Functional Proteomic Analysis of Repressive Histone Methyltransferase Complexes Reveals ZNF518B as a G9A Regulator*

Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP.

[1]  B. Monsarrat,et al.  Comparison of label-free quantification methods for the determination of protein complexes subunits stoichiometry , 2014 .

[2]  Jeannie T. Lee,et al.  Regulatory interactions between RNA and polycomb repressive complex 2. , 2014, Molecular cell.

[3]  A. Singh,et al.  EZH2: Not EZHY (Easy) to Deal , 2014, Molecular Cancer Research.

[4]  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.

[5]  R. Margueron,et al.  The histone H3 lysine 9 methyltransferases G9a and GLP regulate polycomb repressive complex 2-mediated gene silencing. , 2014, Molecular cell.

[6]  A. Giordano,et al.  Roles of enhancer of zeste homolog 2: From skeletal muscle differentiation to rhabdomyosarcoma carcinogenesis , 2014, Cell cycle.

[7]  Jeannie T. Lee,et al.  Tsix RNA and the germline factor, PRDM14, link X reactivation and stem cell reprogramming. , 2013, Molecular cell.

[8]  S. Carr,et al.  EMRE Is an Essential Component of the Mitochondrial Calcium Uniporter Complex , 2013, Science.

[9]  Daniel R. Zerbino,et al.  Ensembl 2014 , 2013, Nucleic Acids Res..

[10]  A. H. Smits,et al.  Cdyl, a New Partner of the Inactive X Chromosome and Potential Reader of H3K27me3 and H3K9me2 , 2013, Molecular and Cellular Biology.

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

[12]  Jacob D. Jaffe,et al.  Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia , 2013, Nature Genetics.

[13]  Alexander Schmidt,et al.  Critical assessment of proteome‐wide label‐free absolute abundance estimation strategies , 2013, Proteomics.

[14]  Nancy A. Jenkins,et al.  EVI1 oncoprotein interacts with a large and complex network of proteins and integrates signals through protein phosphorylation , 2013, Proceedings of the National Academy of Sciences.

[15]  Steven A Carr,et al.  Integrated proteomic analysis of post-translational modifications by serial enrichment , 2013, Nature Methods.

[16]  Jonathan Göke,et al.  A PRC2‐Dependent Repressive Role of PRDM14 in Human Embryonic Stem Cells and Induced Pluripotent Stem Cell Reprogramming , 2013, Stem cells.

[17]  N. Brockdorff.  Noncoding RNA and Polycomb recruitment. , 2013, RNA.

[18]  K. Shirahige,et al.  PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells. , 2013, Cell stem cell.

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

[20]  D. Zheng,et al.  An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting. , 2013, Molecular cell.

[21]  R. Taneja,et al.  G9a, a multipotent regulator of gene expression , 2013, Epigenetics.

[22]  Nir Hacohen,et al.  Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach , 2012, Nature Immunology.

[23]  Hana Kim,et al.  Recruitment and biological consequences of histone modification of H3K27me3 and H3K9me3. , 2012, ILAR journal.

[24]  J. Whetstine,et al.  Histone lysine methylation dynamics: establishment, regulation, and biological impact. , 2012, Molecular cell.

[25]  Damian Szklarczyk,et al.  STRING v9.1: protein-protein interaction networks, with increased coverage and integration , 2012, Nucleic Acids Res..

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

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

[28]  Jian Ye,et al.  Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction , 2012, BMC Bioinformatics.

[29]  Jacob D. Jaffe,et al.  Methods for Quantification of in vivo Changes in Protein Ubiquitination following Proteasome and Deubiquitinase Inhibition* , 2012, Molecular & Cellular Proteomics.

[30]  H. Ouyang,et al.  Structural Basis of the Chromodomain of Cbx3 Bound to Methylated Peptides from Histone H1 and G9a , 2012, PloS one.

[31]  Jing Liang,et al.  Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27* , 2011, The Journal of Biological Chemistry.

[32]  J. Rinn,et al.  lincRNAs act in the circuitry controlling pluripotency and differentiation , 2011, Nature.

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

[34]  Y. Shinkai,et al.  H3K9 methyltransferase G9a and the related molecule GLP. , 2011, Genes & development.

[35]  M. Mann,et al.  Extracting gene function from protein-protein interactions using Quantitative BAC InteraCtomics (QUBIC). , 2011, Methods.

[36]  M. Mann,et al.  Andromeda: a peptide search engine integrated into the MaxQuant environment. , 2011, Journal of proteome research.

[37]  Paul Tempst,et al.  PRC2 Complexes with JARID2, MTF2, and esPRC2p48 in ES Cells to Modulate ES Cell Pluripotency and Somatic Cell Reprograming , 2011, Stem cells.

[38]  Toshiro K. Ohsumi,et al.  Genome-wide identification of polycomb-associated RNAs by RIP-seq. , 2010, Molecular cell.

[39]  B. Bernstein,et al.  Mammalian Polycomb-Like Pcl2/Mtf2 Is a Novel Regulatory Component of PRC2 That Can Differentially Modulate Polycomb Activity both at the Hox Gene Cluster and at Cdkn2a Genes , 2010, Molecular and Cellular Biology.

