Structural insight into the role of CHD4 PHD fingers.

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DESCRIPTION (provided by applicant): Human CHD4 (chromodomain helicase DMA-binding protein 4) is an autoantigen of myositis, an autoimmune disease associated with an increased risk of conjestive heart failure, arrhythmia, myocardial infarction and cardiac arrest. CHD4 is an ATPase and a major subunit of the NuRD (nucleosome remodeling and deacetylase) repression complex, which is involved in transcriptional regulation and development. The role of CHD4 in this complex, however, remains unclear. Our preliminary data suggest that two PHD fingers of CHD4 recognize histone H3, with PHD2 specifically recognizing H3K9me3, a novel interaction. We hypothesize that the tandem PHD fingers of CHD4 bind to the N-terminal tail of histone H3, tethering the CHD4/NuRD complex to chromatin. We seek to elucidate the molecular basis and the functional significance of the CHD4 PHD-H3 interaction. The specific aims are: 1) To establish the molecular mechanism of histone recognition by the CHD4 PHD1 and PHD2 fingers, and 2) To elucidate the role of the tandem PHD (PHD1.2) domain of CHD4 in histone recognition. A set of structural, biochemical and molecular biology approaches, including NMR spectroscopy, X-ray crystallography, mutagenesis and in vivo chromatin immunoprecipitation and fluorescence microscopy will be used to determine the three dimensional structures of the CHD4 PHD fingers and to probe their interaction with histone H3 peptides. Fluorescence spectroscopy and NMR will be used to measure the binding affinities and to determine whether the two PHD fingers bind cooperatively. PUBLIC HEALTH RELEANCE: The results generated in this study will provide a fundamental knowledge about functioning of the CHD4/NuRD complex and will thus aid in determining the etiology of myositis and may help to identify new prognostic and diagnostic markers of this disease.