.…”
Section: Resultsmentioning
confidence: 82%
“…Consistent with this notion, we observed considerable overlap between PCGF6-bound and MAX-bound genes, while the overlap between PCGF6-bound and MYC-bound genes was much less (Figure 4A). Interestingly, MGA, a transcription factor that also forms a heterodimer with MAX and binds the CACGTG E-box motif (Hurlin et al, 1999), is included in the PCGF6 complex determined by us (Figure 1B) and others (Gao et al, 2012; Hauri et al, 2016; Kloet et al, 2016; Ogawa et al, 2002; Qin et al, 2012; Trojer et al, 2011). Therefore, we hypothesized that MAX/MGA heterodimer could play a role in PCGF6-PRC1 recruitment.10.7554/eLife.21064.009Figure 4.The role of MAX/MGA in recruiting PCGF6-PRC1 to its target genes.( A ) Considerable overlap of genes bound by PCGF6 and MAX.…”
Section: Resultsmentioning
confidence: 83%
“…Previous proteomic approaches have repeatedly identified PCGF6 as a component of multimeric protein complexes designated as PCGF6-PRC1 that included MAX, MGA, E2F6, TFDP1, RING1B, RING1A, CBX3, RYBP, L3MBTL2, YAF2 and WDR5 in human cell lines (Gao et al, 2012; Hauri et al, 2016; Ogawa et al, 2002; Trojer et al, 2011). To address the composition of PCGF6 complexes in mouse ESCs, we stably expressed an epitope-tagged form of PCGF6 in mouse ESCs and affinity purified it from nuclear extracts, then used LC-MS/MS analysis to identify associated proteins.…”
Section: Resultsmentioning
confidence: 99%
“…This complex was annotated as PCGF6-PRC1, and was shown to form stable complexes with several other well-known epigenetic factors such as RING1A/B, PCGF6, L3MBTL2 [lethal(3)malignant brain tumor-like 2], RYBP, YAF2, G9A (also known as EHMT2: euchromatic histone-lysine N-methyltransferase 2), GLP (G9a-like protein 1, also known as EHMT1), and CBX1/3 (Gao et al, 2012; Hauri et al, 2016; Kloet et al, 2016; Ogawa et al, 2002; Qin et al, 2012; Trojer et al, 2011). Interestingly, PCGF6-PRC1 also interacts with sequence-specific DNA binding proteins such as E2F6, MAX, MGA and TFDP1 (transcription factor Dp-1) (Gao et al, 2012; Hauri et al, 2016; Kloet et al, 2016; Ogawa et al, 2002; Qin et al, 2012; Trojer et al, 2011). This suggests that such DNA binding proteins could play a role in sequence specific recruitment of PCGF6-PRC1 to target loci; however, this notion has not been experimentally validated.…”
The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.DOI:
http://dx.doi.org/10.7554/eLife.21064.001
“…Polycomb-like proteins (PCLs), including PHF1, MTF2 and PHF19, are PRC2 associated factors that form sub-complexes with PRC2 core components 3 , and have been proposed to modulate PRC2’s enzymatic activity or its recruitment to specific genomic loci 4–13 . Mammalian PRC2 binding sites are enriched in CG content, which correlate with CpG islands that display a low level of DNA methylation 14 .…”
The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression1,2 and plays essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like proteins (PCLs), including PHF1, MTF2 and PHF19, are PRC2 associated factors that form sub-complexes with PRC2 core components3, and have been proposed to modulate PRC2’s enzymatic activity or its recruitment to specific genomic loci4–13. Mammalian PRC2 binding sites are enriched in CG content, which correlate with CpG islands that display a low level of DNA methylation14. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. In this study, we solved the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We found that the extended homologous (EH) regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a manner completely different from the canonical winged-helix motif-DNA recognition. We further showed that the PCL EH domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic cells. Our research provides the first direct evidence demonstrating that PCLs are critical for PRC2 recruitment to CpG islands, thereby further clarifying their roles in transcriptional regulation in vivo.
Almost 100 years after the first descriptions of proteins conjugated to carbohydrates (mucins), several studies suggested that glycoproteins were not restricted to the serum, extracellular matrix, cell surface, or endomembrane system. In the 1980’s, key data emerged demonstrating that intracellular proteins were modified by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc). Subsequently, this modification was identified on thousands of proteins that regulate cellular processes as diverse as protein aggregation, localization, post-translational modifications, activity, and interactions. In this Review, we will highlight critical discoveries that shaped our understanding of the molecular events underpinning the impact of O-GlcNAc on protein function, the role that O-GlcNAc plays in maintaining cellular homeostasis, and our understanding of the mechanisms that regulate O-GlcNAc-cycling.
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