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

摘要/摘要 多梳抑制复合体2(PRC2)是表观遗传学所必需的组蛋白甲基转移酶 发展中的沉默。常可观察到PRC2或其甲基转移酶活性上调 癌症，可能是癌细胞增殖和发展的关键。尽管做出了许多努力来减少 PrC2-核酸在疾病治疗中的表达水平及其催化活性部位的抑制 相互作用作为调节招募和催化活动的重要过程被低估了 PRC2的。最近，我们的团队和其他人证明了RNA结合抑制了催化 PrC2的活性，表明PrC2亚基的RNA结合残基突变可能是一种替代 上调PRC2活性的方法确实在癌症中观察到了这种突变(R34L，K39E， PRC2的催化亚基EZH2中的K491R和R494S)。此外，一些长的非编码(LNC)RNA具有 作为关键的发挥者，从染色质基因座招募或驱逐PRC2来调节特定的基因表达 在发育和疾病中，但对PrC2的RNA结合的生理功能的了解一直是 之前由于缺乏功能分离突变体而受阻，这一点在我最近的 出版(Long等人ELife 2017)。最近，PRC2-DNA的相互作用已被证明推动了 PrC2与核小体的相互作用，DNA结合区位于多个附属亚基上 PRC2复合体包括AEBP2、JARID2、PHF1、MTF2和PHF19。然而，基因的全基因组功能 这些DNA相互作用还没有得到全面的研究。要回答所有这些关键问题 我建议对PrC2的调控进行全面的体外和体内分子机制的研究 以及PRC2-核酸相互作用的细胞功能。具体地说，这项建议旨在：(1)了解如何 RNA相互作用调节PRC2的细胞活动；(2)了解DNA如何通过 与PrC2‘S辅助蛋白的相互作用。在K99阶段，在汤姆·切赫博士和 John Rinn博士，使用体外生化分析和体内基因组范围的综合方法 将利用表征来确定PrC2-核酸的分子机制和功能 互动。在Leslie Leinwand博士的支持下，分析心肌细胞生理学和 将获得行为，以便识别核酸丢失的重要生物学后果 PRC2在心肌细胞中的相互作用，其中PRC2的活动已被证明是关键的。额外的K99 基因组学和计算生物学方面的培训将对拟议的研究至关重要。以上所有培训 将有助于继续研究PRC2-核酸相互作用的功能 R00阶段。这一提议的结果不仅将导致识别重要的PRC2-核酸 相互作用及其分子机制，但也为表征其他表观遗传学提供了一个范例 结合核酸的修饰物，尤其是非编码RNA。

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.