Mechanistic characterization of the cell cycle-dependent DNA repair pathway- Resubmission

项目摘要 基于染色质的DNA损伤反应（DDR）途径是保护细胞免受基因组损伤的基础 不稳定，这是癌症的标志。DDR途径在整个细胞周期中受到严格调节，以确保 DNA修复的时空控制。细胞周期调控的染色质修饰对于编排DNA至关重要 修复.值得注意的是，H4 K20甲基化是一种细胞周期依赖性组蛋白标记，参与DNA双链 断裂（DSB）修复途径的选择。新加入的未经修饰的H4招募TONSL复制受损 H4 K20 me 2在染色质中募集53 BP 1，以执行同源重组（HR）修复;而H4 K20 me 2则在受损的染色质中募集53 BP 1， 染色质主要在G1期以促进非同源末端连接（NHEJ）。知识差距 目前的模型来源于H4 K20 me 1和DSB修复途径选择的作用和调节不清楚。 识别组蛋白H4 K20 me阅读器为染色质修饰如何执行提供了重要的见解 通过在正确的时间将下游效应蛋白募集到受损的染色质来发挥细胞功能。我们确定 ZMYM 3（锌指骨髓增生性和精神发育迟滞，3型），作为HR促进因子， 特异性结合H4 K20甲基化标记。本项目的总体目标是阐明 ZMYM 3对细胞周期调节的H4 K20甲基化的机制调节作用及其如何转化为DNA 复制后染色质上的DSB修复途径选择。具体而言，我们建议：1）确定 通过生物化学测定和遗传研究确定ZMYM 3和H4 K20甲基化之间的联系; 2）表征 ZMYM 3功能复合物在复制后受损染色质上的作用;以及3）阐明 ZMYM 3如何调节细胞周期调节的DSB修复途径选择和。重点查处 DYNLL 1/LC 8，一个最近鉴定的DNA修复蛋白，及其物理，遗传和功能联系 ZMYM 3在DSB修复调控中的作用。我们的长期目标是剖析细胞如何编排DNA的细节 通过染色质修饰进行修复。这些研究有望为H4 K20 me 1和 H4 K20 me 2决定了在复制后染色质修复中HR和NHEJ之间的选择。它还将破译 ZMYM 3如何塑造复制后染色质表观基因组并在受损染色质处招募DDR蛋白。 虽然遗传性DDR缺陷易患癌症的发展，脆弱性是治疗利用 优先杀死肿瘤细胞。因此，DNA损伤剂是主要类别的治疗剂，其包括 放疗由于染色质直接调节DNA修复蛋白在受损染色质上的积累， 表观基因组是用于癌症治疗的药物发现的有吸引力的靶标。这项工作利用了 生物化学、遗传学、表观遗传学和细胞方法来剖析细胞周期的详细机制- 调节表观基因组对基因组完整性维护的影响，可以转化为潜在的生物标志物和药物 癌症治疗的新发现

PROJECT SUMMARY Chromatin-based DNA damage response (DDR) pathway is fundamental for protecting cells from genome instability, which is a hallmark of cancer. The DDR pathway is tightly regulated throughout the cell cycle to ensure spatiotemporal control of DNA repair. Cell cycle-regulated chromatin modification is crucial for orchestrating DNA repair. Notably, H4K20 methylation is a cell cycle-dependent histone mark that is involved in DNA double-strand break (DSB) repair pathway choice. Newly incorporated unmodified H4 recruits TONSL to replicated damaged chromatin to execute homologous recombination (HR) repair; whereas, H4K20me2 recruits 53BP1 to damaged chromatin predominately at G1 phase to promote non-homologous end joining (NHEJ). The knowledge gap for the current model comes from the unclear role and regulation of H4K20me1 and DSB repair pathway choice. Identifying histone H4K20me readers provides important insights into how chromatin modifications execute cellular functions by recruiting downstream effector proteins to damaged chromatin at the right time. We identified ZMYM3 (Zinc finger myeloproliferative and mental retardation, type-3), as an HR promoting factor, which specifically binds to the H4K20 methylation mark. The overall objective of this project is to elucidate the mechanistic regulatory role of ZMYM3 on cell cycle-regulated H4K20 methylation, and how it translates into DNA DSBs repair pathway choice on post-replicative chromatin. Specifically, we propose to 1) determine the connection between ZMYM3 and H4K20 methylation by biochemical assays and genetic studies; 2) characterize the ZMYM3 functional complex(es) on post-replicative damaged chromatin; and 3) elucidate the mechanism of how ZMYM3 regulates cell cycle-regulated DSB repair pathway choice and. We will focus on investigating DYNLL1/LC8, a recently characterized DNA repair protein, and its physical, genetic and functional connections with ZMYM3 in DSB repair regulation. Our long-term goal is to dissect the detail of how cells orchestrate DNA repair via chromatin modifications. These studies are poised to provide critical insights into how H4K20me1 and H4K20me2 dictate the choice between HR and NHEJ on post-replicative chromatin repair. It will also decipher how ZMYM3 shapes the post-replicative chromatin epigenome and recruits DDR proteins at damaged chromatin. Although inherited DDR defects predispose in cancer development, the vulnerability is therapeutically exploited to preferentially kill tumor cells. Thus, DNA damaging agents are a major class of therapeutic agents that include radiotherapy. Since chromatin directly regulates DNA repair proteins accrual at damaged chromatin, the epigenome is an attractive target for drug discovery for cancer treatment. This work exploits a combination of biochemical, genetic, epigenetics and cellular approaches to dissect the detailed mechanism of cell cycle- regulated epigenome on genome integrity maintenance that can translate to potential biomarkers and drug discovery for cancer treatment.