Genomic studies of CHD7 in CHARGE syndrome

描述（由申请人提供）： CHARGE综合征是一种以多器官畸形为特征的先天性疾病。约70%的CHARGE综合征病例是由CHD7基因（编码染色体结构域解旋酶DMA结合蛋白7）的功能丧失从头突变引起的。关于CHD7蛋白的正常功能及其在人类发育和疾病中的作用的信息很少。我们的初步研究表明，CHD7是一种核蛋白，直接结合到多个基因，包括HOX基因（HOXA 5，HOXA 10和HOXA 11）和印记基因（IGF 2和H19），这些基因对正常胚胎发育至关重要。这项研究验证了一个假设，即CHARGE综合征患者的畸形是由特定CHD7靶基因的异常转录引起的。这一假设将在3个特定目标中进行检验。在目标1中，我们将评估CHD7靶基因的子集，以确定CHD7是否直接调节其表达。具体地，在通过RNAi敲低CHD7之前和之后，将在细胞培养物中定量50种CHD7靶标的表达。此外，为了确定CHARGE综合征的异常是否是由于HOX、Igf2和H19的表达失调，我们将分析这些基因在发育中的Chd7突变小鼠中的表达，这些小鼠是CHARGE综合征的优秀模型。在目标2中，我们将研究CHD7被招募到其靶基因的机制，使用体外和体内试验，旨在揭示CHD7和染色质上的各种组蛋白修饰之间的相互作用。在目标3中，我们将使用无偏基因组学方法鉴定在早期发育期间直接依赖于CHD7的CHD7靶标，该方法将微阵列上的染色质免疫沉淀技术（ChIP芯片）与野生型和突变小鼠ES细胞的表达谱相结合。通过鉴定和表征CHD7直接调控的基因，我们希望更多地了解（1）正常的人类发育，（2）构成CHARGE异常谱的孤立出生缺陷的原因，以及（3）这种罕见综合征的病因。除了进一步了解CHD7调控基因的临床意义外，我们还希望通过描述CHD7与其靶基因之间的相互作用，获得大量关于转录调控分子机制的知识。

DESCRIPTION (provided by applicant): CHARGE syndrome is a congenital disease characterized by malformations of multiple organs. ~70% of CHARGE syndrome cases are caused by loss-of-function de novo mutations in the CHD7 gene (coding for chromodomain helicase DMA-binding protein 7). Little information is available about the normal function of the CHD7 protein and its role in human development and disease. Our preliminary studies demonstrate that CHD7 is a nuclear protein that directly binds to multiple genes, including HOX genes (HOXA5, HOXA10, and HOXA11) and imprinted genes (IGF2 and H19) that are essential for normal embryonic development. The proposed research tests the hypothesis that the malformations seen in patients with CHARGE syndrome are caused by aberrant transcription of specific CHD7 target genes. This hypothesis will be tested in 3 Specific Aims. In Aim 1, we will evaluate a subset of the CHD7 target genes to determine if CHD7 directly regulates their expression. Specifically, expression of 50 CHD7 targets will quantified in cell culture before and after knockdown of CHD7 by RNAi. In addition, to determine if anomalies in CHARGE syndrome are due to dysregulated expression of HOX, Igf2, and H19, we will analyze expression of these genes in developing Chd7 mutant mice that are an excellent model CHARGE syndrome. In Aim 2, we will investigate the mechanism by which CHD7 is recruited to its target genes, using in vitro and in vivo assays designed to reveal interactions between CHD7 and various histone modifications on chromatin. In Aim 3, we will identify CHD7 targets that directly depend on CHD7 during early development, using an unbiased genomics approach that combines the technique of chromatin immunoprecipitation on microarrays (ChlP-chip) with expression profiling of wild type and mutant mouse ES cells. By identifying and characterizing the genes that are directly regulated by CHD7, we expect to learn more about (1) normal human development, (2) the causes of the isolated birth defects that make up the spectrum of anomalies in CHARGE, and (3) the etiology of this rare syndrome. In addition to furthering our understanding of the clinical implications of genes regulated by CHD7, we anticipate gaining a significant amount of knowledge about the molecular mechanisms of transcriptional regulation by delineating the interactions between CHD7 and its target genes.