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

使用新型高通量筛选方法鉴定 DNA 修复必需的染色质因子

基本信息

批准号：
10505883
负责人：
Thomas L Clarke
金额：
$ 10.91万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-05 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10505883
关键词：
22q11.2 Address BRCA1 gene Biochemical Biochemistry Biological Assay Biological Models Camptothecin Cells Cellular biology Chromatin Chromatin Structure Complex Coupled DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Pathway DNA biosynthesis Data Development DiGeorge Syndrome Environment Enzymes Event Excision Exposure to Expression Library General Hospitals Genes Genome Stability Genomic Instability Goals Grant Human Hypersensitivity Immunofluorescence Immunologic Immunoprecipitation Innovative Therapy Ionizing radiation Maintenance Mammalian Cell Mass Spectrum Analysis Massachusetts Mediator of activation protein Mentors Methodology Molecular Biology Mutation Analysis Neurodegenerative Disorders Outcome PARP inhibition Pancreatic Ductal Adenocarcinoma Pathology Pathway interactions Patients Phase Post-Translational Protein Processing Process Prognosis Proteins Publications Reagent Research Resolution Resources Role Series Site Source Structure Syndrome Technical Expertise Techniques Therapeutic Intervention Toxic Environmental Substances Ultraviolet Rays Validation Work Writing XCL1 gene age related base cDNA Expression cDNA Library cancer risk chromatin immunoprecipitation experimental study genome integrity helicase high throughput screening homologous recombination in vivo ion source novel nuclease phosphoproteomics preservation protein complex recruit repaired replication stress response screening skills therapy resistant toxicant treatment response

项目摘要

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.

项目概要哺乳动物细胞不断暴露于环境毒物，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疾病的风险增加鉴于我们不断接触环境毒物，阐明基因组的基本原理。完整性维护对于开发针对许多与年龄相关的病理的治疗干预措施至关重要。多年来，一些基于染色质的事件已被证明是有效 DNA 损伤的关键介质反应（DDR），然而，缺乏高通量筛选方法极大地阻碍了为了解决这个问题，该提案将使用新开发的染色质因子。高通量筛选方法，结合预测染色质相互作用子的 cDNA 文库（“ChromORFeome”），以确定在指导（K99）阶段参与 DNA 修复的新染色质因子。根据该提案，候选人将确定新鉴定的染色质相互作用蛋白的重要性， ZNF280A，用于修复 DNA 损伤，确定需要 ZNF280A 的特定修复途径（目标 1）。初步数据表明，ZNF280A 被招募到多种来源引起的 DNA 损伤位点包括电离辐射 (IR)，考生将根据这些数据来确定 ZNF280A 的机械性能。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 表达增加与显着较差的预后相关重要的是，在指导 (K99) 阶段，候选人将利用资源。在马萨诸塞州总医院进行专业发展，通过指导应用这些技能，在非指导/独立研究阶段（R00）期间的数据展示和写作机会。候选人在 K99 阶段开发的项目、技术技能和试剂将用于阐明 ZNF280A 在 22q11.2 缺失人类综合征中的重要性在于 22q11.2 位点和初步数据表明，ZNF280A 的消耗会导致自发性 DNA 损伤。因此，研究 ZNF280A 对于解决 DNA 复制应激的重要性，并确定是否这可以从机制上解释 22q11.2 缺失综合征的一些特征（目标 2C）。已知 DNA 修复后染色质结构和功能如何重建，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参与 DNA 修复后期的染色质因子（目标 3）。早期独立出版物的数据和 R 系列资助（R21、R01）的初步数据。 R00 独立阶段，候选人将通过解决关键机制来发展独立于导师的能力在 DNA 修复的后期阶段支持染色质重建 - 将这些机制研究应用于解释尽管持续暴露于 DNA 破坏性环境毒物中，基因组完整性如何得以保持。