Maier Verena K, Feeney Caitlin M, Taylor Jordan E, Creech Amanda L, Qiao Jana W, Szanto Attila, Das Partha P, Chevrier Nicholas, Cifuentes-Rojas Catherine, Orkin Stuart H, Carr Steven A, Jaffe Jacob D, Mertins Philipp, Lee Jeannie T
From the ‡Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, 185 Cambridge Street, Boston, Massachusetts 02143;
§Proteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, Massachusetts 02142;
Mol Cell Proteomics. 2015 Jun;14(6):1435-46. doi: 10.1074/mcp.M114.044586. Epub 2015 Feb 13.
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.
由组蛋白H3赖氨酸27和赖氨酸9甲基转移酶复合物PRC2和G9A-GLP介导的细胞类型特异性基因沉默在发育过程中和维持细胞特性方面都至关重要。尽管对它们的相互作用伙伴的研究为其功能提供了有价值的见解，但这些因子在网络水平上是如何被调控的仍未完全了解。在这里，我们提出了一种新方法，将定量相互作用蛋白质组学与全局染色质分析相结合，以在功能上表征胚胎干细胞中抑制性染色质修饰蛋白复合物。我们分别确定了PRC2的9个新的和12个已知的相互作用伙伴以及G9A-GLP的10个已知的和29个新的相互作用伙伴的结合化学计量。我们证明PRC2和G9A-GLP在物理上相互作用并共享几个相互作用伙伴，包括锌指蛋白ZNF518A和ZNF518B。通过靶向质谱进行全局染色质分析，我们发现即使是亚化学计量的结合伙伴，如ZNF518B，也可以正向调节全局H3K9me2水平。生化分析表明ZNF518B直接与EZH2和G9A相互作用。我们的系统分析表明，ZNF518B可能介导PRC2和G9A-GLP组蛋白甲基转移酶之间的结构关联，并额外调节G9A-GLP的活性。
