Project 3: Chromatin modifiers of BRCA-related DNA repair Pathways

项目3：BRCA相关DNA修复途径的染色质修饰剂

• 批准号: 10396610
• 负责人: Shridar Ganesan
• 金额: $ 38.82万
• 依托单位: RBHS -CANCER INSTITUTE OF NEW JERSEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396610
• 关键词: Acetylation; Address; Affect; BMI1 gene; BRCA1 gene; BRCA2 gene; Binding; Cell Survival; Cells; Cellular biology; Chromatin; Chromatin Remodeling Factor; Clinical; Computational Biology; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; Defect; Development; Drug Targeting; Engineering; Event; Gene Silencing; Genetic Transcription; Genomics; Histones; Human; Impairment; Lead; Location; Malignant Neoplasms; Mediating; Methods; Methyltransferase; Modification; Molecular Biology; Mus; Mutation; PALB2 gene; Pathway interactions; Phenotype; Phosphorylation; Play; Post-Translational Protein Processing; Proteins; Regulation; Repair Complex; Role; Serine; Signal Pathway; Signal Transduction; Site; System; Transcriptional Regulation; Transferase; Ubiquitination; chromatin modification; clinically relevant; genome editing; histone methylation; histone modification; inhibitor; innovation; insight; knock-down; member; mutant; p53-binding protein 1; recruit; repaired; response; small molecule; small molecule inhibitor; targeted treatment; therapeutic target; treatment response; tumorigenesis

PROJECT SUMMARY/ABSTRACT: Induction of a DNA break initiates DNA damage signaling pathways that lead to coordinated and dynamic modification of the local chromatin landscape, including regulated changes in post-translational modification of histones flanking the site of DNA damage. These chromatin changes are required to recruit repair complexes to the site of damage, and repress transcription of genes flanking the break during repair. We recently found that euchromatic histone methyl transferase 2 (EHMT2, also known as G9a) plays a crucial role in regulating DNA repair. EHMT2 is rapidly recruited to sites of DNA breaks in a manner dependent on its phosphorylation by ATM at serine 569. Interestingly, the methyl-transferase activity of EHMT2 is required for the early, H2AX- independent recruitment of DNA repair factors including RNF8, 53BP1 and BRCA1; We have also recently found that EHMT2 activity is required for normal regulation of key features of ATM-mediated induction of transcriptional silencing near a DNA break. This includes regulation and recruitment of key silencing factors such as BMI1 and CDYL. These findings suggest that EHMT2 has a key role in regulating both DNA repair efficiency and modulating transcriptional regulation near a DNA break. As small molecule inhibitors of EHMT2 are now being developed for clinical use, EHMT2 may be one of the components of the DNA repair pathway that may be therapeutically targetable. We hypothesize that EHMT2 activity during the DNA damage response is 1) required to mediate local transcriptional silencing by regulating both histone and non-histone substrates 2) enables the early, H2AX independent, recruitment of DNA repair factors to sites of DNA damage through the action of key binding partners and substrates and 3) is critical for cell survival in the presence of specific cancer-associated DNA repair defects. These hypotheses will be investigated through the following set of specific aims. Aim 1. Determine the role of EHMT2 in DNA-damage-induced transcriptional silencing. Aim 2: Determine role of EHMT2 in H2AX-indpendent recruitment of DNA repair factors. Aim 3: Define synthetic lethal interactions of EHMT2 in DNA repair and chromatin regulation. To address these aims we will use carefully interrogate the effect of EHMT2 loss and inhibition on multiple aspects of DNA repair and local chromatin landscape near a DNA break. This will be done in cell and mouse systems where induction of DNA damage is engineered at specific genomic locations, and the effect of DNA damage on local chromatin landscape and recruitment of DNA repair factors can be systematically interrogated. We will also determine how EHMT2 inhibition using small molecule catalytic inhibitors perturbs repair pathway and survival in both wt cells and cells harboring specific, clinically relevant mutations in DNA repair pathways, including ATM, BRCA1, PALB2 and BRCA2. By this approach we will gain new insight into the fundamental role of EHMT2 in regulating DNA repair, and develop synthetic lethal strategies to target cancers harboring specific defects in DNA repair or chromatin regulation with EHMT2 inhibitors.

项目总结/摘要： DNA断裂的诱导启动DNA损伤信号通路，导致协调和动态的 局部染色质景观的修饰，包括蛋白质翻译后修饰的调节变化， DNA损伤位点两侧的组蛋白。这些染色质的变化是募集修复复合物所必需的 在修复过程中抑制断裂两侧基因的转录。我们最近发现 常染色质组蛋白甲基转移酶2（EHMT 2，也称为G9 a）在调节 DNA修复EHMT 2以依赖于其磷酸化的方式快速募集到DNA断裂位点 在丝氨酸569处通过ATM。有趣的是，EHMT 2的甲基转移酶活性是早期H2 AX-1所必需的。 DNA修复因子包括RNF 8，53 BP 1和BRCA 1的独立招募;我们最近还 发现EHMT 2活性是ATM介导的诱导细胞凋亡的关键特征的正常调节所必需的。 DNA断裂附近的转录沉默。这包括关键沉默因子的调节和募集 例如BMI 1和CDYL。这些发现表明，EHMT 2在调节DNA修复和DNA修复中起着关键作用。 效率和调节DNA断裂附近的转录调控。作为EHMT 2的小分子抑制剂 EHMT 2可能是DNA修复途径的组成部分之一 这可能是治疗靶向的。我们假设EHMT 2在DNA损伤反应中的活性 1）通过调节组蛋白和非组蛋白底物来介导局部转录沉默所需2） 能够早期，H2 AX独立的，DNA修复因子的招聘到DNA损伤的网站，通过 关键结合配偶体和底物的作用，以及3）在特异性抗体存在下对细胞存活至关重要。 癌症相关的DNA修复缺陷这些假设将通过以下一组 明确的目标。目标1.确定EHMT 2在DNA损伤诱导的转录沉默中的作用。目标二： 确定EHMT 2在DNA修复因子的H2 AX依赖性募集中的作用。目标3：定义合成致死 EHMT 2在DNA修复和染色质调节中的相互作用。为了实现这些目标，我们将谨慎地使用 询问EHMT 2丢失和抑制对DNA修复和局部染色质的多个方面的影响 DNA断裂附近的景观这将在细胞和小鼠系统中完成，其中诱导DNA损伤是 在特定的基因组位置进行工程改造，以及DNA损伤对局部染色质景观和 可以系统地询问DNA修复因子的募集。我们还将确定EHMT 2 使用小分子催化抑制剂的抑制扰乱了WT细胞中的修复途径和存活， 在DNA修复途径中携带特异性临床相关突变的细胞，包括ATM、BRCA 1、PALB 2 BRCA2。通过这种方法，我们将对EHMT 2在调节DNA中的基本作用有新的认识。 修复，并开发合成致死策略，以靶向在DNA修复中具有特定缺陷的癌症， 染色质调控与EHMT 2抑制剂。