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: 8869493
• 负责人: Tej K Pandita
• 金额: $ 32万
• 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/8869493
• 关键词: Chromatin; modifying; factors; control; radiation

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（雄性首先缺失）的人类直系同源物，它与 ATM 的电离辐射（IR）响应功能相互作用并调节。 hMOF 是组蛋白乙酰转移酶 (HAT) MYST 家族的成员，可在赖氨酸 16 (H4K16ac) 处乙酰化组蛋白 H4。组蛋白 H4K16ac 是一种独特的组蛋白修饰，可防止高阶染色质堆积，从而阻碍蛋白质接触 DNA。哺乳动物 MOF 可能作为染色质结构的关键调节因子，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。这种竞争性的更新汇集并扩展了在之前的资助期间从两个单独的 RO1 [R01CA123232（未更新）、R01CA129537（本次更新）] 获得的见解，这将我们的注意力集中在 MOF 依赖性 H4K16ac 水平和 MOF 的磷酸化如何影响 DNA DSB 修复和肿瘤发生，因为我们发现乙酰化 MOF 对 K16 处组蛋白 H4 的修饰是胚胎发生和肿瘤发生中常见的细胞增殖的表观遗传特征。此外，我们的结果表明，先前存在的依赖 MOF 的 H4K16ac 可能会影响 DDR。然而，H4K16ac 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尚不清楚。哺乳动物 DNA 修复领域回答此类问题的一个主要障碍是 DNA 损伤诱导剂（例如：电离辐射）的非特异性，这使得很难描述染色质环境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 H4K16ac 在 DSB 修复中的作用来规避这个问题。我们已经成功生成了高密度全基因组图谱，可识别 H4K16ac 丰富或贫乏的染色体位点。利用这些数据，我们将直接检验染色体上局部 H4K16ac 水平调节 DDR 的假设。为了检查具有确定的 H4K16ac 染色质状态的位点，我们将利用锌指核酸酶 (ZFN) 在我们确定为具有高或低 H4K16ac 水平的区域内产生 DSB，并确定随后 DDR 组件的招募。此外，我们还发现，MOF 本身在照射后的苏氨酸 392 (MOF-T392) 处以 ATM 依赖性方式磷酸化，这对于 DDR 和细胞存活很重要。因此，为了确定 ATM 依赖性 MOF 磷酸化在信号/修复蛋白招募中的作用，我们将生成突变型 MOF 磷酸化 (mMof-T392A) 位点敲入小鼠，并检查 DSB 信号/修复机制以及对基因组完整性和癌症发展的影响。这种方法独特地允许确定 MOF 依赖性局部 H4K16ac 水平和 MOF 磷酸化如何影响 DDR、修复途径选择和肿瘤发生。