[40]  H. Kimura,et al.  Human POGZ modulates dissociation of HP1α from mitotic chromosome arms through Aurora B activation , 2010, Nature Cell Biology.

[41]  Henning Urlaub,et al.  Determination of protein stoichiometry within protein complexes using absolute quantification and multiple reaction monitoring. , 2010, Analytical chemistry.

[42]  Juri Rappsilber,et al.  JARID2 regulates binding of the Polycomb repressive complex 2 to target genes in ES cells , 2010, Nature.

[43]  Gang Li,et al.  Jarid2 and PRC2, partners in regulating gene expression. , 2010, Genes & development.

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

[45]  Arend Sidow,et al.  Jarid2/Jumonji Coordinates Control of PRC2 Enzymatic Activity and Target Gene Occupancy in Pluripotent Cells , 2009, Cell.

[46]  D. Shippen,et al.  Conserved telomere maintenance component 1 interacts with STN1 and maintains chromosome ends in higher eukaryotes. , 2009, Molecular cell.

[47]  F. Mackenzie,et al.  Structural Studies of a Four-MBT Repeat Protein MBTD1 , 2009, PloS one.

[48]  Jonghwan Kim,et al.  Use of in vivo biotinylation to study protein–protein and protein–DNA interactions in mouse embryonic stem cells , 2009, Nature Protocols.

[49]  M. Walsh,et al.  The ZNF217 oncogene is a candidate organizer of repressive histone modifiers , 2009, Epigenetics.

[50]  S. Jacobsen,et al.  UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells , 2008, Nucleic acids research.

[51]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[52]  Guo-Cheng Yuan,et al.  EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. , 2008, Molecular cell.

[53]  H. Cedar,et al.  De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes , 2008, Nature Structural &Molecular Biology.

[54]  F. Boisvert,et al.  Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes , 2008, The Journal of cell biology.

[55]  Y. Shinkai,et al.  Identification of ZNF200 as a novel binding partner of histone H3 methyltransferase G9a , 2007, Genes to cells : devoted to molecular & cellular mechanisms.

[56]  M. Fraga,et al.  The Polycomb group protein EZH2 directly controls DNA methylation , 2007, Nature.

[57]  O. Gangisetty,et al.  Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication. , 2006, Genes & development.

[58]  Nir Hacohen,et al.  Genome-scale loss-of-function screening with a lentiviral RNAi library , 2006, Nature Methods.

[59]  Y. Shinkai,et al.  Zinc Finger Protein Wiz Links G9a/GLP Histone Methyltransferases to the Co-repressor Molecule CtBP* , 2006, Journal of Biological Chemistry.

[60]  Cyrus Martin,et al.  Substrate Preferences of the EZH2 Histone Methyltransferase Complex* , 2006, Journal of Biological Chemistry.

[61]  M. Nakao,et al.  Transcriptional Repression and Heterochromatin Formation by MBD1 and MCAF/AM Family Proteins* , 2005, Journal of Biological Chemistry.

[62]  Tsutomu Ohta,et al.  Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. , 2005, Genes & development.

[63]  K. Luger,et al.  H2A.Z alters the nucleosome surface to promote HP1alpha-mediated chromatin fiber folding. , 2004, Molecular cell.

[64]  Kristian Helin,et al.  Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity , 2004, The EMBO journal.

[65]  Paul Tempst,et al.  Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3. , 2004, Molecular cell.

[66]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[67]  Ya-Li Yao,et al.  The Metastasis-associated Proteins 1 and 2 Form Distinct Protein Complexes with Histone Deacetylase Activity* , 2003, Journal of Biological Chemistry.

[68]  Yang Shi,et al.  Coordinated histone modifications mediated by a CtBP co-repressor complex , 2003, Nature.

[69]  Blagoy Blagoev,et al.  A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling , 2003, Nature Biotechnology.

[70]  H. Kato,et al.  G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. , 2002, Genes & development.

[71]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[72]  R. Jaenisch,et al.  Antisense Transcription through theXist Locus Mediates Tsix Function in Embryonic Stem Cells , 2001, Molecular and Cellular Biology.

[73]  Weidong Wang,et al.  NURD, a novel complex with both ATP-dependent chromatin-remodeling and histone deacetylase activities. , 1998, Molecular cell.

[74]  Hana Kim,et al.  AEBP2 as a potential targeting protein for Polycomb Repression Complex PRC2 , 2009 .

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

[76]  John H. White,et al.  Ligand-dependent nuclear receptor corepressor LCoR functions by histone deacetylase-dependent and -independent mechanisms. , 2003, Molecular cell.

[77]  M. Surani,et al.  The Polycomb-Group Gene Ezh2 Is Required for Early Mouse Development , 2001 .

[78]  Brendan MacLean,et al.  Bioinformatics Applications Note Gene Expression Skyline: an Open Source Document Editor for Creating and Analyzing Targeted Proteomics Experiments , 2022 .

[79]  A. Bird,et al.  Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins , 2022 .