Regulation of a chromatin modifier (Polycomb Repressive Complex 2) by nucleic acids interactions

总结/摘要 多梳抑制复合物2（PRC 2）是表观遗传学所需的重要组蛋白甲基转移酶。 发展中的沉默PRC 2或其甲基转移酶活性的上调经常在哺乳动物中观察到。 癌症，并且对于癌细胞增殖和进展是至关重要的。尽管作出了许多努力， PRC 2的表达水平和在疾病治疗中抑制其催化活性位点，PRC 2-核酸 相互作用被低估为规范招募和催化活动的重要过程 在PRC 2。我们的研究小组和其他人最近已经证明，RNA结合抑制了催化活性， PRC 2的活性，表明PRC 2亚基的RNA结合残基的突变可能是一种替代方法 上调PRC 2活性的方法，并且实际上已经在癌症中观察到这种突变（R34 L，K39 E， EZH 2中的K491 R和R494 S，PRC 2的催化亚基）。此外，一些长的非编码（lnc）RNA具有 作为关键的参与者出现，从染色质位点招募或驱逐PRC 2，以调节特定的基因表达 在发育和疾病中的作用，但了解PRC 2的RNA结合的生理功能一直是 以前由于缺乏功能分离突变体而受到阻碍，这在我最近的研究中终于被发现。 出版物（Long et al. eLife 2017）。最近，PRC 2-DNA相互作用已被证明可以驱动DNA的降解。 PRC 2与核小体的相互作用，以及位于PRC 2的多个辅助亚基上的DNA结合结构域， PRC 2复合物包括AEBP 2、JARID 2、PHF 1、MTF 2和PHF 19。然而， 这些DNA相互作用尚未得到全面研究。为了回答所有这些关键问题， PRC 2调节，我提出了一个全面的体外和体内研究的分子机制 和PRC 2-核酸相互作用的细胞功能。具体而言，本建议旨在：（1）了解如何 RNA相互作用调节PRC 2的细胞活动;（2）了解DNA如何通过 与PRC 2辅助蛋白的相互作用。在K99阶段，在Tom Cech博士的指导下， 博士John Rinn，体外生化分析和体内全基因组分析的综合方法 表征将用于确定PRC 2-核酸的分子机制和功能 交互.在Leslie Leinwand博士的支持下， 将获得行为，以确定核酸丢失的重要生物学后果 PRC 2在心肌细胞中的相互作用，其中PRC 2活性已被证明是关键的。其他K99 基因组学和计算生物学方面的培训对拟议的研究至关重要。以上所有培训 这将有助于在独立的PCR过程中继续研究PRC 2-核酸相互作用的功能。 R 00阶段。该提案的结果不仅将导致重要的PRC 2-核酸的鉴定， 相互作用及其分子机制，但也提供了一个范例，表征其他表观遗传 结合核酸特别是非编码RNA的修饰剂。

Summary/abstract Polycomb repressive complex 2 (PRC2) is an essential histone methyltransferase required for epigenetic silencing in development. Upregulation of PRC2 or its methyltransferase activity is frequently observed in cancer and can be critical for cancer cell proliferation and progression. Despite many efforts in reducing the expression level of PRC2 and inhibiting its catalytic active site in treatment of diseases, PRC2-nucleic acid interactions have been underestimated as important processes to regulate recruitment and catalytic activities of PRC2. It has been recently demonstrated by our group and others that RNA binding inhibits catalytic activities of PRC2, indicating that mutations of RNA binding residues of PRC2 subunits could be an alternative way to upregulate PRC2 activity and indeed such mutations have been observed in cancer (R34L, K39E, K491R and R494S in EZH2, the catalytic subunit of PRC2). Moreover, a few long noncoding (lnc)RNAs have emerged as key playmakers to recruit or evict PRC2 from chromatin loci to regulate specific gene expression in development and diseases, but understanding the physiological functions of RNA binding of PRC2 has been hampered previously by the lack of separation-of-function mutant, which has been finally identified in my recent publication (Long et al. eLife 2017). More recently, PRC2-DNA interaction has been shown to drive the interaction of PRC2 with nucleosome, and DNA-binding domains reside on multiple accessory subunits of PRC2 complex including AEBP2, JARID2, PHF1, MTF2 and PHF19. However, the genome-wide function of these DNA interactions has not been comprehensively investigated. To answer all these critical questions on PRC2 regulation, I propose a comprehensive in vitro and in vivo investigation of the molecular mechanisms and cellular functions of PRC2-nucleic acid interactions. Specifically, this proposal aims to: (1) understand how RNA interactions regulate cellular activities of PRC2; (2) understand how DNA regulates PRC2 through interaction with PRC2’s accessory proteins. During the K99 phase, under the mentorship of Dr. Tom Cech and Dr. John Rinn, comprehensive approaches using in vitro biochemical analysis and in vivo genome-wide characterization will be used to determine molecular mechanism and function of PRC2-nucleic acid interactions. With support from Dr. Leslie Leinwand, expertise in analyzing cardiomyocyte physiology and behavior will be acquired in order to identify important biological consequences of loss of nucleic acid interaction of PRC2 in cardiomyocytes, in which PRC2 activities have been shown to be critical. Additional K99 training in genomics and computational biology will be essential for the proposed research. All training above will facilitate continued investigations of functions of PRC2-nucleic acid interactions during the independent R00 phase. The results of this proposal will not only lead to identification of important PRC2-nucleic acid interactions and their molecular mechanisms, but also provide a paradigm for characterizing other epigenetic modifiers that bind nucleic acids especially noncoding RNAs.