Developmental Mechanisms of the Chromodomain Gene Chd7

描述（由申请人提供）：这些研究与提高我们对与Chd 7缺乏相关的发育缺陷的理解直接相关。基因表达的精确调控对于大多数组织和器官的正常发育至关重要。染色体结构域蛋白，其特征在于DNA结合和ATP依赖性解旋酶结构域，在染色质重塑中的作用知之甚少，并对基因表达产生表观遗传影响。在人类中，CHD 7（第三个染色体结构域蛋白亚家族的成员）的单倍不足导致CHARGE综合征。CHARGE是一种多发性异常疾病，其特征为眼缺损、心脏缺陷、软骨闭锁、生长发育迟缓、生殖器发育不全和耳异常（包括耳聋和前庭疾病）。我们产生了基因捕获无效等位基因（Chd 7 Gt/+）的杂合子小鼠。这些小鼠表现出许多与人类CHARGE表型相似的特征，包括严重的内耳缺陷和出生后生长延迟。Chd 7 Gt/+小鼠也在Chd 7阳性组织中表达半乳糖苷酶报告基因，可用于追踪Chd 7缺陷细胞的命运。CHARGE在人类和小鼠中的临床特征是可变的和不完全渗透的，并且在人类和小鼠中具有CHD 7功能丧失的最常见的发育畸形是半规管发育不全或发育不全。Chd 7缺乏的小鼠内耳缺陷是复杂的，包括半规管畸形和感觉上皮的神经支配。特定的组织和细胞类型似乎对Chd 7剂量唯一敏感，但细胞Chd 7表达模式和Chd 7缺乏在细胞增殖，分化和存活中的机制尚不清楚。此外，CHD 7相互作用伙伴和下游目标在耳朵和神经细胞没有特征。这些知识将有助于指导内耳缺陷治疗的合理发展。我们的总体假设是CHD 7在转录复合物中发挥作用，以抑制或激活发育中的耳和神经组织中的下游靶基因。我们有三个具体目标： (1)表征Chd 7突变小鼠中的细胞增殖、存活和分化， (2)确定CHD 7表达组织对内耳发育的贡献， (3)鉴定来自野生型和Chd 7突变胚胎的培养的内耳和神经干细胞，并使用它们来确定CHD 7是否直接结合候选靶基因的启动子。 拟议的实验结果将带来新的见解染色质重塑和转录调控的基本过程。对Chd 7组织特异性需求的进一步了解也可能改善CHARGE相关内耳和其他发育缺陷的诊断和治疗。公共卫生相关性：该项目与理解CHD 7缺陷引起的儿童和成人CHARGE综合征听力和平衡障碍的机制直接相关。CHARGE综合征是一种多发性异常疾病，影响全球1/10，000的新生儿。本提案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.