Structural insight into the role of CHD4 PHD fingers.

描述(由申请人提供)：人类CHD4(染色域解旋酶DMA结合蛋白4)是肌炎的自身抗原，肌炎是一种自身免疫性疾病，与结节性心力衰竭、心律失常、心肌梗死和心脏骤停的风险增加有关。CHD4是一种ATPase，是核小体重塑和去乙酰化酶抑制复合体的一个主要亚基，参与转录调控和发育。然而，CHD4在这个复合体中的作用尚不清楚。我们的初步数据表明，CHD4的两个PHD手指识别组蛋白H3，而PHD2特异识别H3K9me3，这是一种新的相互作用。我们假设CHD4的串联PHD手指与组蛋白H3的N端结合，将CHD4/NuRD复合体与染色质捆绑在一起。我们试图阐明CHD4 PHD-H3相互作用的分子基础和功能意义。其具体目的是：1)建立CHD4 PhD1和PHD2手指识别组蛋白的分子机制；2)阐明CHD4的串联PHD(PHD1.2)结构域在组蛋白识别中的作用。我们将利用核磁共振光谱、X射线结晶学、诱变、体内染色质免疫沉淀和荧光显微镜等一系列结构、生化和分子生物学方法来确定CHD4 PhD手指的三维结构，并探讨它们与组蛋白H3多肽的相互作用。荧光光谱和核磁共振将被用来测量结合亲和力，并确定两个PHD手指是否协同结合。公共卫生结果：这项研究的结果将提供有关CHD4/NuRD复合体功能的基础知识，从而有助于确定肌炎的病因，并可能有助于确定这种疾病的新的预后和诊断标记物。

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.