A novel role for 53BP1 independent of damage that controls chromatin structure

 描述(由申请人提供)：AID酶在类开关重组(CSR)过程中启动DNA双链断裂(DSB)的引入，从而使B细胞暴露在基因组不稳定的高机会下。事实上，95%的侵袭性淋巴瘤起源于B细胞，CSR期间涉及IGH的易位是弥漫性大B细胞淋巴瘤等疾病的主要原因。因此，对这一危险的程序性重组事件有一个完整的了解是至关重要的，以便更好地了解哪些条件容易导致这些癌症的发生，以及如何潜在地治愈它们。尽管在理解双链断裂在CSR过程中是如何产生的方面已经取得了相当大的进展，但对双链断裂的引入和修复的动态的机械性洞察仍然不清楚。在这项提议中，我将 回答其中的一些问题。为了解决这些问题，我开发了一种单基因座分析方法来研究DNA断裂是如何在CSR过程中引入的。我的初步数据显示，在招募艾滋病的两个地点中，最上游的地区首先被瞄准。负责这种有序引入DNA断裂的机制依赖于53BP1。这是令人惊讶的，因为53BP1是一种已知在DNA断裂修复中发挥作用的蛋白质，因此在决定断裂在哪里引入方面的作用是没有预料到的。此外，这一过程似乎不需要参与DNA修复的其他蛋白质，从而支持53BP1能够在DNA损伤之前指导AID靶向的理论。这一点具有很高的相关性，因为53BP1在CSR过程中参与DNA修复的所有蛋白质中具有最极端的缺陷，因此表明有序地引入中断是成功重组事件所必需的。在这项提案中，我将研究53BP1被招募到DNA损伤上游的机制(目的1)。然后，我将通过改变IGH染色质架构(AIM 2)来表征53BP1如何影响Break引入的顺序。在独立阶段，我将把这些发现扩展到其他基因座和细胞类型。具体地说，我将调查53BP1是否在沉默转座子(AIM 3)的上游损伤是重要的。这一点非常重要，因为在小鼠和人类基因组中，转座元件被估计负责10%的从头突变活性。这一奖项的K99阶段将是我完成博士后培训的关键。它会让我 继续在简·斯科克博士的监督下工作，我将要求她完成目标1，在那里她关于DNA FISH和DNA损伤修复的知识将是有用的。最重要的是，我的顾问委员会的其他3名成员将需要对我想要作为独立研究员建立的实验进行培训。丹尼·莱因伯格博士将帮助我进行芯片序列和DNA甲基化分析。Jef Boeke博士将向我介绍转座子生物学领域。理查德·博诺博士将指导我分析和整合不同类型的全基因组数据集分析。最后，纽约大学的环境和机会(研讨会、会议、课程等)将为我博士后培训的最后阶段提供理想的环境。

 DESCRIPTION (provided by applicant): The AID enzyme initiates introduction of DNA double strand breaks (DSBs) during class switch recombination (CSR) thus exposing B cells to high chances of genomic instability. In fact, 95% of aggressive lymphomas are of B cell origin and translocations involving Igh during CSR are the main cause of diseases such as diffuse large B cell lymphoma. It is therefore crucial to have a complete understanding of this dangerous programed recombination event in order to better understand what conditions predispose to the occurrence of these cancers and how they can potentially be cured. Even though considerable progress has been made in understanding how DSBs are generated during CSR, mechanistic insight into the dynamics of DSBs introduction and repair is still unclear. In this proposal I will address some of these questions. To address these questions, I developed a single locus assay to investigate how DNA breaks are introduced during CSR. My preliminary data shows that of the two locations to which AID is recruited, the most upstream region is targeted first. The mechanism responsible for this ordered introduction of DNA breaks relies on 53BP1. This was surprising as 53BP1 is a protein known to play a role in DNA break repair and therefore a role in deciding where breaks are introduced was not expected. In addition, no other protein involved in DNA repair seems to be required for this process thus supporting the theory that 53BP1 is able to direct AID targeting, before DNA damage. This is of high relevance as 53BP1 has the most extreme defect of all proteins involved in DNA repair during CSR thus suggesting that ordered introduction of breaks is necessary for a successful recombination event. In this proposal I will study the mechanisms by which 53BP1 is recruited to DNA upstream of damage (AIM 1). I will then characterize how 53BP1 impacts order of break introduction by altering Igh chromatin architecture (AIM 2). In the independent phase I will extend these findings to other loci and cell types. Specifically, I will investigate whether 53BP1 is important, upstream of damage to silence transposons (AIM 3). This is highly important as transposable elements are estimated to be responsible for 10% of the de novo mutagenic activity in mouse and human genomes. The K99 phase of this award will be essential for me to finalize my postdoctoral training. It will allow me to continue to work under supervision of Dr. Jane Skok, which I will require to finalize Aim 1 where her knowledge of DNA FISH and DNA damage repair will be instrumental. Most importantly, training by the other 3 members of my advisory board will be required for the experiments I want to set up as an independent researcher. Dr Danny Reinberg will help me with the ChIP-seq and DNA methylation assays. Dr Jef Boeke will introduce me to the field of transposon biology. Dr. Richard Bonneau will guide me in the analysis and integration of the different types of genome-wide dataset analysis. Finally, the environment and opportunities (workshops, conferences, courses, etc) at NYU will provide the ideal setting for the final stages of my postdoctoral training.