title: Functional Proteomic Analysis of Repressive Histone Methyltransferase Complexes Reveals ZNF518B as a G9A Regulator.
abstract: 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.
authors: From the ‡Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, 185 Cambridge Street, Boston, Massachusetts 02143;
authors: Verena K
authors: VK
authors: Maier
authors: §Proteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142;
authors: Caitlin M
authors: CM
authors: Feeney
authors: §Proteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142;
authors: Jordan E
authors: JE
authors: Taylor
authors: §Proteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142;
authors: Amanda L
authors: AL
authors: Creech
authors: §Proteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142;
authors: Jana W
authors: JW
authors: Qiao
authors: From the ‡Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, 185 Cambridge Street, Boston, Massachusetts 02143;
authors: Attila
authors: A
authors: Szanto
authors: ¶Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, 02115;
authors: Partha P
authors: PP
authors: Das
authors: ‖FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts 02138.
authors: Nicholas
authors: N
authors: Chevrier
authors: From the ‡Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, 185 Cambridge Street, Boston, Massachusetts 02143;
authors: Catherine
authors: C
authors: Cifuentes-Rojas
authors: ¶Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, 02115;
authors: Stuart H
authors: SH
authors: Orkin
authors: §Proteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142;
authors: Steven A
authors: SA
authors: Carr
authors: §Proteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142;
authors: Jacob D
authors: JD
authors: Jaffe
authors: §Proteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142; pmertins@broadinstitute.org lee@molbio.mgh.harvard.edu.
authors: Philipp
authors: P
authors: Mertins
authors: From the ‡Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, 185 Cambridge Street, Boston, Massachusetts 02143;
authors: Jeannie T
authors: JT
authors: Lee
mesh_terms: D000818:Animals; D053595:Embryonic Stem Cells / Q000378:metabolism; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D051379:Mice; D040901:Proteomics; D016335:Zinc Fingers / Q000502:physiology*
chemical_list: C533711:G9a protein, mouse; C513479:GLP protein, mouse; D011495:Histone-Lysine N-Methyltransferase