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Characterization of the modifying enzymes and binding effectors for a novel histone modification site during DNA damage response
DNA double-stranded break (DSB) is one of the leading causes of genomic instability and carcinogenesis including breast cancer. The involvement of two breast cancer susceptibility genes (BRCA1 and BRCA2) in DSB repair further underscores the importance of DSB in breast cancer development. The hallmark in mammalian DSB response is the formation of IRradiation Induced Foci (IRIF), composed of compacted chromatin structure and numerous DNA repair and checkpoint proteins. The specialized forms of chromatin formed at IRIF aren’t well understood and neither is their role in the regulation of DSB response. Recent studies demonstrated phosphorylation of H2A.X (a specialized histone H2A variant) at Ser139 is one of the earliest events at IRIF. In support, H2A.X-deficiency predisposes mice to profound genomic instability and turmorigenesis. Despite its critical role in preventing genomic instability and cancer, how H2A.X engages DSB repair pathways remains elusive. My preliminary studies demonstrate that Tyr142 is a novel phosphorylation mark on H2A.X, which plays critical roles in DSB response. Objective/Hypothesis: The objective of this proposal is to understand the molecular pathways preventing genomic instability and caner development by elucidating and characterizing the modification enzymes and novel effector binding proteins of phosphorylated H2A.X C-tail. I hypothesize that: 1. H2A.X Tyr142 phosphorylation is regulated by unidentified nuclear tyrosine kinase(s) and phosphatase(s) in DSB response; 2. The “phosphorylation fingerprint” of Tyr142 and Ser139 (single-Phos, respectively or double-Phos) regulates the recruitment of distinct protein factors, which may be critical mediators for DNA repair pathway and checkpoint control. Specific Aims: I propose to: 1. Identify the enzymes (tyrosine kinase(s) and phosphastase(s)) and/or enzyme complexes for Tyr142 of H2A.X by genomic-wide RNAi screens and biochemical purifications. 2. Identify binding effectors that interact with singly or doubly phosphorylated H2A.X tails by in vitro and in vivo approaches. The functions of the enzymes and binding proteins in mediating signal transduction pathway leading to DNA repair and checkpoint control will be characterized. Potential outcomes and benefits: Functional characterization of enzymes modifying and factors binding to this novel modification mark will provide valuable insights on DSB repair, genomic instability and breast tumorigenesis.
DNA damage predisposes cells to genomic instability and carcinogenesis including breast cancer. The involvement of two breast cancer susceptibility genes (BRCA1 and BRCA2) in DNA damage repair further underscores the importance of DNA repair pathway in breast cancer development. The hallmark in mammalian DNA damage response is the formation of compacted speckles in the nucleus around the damaged site, composed of chromatin (see below) and numerous DNA repair and checkpoint factors. Chromatin is the physiological form of our genome, composed of DNA and histone proteins. Interestingly, human cells introduce H2A.X, a special variant histone protein to mark DNA damage foci in chromatin minuates after DNA damage. In support, the H2A.X gene in the human genome is frequently deleted in a variety of human cancers, including breast carcinoma. Thus, H2A.X has been described as a ‘caregiver’ of our genome. While H2A.X may participate in response to DNA damage by creating a specialized chromatin, little is known about its function(s) and mechanisms. My preliminary studies discovered a novel modification of H2A.X. This mark is constitutively on in normal cells and becomes off in response to DNA damage. Further investigation also demonstrated that this mark plays a critical role in regulating DNA damage response in a variety of human cells, including breast cancer cell lines. The objective of the proposed study is to understand the molecular pathways preventing genomic instability and caner by elucidating and characterizing the modification enzymes and interacting proteins for this mark. I hypothesize that this mark is regulated by specific enzymes during DNA damage response and additional factors directly interact with this mark to mediate its function. Specific Aims : 1. Identify the enzymes adding or removing this mark. 2. Identify binding effectors that directly interact with this mark. Potential outcomes and benefits : Despite its direct involvement in cancer, the function of H2A.X is elusive. C haracterization of enzymes modifying and factors binding to this novel modification mark of H2A.X will provide valuable insights on DSB repair, genomic instability and breast tumorigenesis.