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

• 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
• 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

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.

项目摘要 哺乳动物细胞不断暴露于环境有毒物质，包括紫外线辐射和各种来源 电离辐射（IR）威胁基因组完整性，导致癌症和神经退行性的风险增加 疾病。鉴于我们不断暴露于环境有毒物质，阐明了基因组的基本原理 完整性维持对于为多种与年龄相关的病理发展开发治疗干预措施至关重要。最近 多年来，已经证明几个基于染色质的事件是有效DNA损伤的关键介体 响应（DDR），但是缺乏高通量筛选方法已显着阻碍 鉴定DNA修复必不可少的染色质因子。为了解决这个问题，该建议将使用新开发 高通量筛选方法，再加上预测染色质相互作用的cDNA库 （“ Chromorfeome”），以确定与DNA修复有关的新型染色质因子。在此问题（K99）阶段 该提议，候选人将确定新确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（AIM 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）。候选人将基于此数据，以机械地确定Znf280a的方式 编排DNA修复（AIM 2A），并确定这是否有助于胰腺导管的治疗性耐药性 腺癌（PDAC），Znf280a的表达增加与预后明显差的相关 患者（AIM 2B）。重要的是，在此问题（K99）阶段，候选人将利用资源 可在马萨诸塞州综合医院获得专业发展，通过心理运用这些技能， 数据演示和写作机会。在未进行的/独立研究阶段（R00）期间 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。 Znf280a在22q11.2基因座的住宅和 初步数据表明，ZNF280A的耗竭会导致发起的DNA损伤。候选人会 因此，研究ZnF280A对于解决DNA复制应力的重要性，并确定是否是否 这可以机械解释22Q11.2缺失综合征（AIM 2C）的某些特征。另外，很少 关于在DNA修复后如何重新建立染色质结构和功能。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颖 DNA修复晚期涉及的染色质因子（AIM 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发展独立 在DNA修复的后期阶段，基础染色质重建 - 将这些机械研究应用于 解释如何保留基因组完整性的目的地继续暴露于DNA破坏环境有毒物质。