Role of Gene Silencing in the DNA Damage Response

描述（由申请人提供）：在定义控制DNA损伤反应的分子和细胞机制以及该途径如何决定抗癌药物的功效方面已经花费了相当大的努力。这些进步推动了药物开发，有朝一日可能帮助医生针对特定疾病量身定制癌症治疗方案。DNA损伤反应需要一个协调的核-细胞质级联事件，最终汇聚到染色质中受损的DNA。介导染色质重塑的蛋白质和介导这种损伤反应的蛋白质之间的联系很少。我们研究了DNA损伤诱导的KAP1磷酸化，KAP1是所有蟹黄锌指蛋白的专用共同抑制因子。该提案将利用三种现有技术，并寻求以一种新颖的方式应用它们。首先，我们将利用ChIP-Chip来确定DNA损伤后KAP1是否仍与已知结合位点相关。如果KAP1在损伤后改变了结合伙伴，那么用全基因组平铺阵列检测到的KAP1结合的DNA区域应该发生改变。如果KAP1仍然与KRAB-ZFPs（或其他尚未确定的锚点）相关联，那么KAP1结合的检测位点在损伤后应该保持不变。这是首次使用全基因组平铺阵列来确定一个关键的染色质重塑因子是否在其专用结合位点附近局部参与DNA损伤反应，还是在受损位点出现时全局参与DNA损伤反应。该项目的这一部分是Rauscher和Farnham实验室所做工作的逻辑延伸，并利用了双方在KAP1/KRAB-ZFP范式方面取得的重大进展。其次，我们将应用现有的蛋白质半合成方法来创建磷酸化KAP1的纯池。以这一改良的KAP1为诱饵，我们将尝试分离出一组新的KAP1损伤相关因子。第三，我们将修改目前用于转录实时成像的串联阵列，用于观察和量化双链断裂处修复因子的聚集。我们将第一次能够观察到单个癌细胞中修复机制的协调组装。此外，它将填补我们知识中的一个重要空白，即染色质在DNA损伤位点周围如何在修复之前快速重塑。这个项目代表了Rauscher和Janicki实验室一个新的和令人兴奋的研究方向。根据我们之前的研究和引用的文献，我们认为KAP1的磷酸化可能在DNA修复机制的某些元件的定位和组装中至关重要。本研究不仅试图阐明KAP1在DNA修复中的作用，而且还试图确定DNA损伤后KAP1/KRAB-ZFP沉默复合体是否发生实质性重组。

DESCRIPTION (provided by applicant): Considerable effort has been expended in defining the molecular and cellular mechanisms governing the DNA damage response and how this pathway determines the efficacy of anti-cancer drugs. These strides have furthered drug development and someday may help physicians tailor their cancer treatment to specific diseases. The DNA damage response requires a coordinated nucleo-cytoplasmic cascade of events which ultimately converge on damaged DNA packed in chromatin. Few connections between the proteins that mediate chromatin remodeling and the proteins that mediate this damage response have been demonstrated. We have investigated the DNA damage-induced phosphorylation of KAP1, the dedicated co-repressor for all KRAB-zinc finger proteins. This proposal will utilize three existing technologies and seek to apply them in a novel way. First, we will utilize ChIP-Chip to determine whether the KAP1 remains associated to known binding sites after DNA damage. If KAP1 changed binding partners after damage, then the regions of DNA bound by KAP1 as detected with a whole genome tiling array should become altered. If KAP1 remains associated to KRAB-ZFPs (or another yet to be determined anchor), then the detected sites bound by KAP1 should remain unchanged following damage. This is the first time a whole genome tiling array will be used to determine whether a key chromatin remodeling factor participates in the DNA damage response locally near its dedicated binding site, or globally as damaged sites arise. This part of the project is a logical extension of work done in both Rauscher and Farnham laboratories and takes advantages in major strides made by both parties in the KAP1/KRAB-ZFP paradigm. Second, we will apply an existing method of protein semisynthesis to create a pure pool of phosphorylated KAP1. Using this modified KAP1 as bait, we will attempt to isolate a new group of damage induced KAP1-associating factors. Third, we will modify a tandem array that is currently used for real-time imaging of transcription for use to observe and quantitate the aggregation of repair factors at double stranded breaks. For the first time, we will be capable of observing the coordinated assembly of repair machinery in individual cancer cells. Moreover, it will fill an important gap in our knowledge, namely, how chromatin is rapidly remodeled around sites of DNA damage prior to its repair. This project represents a new and exciting research direction for the Rauscher and Janicki laboratories. Based on our previous studies and the literature cited, we believe that the phosphorylation of KAP1 may be critical in the localization and assembly of some elements of the DNA repair machinery. This proposal not only seeks to clarify the role of KAP1 in DNA repair, but also to determine whether a substantial reorganization of the KAP1/KRAB-ZFP silencing complex occurs after DNA damage.