Shaping DNA Damage Response Networks Via Histone H2A Variants

 描述(申请人提供)：基于染色质的DNA损伤反应(DDR)途径保护细胞免受基因组和表观基因组不稳定性的影响，这是癌症的特征，被认为是推动癌症进展的因素。这项建议解决了与癌症有关的表观遗传和遗传机制的根本重要性问题。组蛋白H2A变体是染色质的关键成分，但它们如何促进表观基因组和基因组稳定性尚不清楚。对于这项提议，我们的目标是使用遗传系统，使用我们的组蛋白H2A变体敲除/敲除工程人类细胞，并对组蛋白变体进行免费的蛋白质组学分析，以回答组蛋白H2A变体如何促进DDR以维持基因组稳定的基本问题。我们还旨在提供对组蛋白H2A变体如何调节辐射反应的见解，这是一种经常用于癌症治疗的疗法。这些研究为这些问题提供了创新和新的方法，可以为组蛋白H2A变体介导的基于染色质的DNA损伤反应提供变革性的见解。我们将首先分析组蛋白H2 A变体对辐射的DDR功能，方法是使用删除或耗尽组蛋白H2 A变体的人类细胞，包括H2 AX、H2 AZ、宏H2 A和H2 A.bbd。我们的初步结果表明，组蛋白H_2A变异体在这些途径中发挥着重要而独特的作用。这些研究准备为组蛋白H2A变体如何调节DDR和对IR的反应提供关键的见解。然后，我们将利用我们的初步分析，确定每个组蛋白H2A变体的候选蛋白质相互作用因子，以确定 组蛋白H_2A变种的DDR效应蛋白。最后，我们将测试我们的工作假设，即宏H2A如何通过与我们已经确定的一种新的DDR因子相互作用促进HR的DNA修复。这些研究将提供一个无与伦比的视角，了解组蛋白H2A变体如何塑造DDR，以保护人类细胞中结构和功能不同的染色质景观中的基因组完整性。DNA损伤剂是包括放射治疗在内的一类主要的化疗药物，表观基因组代表着令人兴奋的药物发现的新线索。因此，这项工作利用人类细胞中的遗传、生化和细胞方法的组合来确定促进基因组和表观基因组完整性的关键DDR途径，以及对包括辐射和PARP抑制剂在内的癌症相关治疗的反应，这些治疗可以转化为改善对人类癌症的治疗。

 DESCRIPTION (provided by applicant): Chromatin-based DNA damage response (DDR) pathways protect cells from genome and epigenome instability, which are hallmarks of cancer and are thought to drive cancer progression. This proposal addresses questions of fundamental importance for epigenetic and genetic mechanisms involved in cancer. Histone H2A variants are critical components of chromatin but how they promote epigenome and genome stability are poorly understood. For this proposal, we aim to use genetic systems using our histone H2A variant knockout/knockdown engineered human cells with complimentary proteomic analyses of histone variants to answer fundamental questions for how histone H2A variants promote the DDR to maintain genome stability. We also aim to provide insights into how histone H2A variants regulate radiation responses, a therapy often used in cancer treatments. These studies provide innovative and new approaches to these questions that can provide transformative insights into chromatin-based DNA damage responses mediated by histone H2A variants. We will first analyze the DDR function of histone H2A variants in response to radiation, by using human cells deleted or depleted for histone H2A variants including H2AX, H2AZ, macroH2A and H2A.Bbd. Our preliminary results indicate that histone H2A variants play vital and unique roles in these pathways. These studies are poised to provide critical insights into how histone H2A variants regulate the DDR and response to IR. We will then exploit our preliminary analyses identifying candidate protein-interacting factors for each histone H2A variant to identify DDR effector proteins for individual histone H2A variants. Finally, we will test our working hypothesis for how macroH2A promotes DNA repair by HR through the interactions with a new DDR factor that we have identified. These studies will provide an unparalleled view of how histone H2A variants shape the DDR to protect the genome integrity across structurally and functionally diverse chromatin landscapes in human cells. DNA damaging agents are a major class of chemotherapeutic agents including radiotherapy and the epigenome represents exciting new leads for drug discovery. Thus, this work exploits a combination of genetic, biochemical and cellular approaches in human cells to identify key DDR pathways that promote genome and epigenome integrity, as well as responses to cancer-relevant therapies including radiation and PARP inhibitors that can translate to improved treatments for human cancers.