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

项目摘要/摘要： DNA断裂的诱导启动了DNA损伤信号通路，导致协调和动态的 对当地染色质景观的修饰，包括对翻译后修饰的调节变化 DNA损伤部位两侧的组蛋白。这些染色质的变化是招募修复复合体所必需的 损伤部位，并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(EHMT2，又称G9a)在调节 DNA修复。EHMT2以依赖于其磷酸化的方式被迅速招募到DNA断裂的位置 被自动取款机用丝氨酸569。有趣的是，EHMT2的甲基转移酶活性对于早期的H_2AX- 独立招募DNA修复因子包括RNF8、53BP1和BRCA1；我们最近也 发现EHMT2活性是ATM介导的诱导的关键特征的正常调节所必需的 DNA断裂附近的转录沉默。这包括管制和招募关键的沉默因素。 如BMI1和CDYL。这些发现表明，EHMT2在调节这两种DNA修复中起着关键作用 DNA断裂附近的效率和调控转录调控。作为EHMT2的小分子抑制剂 EHMT2可能是DNA修复途径的组成部分之一 这在治疗上可能是有针对性的。我们假设在DNA损伤反应中EHMT2的活性 1)需要通过调节组蛋白和非组蛋白底物来调节局部转录沉默 使早期的，非依赖于H_2AX的DNA修复因子通过 关键结合伙伴和底物的作用以及3)在存在特定的 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。目的1.确定EHMT2在DNA损伤诱导的转录沉默中的作用。目标2： 确定EHMT2在H_2AX依赖的DNA修复因子募集中的作用。目标3：定义合成致命性 EHMT2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 探讨EHMT2缺失和抑制对DNA修复和局部染色质的多方面影响 DNA断裂附近的风景。这将在诱导DNA损伤的细胞和小鼠系统中完成。 在特定的基因组位置进行工程，以及DNA损伤对局部染色质景观和 DNA修复因子的招募可以被系统地询问。我们还将确定EHMT2如何 使用小分子催化抑制剂的抑制扰乱了wt细胞和 在DNA修复途径中存在特定的、临床相关的突变的细胞，包括ATM、BRCA1、PALB2 和BRCA2。通过这种方法，我们将对EHMT2在调节DNA中的基础作用有新的认识 修复，并开发合成致命策略，以针对在DNA修复或 EHMT2抑制剂对染色质的调节。

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.