Chromatin modifying factors control radiation response and genomic stability

染色质修饰因子控制辐射反应和基因组稳定性

• 批准号: 8920046
• 负责人: Tej K Pandita
• 金额: $ 31.14万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8920046
• 关键词: ATM function; Acetylation; Adverse effects; Affect; Attention; Cancer Patient; Cell Proliferation; Cell Survival; Cells; ChIP-on-chip; Chromatin; Chromatin Structure; Chromosomes; Chromosomes, Human, Pair 5; Cleaved cell; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA lesion; Data; Development; Double Strand Break Repair; Drosophila genus; Embryo; Embryonic Development; Environment; Epigenetic Process; Event; Family; Fibroblasts; Frequencies; Funding; Genes; Genome Stability; Genomic Instability; Genomic Segment; Genomics; Goals; Health; Histone H4; Histones; Human; In Vitro; Individual; Investigation; Ionizing radiation; Kinetics; Knock-in Mouse; Lesion; Location; Lysine; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Mediating; Meiotic Recombination; Methodology; Molecular; Mus; Mutant Strains Mice; Mutation; Neoplastic Cell Transformation; Nonhomologous DNA End Joining; Oncogenic; Orthologous Gene; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Predisposition; Process; Progress Reports; Proteins; Radiation Tolerance; Radiation therapy; Reporter Genes; Reporting; Research; Role; Signal Transduction; Site; System; Testing; Threonine; Time; Zinc Fingers; base; cancer cell; cancer therapy; cell killing; chromatin immunoprecipitation; chromatin protein; density; endodeoxyribonuclease SceI; genome integrity; genome-wide; histone acetyltransferase; histone modification; homologous recombination; improved; in vivo; innovation; insight; irradiation; male; member; mutant; new technology; novel; novel strategies; nuclease; prevent; radiation response; reconstitution; repaired; response; tumor; tumorigenesis

DESCRIPTION (provided by applicant): We identified a chromatin-modifying factor "hMOF", the human ortholog of the Drosophila MOF gene (Males absent On the First) that interacts with and regulates the ionizing radiation (IR) response function of ATM. hMOF is a member of the MYST family of histone acetyltransferases (HATs) and acetylates histone H4 at lysine 16 (H4K16ac). Histone H4K16ac is a unique histone modification that prevents higher order chromatin packing, which can impede protein access to DNA. Mammalian MOF impacts multiple points in the cellular DNA damage response (DDR) and double-strand break (DSB) repair pathways probably as a critical regulator of chromatin structure. This competing renewal brings together and expands insights gained during the previous funding period from two separate RO1's [R01CA123232 (not being renewed), R01CA129537 (this renewal)] that focused our attention on how MOF-dependent H4K16ac levels and the phosphorylation of MOF influence DNA DSB repair and oncogenesis, as we found that acetylation of histone H4 at K16 by MOF is an epigenetic signature of cellular proliferation common to both embryogenesis and oncogenesis. In addition, our results suggest that preexisting MOF-dependent H4K16ac may influence the DDR. However the precise effect of H4K16ac chromatin status as well as most other chromatin proteins/modifications on the DDR is largely unknown. A major impediment in the mammalian DNA repair field to answering this type of question has been the non-specificity of DNA damage inducing agents (for example: ionizing radiations), making it difficult to characterize how specific differences in chromatin environment impact DNA lesion signaling/repair. We will circumvent this problem by using site-specific repair systems to determine the role of histone H4K16ac in DSB repair. We have successfully generated a high-density genome wide map identifying H4K16ac rich or poor chromosomal sites. Utilizing this data, we will directly test the hypothesis that local H4K16ac levels on chromosomes regulate DDR. To examine sites with defined H4K16ac chromatin status, we will utilize zinc finger nucleases (ZFNs) to produce DSBs within regions we have identified as having high or low H4K16ac levels and determine the subsequent recruitment of DDR components. Furthermore, we have shown that MOF itself is phosphorylated at threonine 392 (MOF-T392) post irradiation in an ATM-dependent manner that is important for the DDR and cell survival. Therefore, to determine the role of ATM dependent MOF phosphorylation in signaling/repair protein recruitment, we will generate mutant MOF-phosphorylation (mMof- T392A) site knock-in mice and examine the DSB signaling/repair mechanism and consequences for genomic integrity and cancer development. This approach uniquely allows for the determination of how MOF-dependent local H4K16ac levels and MOF phosphorylation affect the DDR, repair pathway choice and oncogenesis.

描述（由申请人提供）：我们鉴定了一种染色质修饰因子“hMOF”，它是果蝇MOF基因的人类直系同源物（雄性在第一次缺失），与ATM的电离辐射（IR）反应功能相互作用并调节其功能。hMOF是组蛋白乙酰转移酶（HAT）的MYST家族的成员，并且在赖氨酸16（H4 K16 ac）处乙酰化组蛋白H4。组蛋白H4 K16 ac是一种独特的组蛋白修饰，可以阻止高级染色质包装，这可能会阻碍蛋白质进入DNA。哺乳动物的MOF可能作为染色质结构的关键调节剂影响细胞DNA损伤反应（DDR）和双链断裂（DSB）修复途径中的多个点。此次竞争性更新将上一个资助期内从两个独立的RO 1 [R 01 CA 123232]中获得的见解结合在一起并加以扩展（未更新），R 01 CA 129537（这次更新）]，使我们的注意力集中在MOF依赖性H4 K16 ac水平和MOF的磷酸化如何影响DNA DSB修复和肿瘤发生，因为我们发现组蛋白H4在K16处被MOF乙酰化是胚胎发生和肿瘤发生共同的细胞增殖的表观遗传标记。此外，我们的研究结果表明，预先存在的MOF依赖的H4 K16 ac可能会影响DDR。然而，H4 K16 ac染色质状态以及大多数其他染色质蛋白/修饰对DDR的精确影响在很大程度上是未知的。在哺乳动物DNA修复领域回答这类问题的主要障碍是DNA损伤诱导剂（例如：电离辐射）的非特异性，使得难以表征染色质环境中的特定差异如何影响DNA损伤信号传导/修复。我们将通过使用位点特异性修复系统来确定组蛋白H4 K16 ac在DSB修复中的作用来规避这个问题。我们已经成功地产生了一个高密度的全基因组图谱，识别H4 K16 ac丰富或贫穷的染色体位点。利用这些数据，我们将直接测试染色体上的局部H4 K16 ac水平调节DDR的假设。为了检查具有确定的H4 K16 ac染色质状态的位点，我们将利用锌指核酸酶（ZFN）在我们已经鉴定为具有高或低H4 K16 ac水平的区域内产生DSB，并确定DDR组分的后续募集。此外，我们已经表明，MOF本身在照射后以ATM依赖性方式在苏氨酸392（MOF-T392）处磷酸化，这对于DDR和细胞存活是重要的。因此，为了确定ATM依赖性MOF磷酸化在信号传导/修复蛋白募集中的作用，我们将产生突变的MOF磷酸化（mMof-T392 A）位点敲入小鼠，并检查DSB信号传导/修复机制以及对基因组完整性和癌症发展的后果。这种方法独特地允许确定MOF依赖性局部H4 K16 ac水平和MOF磷酸化如何影响DDR、修复途径选择和肿瘤发生。