Developmental Mechanisms of the Chromodomain Gene Chd7

描述(由申请人提供)：这些研究对于提高我们对与CHD7缺乏相关的发育缺陷的理解有直接的相关性。基因表达的精确调控对大多数组织和器官的正常发育至关重要。染色域蛋白以DNA结合和依赖于ATP的解旋酶结构域为特征，但对染色质重塑和对基因表达的表观遗传影响知之甚少。在人类中，CHD7的单倍性不足会导致电荷综合征。CHD7是第三个色域蛋白亚家族的成员。Charge是一种以眼部缺损、心脏缺陷、后鼻孔闭锁、生长发育迟缓、生殖器发育不全以及包括耳聋和前庭功能障碍在内的耳朵异常为特征的多发性畸形。我们产生了一种捕获零等位基因(Chd7Gt/)的杂合子小鼠。这些小鼠表现出许多与人类相似的充电表型，包括严重的内耳缺陷和出生后生长迟缓。ChD7Gt/小鼠在CHD7阳性组织中也表达β-半乳糖苷酶报告基因，可用于追踪CHD7缺陷细胞的命运。人类和小鼠的Charge临床特征多种多样且不完全穿透，CHD7功能丧失的人和小鼠最常见的发育畸形是半规管发育不全或发育不良。CHD7缺乏的小鼠内耳缺陷复杂，包括半规管畸形和感觉上皮神经支配。特定的组织和细胞类型似乎对CHD7的剂量唯一敏感，但CHD7在细胞增殖、分化和存活中缺乏的细胞CHD7表达模式和机制尚不清楚。此外，CHD7在耳朵和神经细胞中的相互作用伙伴和下游靶点也没有被描述。这些知识将有助于指导合理开发内耳缺陷的治疗方法。我们的全球假设是CHD7在转录复合体中发挥作用，在发育中的耳朵和神经组织中抑制或激活下游的靶基因。我们有三个具体目标： (1)研究CHD7突变小鼠的细胞增殖、存活和分化情况， (2)确定CHD7表达组织对内耳发育的贡献； (3)从野生型和CHD7突变的胚胎中鉴定培养的内耳和神经干细胞，并利用它们来确定CHD7是否直接与候选靶基因的启动子结合。 拟议的实验结果将为染色质重塑和转录调控的基本过程带来新的见解。提高对CHD7的组织特异性要求的理解也可能改善与电荷相关的内耳和其他发育缺陷的诊断和治疗。公共卫生相关性：该项目对于理解CHD7缺乏导致儿童和成人Charge综合征听力和平衡障碍的机制具有直接相关性。Charge综合征是一种多发性异常疾病，全世界每10,000名新生儿中就有1名受到影响。这项建议中描述的实验结果将提高我们对调节基本染色质生物学的遗传和表观遗传因素及其对发育的影响的理解。

DESCRIPTION (provided by applicant): These studies have direct relevance for improving our understanding of the developmental defects associated with Chd7 deficiency. Precise regulation of gene expression is critical for normal development of most tissues and organs. Chromodomain proteins, characterized by DNA binding and ATP-dependent helicase domains, have poorly understood roles in chromatin remodeling and exert epigenetic influences on gene expression. In humans, haploinsufficiency for CHD7, a member of the third subfamily of chromodomain proteins, causes CHARGE syndrome. CHARGE is a multiple anomaly disorder characterized by ocular coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, and ear anomalies including deafness and vestibular disorders. We generated mice heterozygous for a gene trapped null allele (Chd7Gt/+). These mice exhibit many features similar to human CHARGE phenotypes, including severe inner ear defects and postnatal growth delays. Chd7Gt/+ mice also express a ¿-galactosidase reporter in Chd7-positive tissues, and can be used for tracking the fates of Chd7 deficient cells. The clinical features of CHARGE in humans and mice are variable and incompletely penetrant, and the most common developmental malformation in both humans and mice with loss of CHD7 function is semicircular canal dysgenesis or hypoplasia. Inner ear defects with Chd7 deficiency in mice are complex, and include malformations of the semicircular canals and innervation of sensory epithelia. Specific tissues and cell types appear uniquely sensitive to Chd7 dosage, but the cellular Chd7 expression patterns and mechanisms of Chd7 deficiency in cell proliferation, differentiation, and survival are not known. Moreover, CHD7 interacting partners and downstream targets in the ear and neural cells are not characterized. Such knowledge would help guide rational development of therapies for inner ear defects. Our global hypothesis is that CHD7 functions in a transcriptional complex to repress or activate downstream target genes in developing ear and neural tissues. We have three specific aims: (1) Characterize cell proliferation, survival, and differentiation in Chd7 mutant mice, (2) Determine the contributions of CHD7-expressing tissues to inner ear development, and (3) Characterize cultured inner ear and neural stem cells from wildtype and Chd7 mutant embryos, and use them to determine whether CHD7 directly binds the promoters of candidate target genes. Results of proposed experiments will bring novel insights into fundamental processes of chromatin remodeling and transcriptional regulation. Improved understanding of tissue-specific requirements for Chd7 might also improve the diagnosis and treatment of CHARGE-related inner ear and other developmental defects. PUBLIC HEALTH RELEVANCE: This project has direct relevance for understanding the mechanisms of CHD7 deficiency-induced hearing and balance disorders in children and adults with CHARGE syndrome. CHARGE syndrome is a multiple anomaly condition that affects 1 in 10,000 newborn children worldwide. Results of experiments described in this proposal will improve our understanding of genetic and epigenetic factors regulating basic chromatin biology, and their impact on development.