Regulation of PRC2 functions by PARP1

项目概要 聚 (ADP-核糖) 聚合酶 —PARP — 活性在 DNA 修复和 DNA 修复抑制剂中发挥着必要的作用 PARP1 已被广泛用于靶向 DNA 修复机制受损的癌症。在过去的几年里 然而，PARP1 也已成为通过染色质调节基因表达的重要因素。 靶向表观遗传学是治疗癌症的一种有前途的方法，因为存在许多药物可以改变表观遗传修饰。 然而，对于 PARP 抑制剂，它们作为表观遗传药物的应用需要更全面的了解 PARP1 的精确功能及其调节染色质结构的作用机制。 我们之前发现，抑制 PARP1 活性会显着改变 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 Polycomb 的水平增加 抑制复合物 2 催化亚基 EZH2 负责 PARP 抑制引起的一些影响。 在这里，我们首次报道 (1) PARP1 和 EZH2 占用率在整个系统中呈负相关。 基因组； (2) PARP1可以直接修改EZH2； (3) PARylation 改变 EZH2 的酶活性。 基于这些数据，我们假设 PARP1 和 PARylation 发挥着重要但未被充分认识的作用 EZH2 活性，PARP 抑制剂可以改变 PRC2 介导的基因抑制。 我们的研究结果可能具有重大的转化意义，因为异常的 EZH2 活性有助于 癌症和PARP1抑制剂正在进行临床试验；然而，它们的影响和相互作用尚未得到证实 在功能和机械层面进行了探索。在这里，我们建议建立 PARP1 和 PARylation 作为 EZH2 调节的新机制并确定 PARP-的机制和功能相关性 介导的 EZH2 抑制。 为了检验我们的假设，我们提出：（目标 1）定义 PARP1 结合和 PARylation 的机制 拮抗 EZH2 介导的染色质修饰，我们将评估 EZH2 结合和 H3K27me3 PARP 活性变化后在基因组中沉积； （目标 2）建立机制 PARylation 抑制 EZH2 功能 我们将确定 PARylation 对 EZH2 对组蛋白亲和力的影响 以及其他 PRC2 蛋白； （目标 3）为了评估 EZH2 PARylation 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 PARylation 会从染色质中去除 EZH2，并且阻止 PARylation 会导致 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，提供了 更好地了解 PARP1 在染色质调控中的整体作用。通过表征机制和 PARP1 活动与 EZH2 功能的相关性，我们将为将 PARP1 作为目标提供更好的理由 癌症治疗。从长远来看，这项工作有可能改善治疗应用 PARP1 抑制剂用于治疗癌症并确定可以从此类药物中受益的癌症患者。

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.