The dynamic interactome and genomic targets of Polycomb complexes during stem cell differentiation

Although the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found that the stoichiometry and genome-wide binding of PRC1 and PRC2 were highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from chromatin marked with trimethylated histone H3 K27 (H3K27me3) during differentiation, whereas PRC1 was retained at these sites. Additionally, we found PRC1 at enhancer and promoter regions independently of PRC2 binding and H3K27me3. Finally, overexpression of NPC-specific PRC1 interactors in ESCs led to increased Ring1b binding to, and decreased expression of, NPC-enriched Ring1b-target genes. In summary, our integrative analyses uncovered dynamic PcG subcomplexes and their widespread colocalization with active chromatin marks during differentiation.

[1]  Esther Willems,et al.  Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification* , 2015, Molecular & Cellular Proteomics.

[2]  J. Rinn,et al.  Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs , 2010, Nature biotechnology.

[3]  S. Orkin,et al.  Jumonji Modulates Polycomb Activity and Self-Renewal versus Differentiation of Stem Cells , 2009, Cell.

[4]  Juri Rappsilber,et al.  A model for transmission of the H3K27me3 epigenetic mark , 2008, Nature Cell Biology.

[5]  D. Duboule,et al.  Chromatin organization and global regulation of Hox gene clusters , 2013, Philosophical Transactions of the Royal Society B: Biological Sciences.

[6]  M. Vidal,et al.  Role of histone H2A ubiquitination in Polycomb silencing , 2004, Nature.

[7]  Colin W. Combe,et al.  xiNET: Cross-link Network Maps With Residue Resolution , 2015, Molecular & Cellular Proteomics.

[8]  Ruedi Aebersold,et al.  Molecular architecture of human polycomb repressive complex 2 , 2012, eLife.

[9]  K. Helin,et al.  Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide. , 2014, Molecular cell.

[10]  H. Stunnenberg,et al.  Non-canonical PRC1.1 Targets Active Genes Independent of H3K27me3 and Is Essential for Leukemogenesis. , 2016, Cell reports.

[11]  B. Bruneau,et al.  Polycomb Regulates Mesoderm Cell Fate-Specification in Embryonic Stem Cells through Activation and Repression Mechanisms. , 2015, Cell stem cell.

[12]  Jürg Müller,et al.  Transcriptional repression by PRC1 in the absence of H2A monoubiquitylation , 2015, Genes & development.

[13]  M. Vermeulen,et al.  Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression , 2014, Nature Structural &Molecular Biology.

[14]  Anthony A. Hyman,et al.  Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics , 2012, Nucleic acids research.

[15]  D. Reinberg,et al.  An AUTS2–Polycomb complex activates gene expression in the CNS , 2014, Nature.

[16]  M. Mann,et al.  Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips , 2007, Nature Protocols.

[17]  Hengbin Wang,et al.  Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing , 2002, Science.

[18]  Kristian Helin,et al.  Chromatin repressive complexes in stem cells, development, and cancer. , 2014, Cell stem cell.

[19]  ENCODEConsortium,et al.  An Integrated Encyclopedia of DNA Elements in the Human Genome , 2012, Nature.

[20]  T. Mikkelsen,et al.  Genome-wide maps of chromatin state in pluripotent and lineage-committed cells , 2007, Nature.

[21]  Karl Mechtler,et al.  BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals , 2008, Nature Methods.

[22]  A. H. Smits,et al.  Dynamic Readers for 5-(Hydroxy)Methylcytosine and Its Oxidized Derivatives , 2013, Cell.

[23]  A. H. Smits,et al.  Characterizing Protein-Protein Interactions Using Mass Spectrometry: Challenges and Opportunities. , 2016, Trends in biotechnology.

[24]  Haruhiko Koseki,et al.  Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. , 2004, Developmental cell.

[25]  Robert S Illingworth,et al.  The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development , 2015, Genes & development.

[26]  Jacob D. Jaffe,et al.  Functional Proteomic Analysis of Repressive Histone Methyltransferase Complexes Reveals ZNF518B as a G9A Regulator* , 2015, Molecular & Cellular Proteomics.

[27]  N. Brockdorff,et al.  Variant PRC1 Complex-Dependent H2A Ubiquitylation Drives PRC2 Recruitment and Polycomb Domain Formation , 2014, Cell.

[28]  L. Szekely,et al.  Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts. , 1994, Oncogene.

[29]  P. Defossez,et al.  MBD5 and MBD6 interact with the human PR‐DUB complex through their methyl‐CpG‐binding domain , 2014, Proteomics.

[30]  Natalie I. Tasman,et al.  A Cross-platform Toolkit for Mass Spectrometry and Proteomics , 2012, Nature Biotechnology.

[31]  Robert E. Kingston,et al.  Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. , 2013, Molecular cell.

[32]  M. Wilm,et al.  Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB , 2010, Nature.

[33]  Renato Paro,et al.  A chromatin-modifying function of JNK during stem cell differentiation , 2011, Nature Genetics.

[34]  Y. Schwartz,et al.  A new world of Polycombs: unexpected partnerships and emerging functions , 2013, Nature Reviews Genetics.

[35]  D. Reinberg,et al.  PRC2 binds to active promoters and contacts nascent RNAs in embryonic stem cells , 2013, Nature Structural &Molecular Biology.

[36]  J. Rinn,et al.  Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs , 2010, Nature Biotechnology.

[37]  Youngchang Kim,et al.  Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. , 2003, Genes & development.

[38]  L. Cozzuto,et al.  Nonoverlapping functions of the Polycomb group Cbx family of proteins in embryonic stem cells. , 2012, Cell stem cell.

[39]  Brigitte Wild,et al.  Histone Methyltransferase Activity of a Drosophila Polycomb Group Repressor Complex , 2002, Cell.

[40]  M. Dong,et al.  Identification of cross-linked peptides from complex samples , 2012, Nature Methods.

[41]  K. Helin,et al.  Transcriptional regulation by Polycomb group proteins , 2013, Nature Structural &Molecular Biology.

[42]  Edith Heard,et al.  MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation , 2012, Cell stem cell.

[43]  Yuval Kluger,et al.  PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes. , 2012, Molecular cell.

[44]  P. Kharchenko,et al.  Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification , 2014, Proceedings of the National Academy of Sciences.

[45]  A. Diallo,et al.  Investigation of genes important in neurodevelopment disorders in adult human brain , 2015, Human Genetics.

[46]  Cory Y. McLean,et al.  GREAT improves functional interpretation of cis-regulatory regions , 2010, Nature Biotechnology.

[47]  Austin G Smith,et al.  Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell , 2005, PLoS biology.

[48]  M. Selbach,et al.  Global quantification of mammalian gene expression control , 2011, Nature.

[49]  J. Min,et al.  Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27. , 2003, Genes & development.

[50]  N. Brockdorff,et al.  Chromatin Sampling—An Emerging Perspective on Targeting Polycomb Repressor Proteins , 2013, PLoS genetics.

[51]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.