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.
Research investigating the DNA damage response continues to aid further advancements in cancer therapy. Understanding how the proteins that repair breaks in DNA are linked to the proteins that govern gene repression is important for not only for increasing our knowledge base in this critical area, but also for helping to broaden our pool of possible targets for cancer therapeutics.
|Yao, Qing; Zhang, Li; Wan, Xiaobo et al. (2014) Structure and specificity of the bacterial cysteine methyltransferase effector NleE suggests a novel substrate in human DNA repair pathway. PLoS Pathog 10:e1004522|
|Peng, Hongzhuang; Talebzadeh-Farrooji, Mehdi; Osborne, Michael J et al. (2014) LIMD2 is a small LIM-only protein overexpressed in metastatic lesions that regulates cell motility and tumor progression by directly binding to and activating the integrin-linked kinase. Cancer Res 74:1390-1403|
|Prokop, Jeremy W; Rauscher 3rd, Frank J; Peng, Hongzhuang et al. (2014) MAS promoter regulation: a role for Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism. Clin Sci (Lond) 126:727-38|
|Chen, Jiangzhi; Xu, Hong; Zou, Xiuqun et al. (2014) Snail recruits Ring1B to mediate transcriptional repression and cell migration in pancreatic cancer cells. Cancer Res 74:4353-63|
|Prokop, Jeremy W; Liu, Yuanjie; Milsted, Amy et al. (2013) A method for in silico identification of SNAIL/SLUG DNA binding potentials to the E-box sequence using molecular dynamics and evolutionary conserved amino acids. J Mol Model 19:3463-9|
|White, David; Rafalska-Metcalf, Ilona U; Ivanov, Alexey V et al. (2012) The ATM substrate KAP1 controls DNA repair in heterochromatin: regulation by HP1 proteins and serine 473/824 phosphorylation. Mol Cancer Res 10:401-14|
|Hou, Zhaoyuan; Peng, Hongzhuang; White, David E et al. (2010) 14-3-3 binding sites in the snail protein are essential for snail-mediated transcriptional repression and epithelial-mesenchymal differentiation. Cancer Res 70:4385-93|