Regulation of PRC2 functions by PARP1

项目总结 多聚(ADP-核糖)聚合酶-PARP活性在DNA修复和抑制中发挥重要作用 PARP1已被广泛用于靶向DNA修复机制受损的癌症。在过去几年里 然而，PARP1也已经成为通过染色质调节基因表达的一个重要因素。 靶向表观遗传学是治疗癌症的一种很有前途的方法，因为有许多药物可以改变表观遗传学的修饰。 然而，对于PARP抑制剂来说，它们作为表观遗传药物的应用需要更全面的了解 PARP1的确切功能及其调节染色质结构的机制。 我们之前发现抑制PARP1活性显著改变了PARP1的表达水平 数百个基因，包括与癌症有关的基因。我们发现，聚合体水平的增加 抑制复合体2催化亚基EZH2参与了PARP抑制所引起的某些效应。 在这里，我们首次报告了(1)PARP1和EZH2的入住率在整个 (2)PARP1可直接修饰EZH2；(3)PAR化改变EZH2的酶活性。 基于这些数据，我们假设PARP1和PAR化起着重要的、被低估的作用 在EZH2活性中，PARP的抑制剂可以改变PRC2介导的基因抑制。 我们的发现可能具有重要的翻译意义，因为异常的EZH2活动有助于 癌症和PARP1抑制剂正在进行临床试验；然而，它们的作用和相互作用还没有得到 在功能和机制层面进行了探索。在这里，我们建议将PARP1和PAR化建立为 EZH2调节的新机制及确定PARP-2的机制和功能相关性 介导的EZH2抑制。 为了验证我们的假设，我们提出：(目标1)定义PARP1结合和PAR化的机制 拮抗EZH2介导的染色质修饰我们将评估EZH2结合和H3K27me3 在PARP活性改变后跨越基因组的沉积；(目标2)建立通过什么机制 PAR化抑制EZH2功能我们将确定PAR化对EZH2与组蛋白亲和力的影响 和其他PRC2蛋白；(目标3)为了评估EZH2 PAR化的相关性，我们将表征 PAR抗性EZH2突变体对染色质组成和PrC2功能的影响。我们 假设EZH2的PAR化将EZH2从染色质中移除，并且阻止EZH2的PAR化导致 持续的EZH2结合和基因抑制。 这项工作的影响超出了PARP1和异染色质形成的影响，以提供 更好地了解PARP1在染色质调节中的全球作用。通过对机制和 PARP1活性与EZH2功能的相关性我们将提供更好的理由将PARP1作为一种 癌症的治疗。从长远来看，这项工作有潜力提高治疗应用 用于治疗癌症的PARP1抑制剂，并确定哪些癌症患者可以受益于这些类别的药物。

Project summary Poly (ADP-ribose) polymerase —PARP— activity plays a necessary role in DNA repair and inhibitors of PARP1 have been widely used to target cancers with impaired DNA repair machinery. In the past few years however PARP1 has also emerged as an important factor in regulating gene expression through chromatin. Targeting epigenetics is a promising approach in cancer as many drugs exist to alter epigenetic modifications. For PARP inhibitors, their application however as epigenetic drugs requires a more complete understanding of the precise function of PARP1 and its mechanism of action in regulating chromatin structure. We previously discovered that inhibition of PARP1 activity dramatically changes the expression levels of hundreds of genes, including genes involved in cancer. We found that increased levels of the Polycomb Repressive Complex 2 catalytic subunit EZH2 are responsible for some effects caused by PARP inhibition. Here we reported for the first time that (1) PARP1 and EZH2 occupancy negatively correlate across the genome; (2) PARP1 can directly modify EZH2; and (3) PARylation alters the enzymatic activity of EZH2. Based on these data we hypothesize that PARP1 and PARylation play an important and underappreciated role in EZH2 activity, and inhibitors of PARP can alter PRC2-mediated gene repression. Our findings may have significant translational implications since aberrant EZH2 activity contributes to cancer and PARP1 inhibitors are in clinical trials; however, their effects and interactions have not been explored at the functional and mechanistic levels. Here we propose to establish PARP1 and PARylation as a novel mechanism of EZH2 regulation and to determine the mechanisms and the functional relevance of PARP- mediated EZH2 inhibition. To test our hypothesis we propose : (Aim 1) To define mechanisms of PARP1 binding and PARylation in antagonizing EZH2-mediated chromatin modification we will assess EZH2 binding and H3K27me3 deposition across the genome after changes in PARP activity; (Aim 2) To establish mechanisms by which PARylation inhibits EZH2 functions we will determine the effect of PARylation on EZH2 affinity for histone and for other PRC2 proteins; (Aim 3) To evaluate the relevance of EZH2 PARylation we will characterize the effect of PAR-resistant EZH2 mutant on chromatin composition and PRC2 functions. We hypothesize that PARylation of EZH2 removes EZH2 from chromatin and that preventing PARylation results in persistent EZH2 binding and gene repression. The impact of this work extend beyond the effect of PARP1 and heterochromatin formation to provide a better insight into the global role of PARP1 in chromatin regulation. By characterizing the mechanism and the relevance of PARP1 activity on EZH2 functions we will provide a better rationale for targeting PARP1 as a treatment in cancer. In the long run, this work has the potential to improve the therapeutic application of PARP1 inhibitors for treating cancer and to identify cancer patients who can benefit by these class of drugs.