DNA damage induces a variety of responses that are initiated by the ataxia- telangiectasia mutated (ATM) and ataxia- telangiectasia and Rad 3-related (ATR) kinases. The identification of a diverse group of ATM/ATR substrates indicates that the DNA damage response mediated by these proteins is extensive and affects a majority of physiological pathways at the cellular level. Despite a wealth of knowledge on DNA damage signaling at the cellular level, the recognition, amplification, and signal transduction of DNA damage in the context of chromatin is not completely understood. The Chromodomain helicase DNA binding (CHD) proteins have distinct structural motifs that implicate functional roles in chromatin remodeling and transcriptional regulation. In an effort to understand the role of the CHD family member, CHD2, in mammalian development and physiology, we generated a Chd2 mutant mouse model. Our data suggest that the Chd2 mutant mouse model photocopies'a variety of physiological defects exhibited by the ATM deficient mice. Furthermore, the CHD2 protein is phosphorylated after DNA damage, consists of ten putative ATM phosphorylation sites, and interacts with phosphorylated H2AX (?H2AX). In this proposal we will test the hypothesis that the ATM kinase phosphorylates CHD2 in response to DNA damage and modulates its function at the chromatin level.
Specific Aim 1 : Determine if CHD2 is a substrate of ATM. Hypothesis: ATM phosphorylates CHD2 and regulates its function. We will determine ATM specific phosphorylation of CHD2 using normal and A-T cells. Phosphorylation of CHD2 by other PIKK kinases will be ascertained using kinase specific inhibitors and DNA damage inducers. The identity of ATM specific SQ motifs within CHD2 will be determined using recombinant peptides and in vitro assays. In addition, we will also determine ATM-CHD2 interactions using coimmunoprecipitation analyses.
Specific Aim 2 : Determine the role of ATM in the modulation of CHD2 levels and CHD2 function. Hypothesis: Phosphorylation of CHD2 by ATM is necessary for its induction and functional modulation after DNA damage. We will determine the induction and stability of CHD2 in normal and A-T cells in response to DNA damage. We will also determine if ATM specific phosphorylation affects the stability of CHD2 and determine if ATM mediated phosphorylation is necessary for CHD2 to interact with ?H2AX. Finally we will determine if CHD2 and ATM cooperate to induce cell cycle arrest and apoptosis in response to DNA damage induction.
This project will determine if the ATM protein interacts and phosphorylates the chromatin remodeling protein, Chromodomain helicase DNA binding protein-2 (CHD2). Determining the role of ATM in modulating chromatin remodeling will allow us to formulate therapeutic approaches aimed at affecting chromatin remodeling in AT patients in the future.
| Rajagopalan, Sangeetha; Nepa, Justin; Venkatachalam, Sundaresan (2012) Chromodomain helicase DNA-binding protein 2 affects the repair of X-ray and UV-induced DNA damage. Environ Mol Mutagen 53:44-50 |
