Identifying chromatin factors essential for DNA repair using a novel high-throughput screening methodology

项目总结 哺乳动物细胞持续暴露在环境毒物中，包括紫外线辐射和各种来源的 电离辐射(IR)威胁基因组完整性，导致癌症和神经退行性变风险增加 疾病。鉴于我们不断接触环境毒物，阐明基因组的基本原理 保持完整性对于开发针对一系列与年龄相关的病理的治疗干预措施至关重要。在最近 多年来，一些以染色质为基础的事件已被证明是有效的DNA损伤的关键介质 应对(DDR)，然而，缺乏高通量筛查方法严重阻碍了 DNA修复所需染色质因子的鉴定。为了解决这个问题，这项提案将使用一种新开发的 高通量筛选方法，结合预测的染色质相互作用蛋白的cDNA库 (“ChromORFeome”)，以确定参与DNA修复的新的染色质因子。在指导(K99)阶段 在这项提案中，候选人将决定一种新发现的染色质相互作用蛋白的重要性， ZNF280A，用于修复DNA损伤，识别需要ZNF280A的特定修复途径(目标1)。 初步数据表明，ZNF280A被招募到由多种来源引起的DNA损伤部位 包括电离辐射(IR)。候选人将基于这些数据来机械地确定ZNF280A 协调DNA修复(目标2A)，并确定这是否有助于胰腺导管的治疗抵抗 腺癌(PDAC)，其中ZNF280A的表达增加与显著不良的预后相关 患者(目标2B)。重要的是，在指导(K99)阶段，候选人将利用资源 可在马萨诸塞州综合医院进行职业发展，通过指导应用这些技能， 数据展示和写作机会。在非指导/独立研究阶段(R00) 候选人在K99阶段开发的项目、技术技能和试剂将被用来阐明 ZNF280A在22q11.2缺失人类综合征中的重要性ZNF280A位于22q11.2基因座， 初步数据显示，ZNF280A的缺失会导致自发的DNA损伤。候选人将会 因此，研究ZNF280A对于解决DNA复制应激的重要性，并确定 这可以从机制上解释22q11.2缺失综合征(目标2C)的一些特征。此外，几乎没有什么是 了解DNA修复后染色质的结构和功能是如何重建的。因此，在R00阶段 候选人将扩展这些方法，并利用高通量筛选方法来识别新的 染色质因子参与DNA修复的晚期阶段(目标3)。这些实验将为候选人提供 早期独立出版物的数据和R系列赠款的初步数据(R21，R01)。重要的是，在 R00独立阶段，候选人将通过解决关键机制来独立于他们的导师 DNA修复后期染色质重建的基础--将这些机制研究应用于 解释基因组完整性是如何在持续暴露于DNA破坏性环境毒物的情况下得到保护的。

PROJECT SUMMARY Mammalian cells are continually exposed to environmental toxicants including UV-radiation and various sources of ionizing radiation (IR) threatening genomic integrity, leading to an increased risk of cancer and neurodegenerative disease. Given our constant exposure to environmental toxicants, elucidating fundamental principles of genome integrity maintenance is critical for developing therapeutic interventions for a host of age-related pathologies. In recent years, several chromatin-based events have been shown to be critical mediators of an effective DNA damage response (DDR), however the lack of high-throughput screening methodologies have significantly hampered the identification of chromatin factors essential for DNA repair. To address this, this proposal will use a newly developed high-throughput screening methodology, coupled with a cDNA library of predicted chromatin interactors (“ChromORFeome”), to identify novel chromatin factors involved in DNA repair. During the mentored (K99) phase of this proposal, the candidate will determine the importance of a newly identified chromatin-interacting protein, ZNF280A, for the repair of DNA damage, identifying specific repair pathways which require ZNF280A (Aim 1). Preliminary data demonstrates that ZNF280A is recruited to sites of DNA damage induced by a variety of sources including ionizing radiation (IR). The candidate will build upon this data to determine mechanistically how ZNF280A orchestrates DNA repair (Aim 2A) and ascertain whether this contributes to therapy resistance in pancreatic ductal adenocarcinoma (PDAC), where increased expression of ZNF280A correlates with significantly poorer outcome in patients (Aim 2B). Importantly, while in the mentored (K99) phase, the candidate will take advantage of the resources available at Massachusetts General Hospital for professional development, applying these skills through mentoring, data presentation and writing opportunities. During the non-mentored/independent research phase (R00) of the project, technical skills and reagents developed by the candidate during the K99 phase will be used to elucidate the importance of ZNF280A in the 22q11.2 deletion human syndrome. ZNF280A resides at the 22q11.2 locus and preliminary data demonstrates that depletion of ZNF280A results in spontaneous DNA damage. The candidate will therefore investigate the importance of ZNF280A for the resolution of DNA replication stress and determine whether this can mechanistically explain some of the features of 22q11.2 deletion syndrome (Aim 2C). In addition, very little is known about how chromatin structure and function is re-established following DNA repair. Therefore, in the R00 phase the candidate will extend these approaches and utilize the high-throughput screening methodology to identify novel chromatin factors involved in the late stages of DNA repair (Aim 3). These experiments will provide the candidate with data for an early independent publication and preliminary data for R-series grants (R21, R01). Importantly, during the R00 independent phase, the candidate will develop independence from their mentor by addressing key mechanisms underpinning chromatin re-establishment in the late stages of DNA repair - applying these mechanistic studies to explain how genome integrity is preserved despite continued exposure to DNA damaging environmental toxicants.