Analysis of craniofacial transcription factor and coactivator functional networks

描述（由申请人提供）：颅面畸形是美国常见的出生缺陷，仅口面裂每年就影响6,800名婴儿。在小鼠中，超过600个基因的突变，其中许多编码转录调节因子，与颅面异常有关。CREB结合蛋白（CBP）和密切相关的p300（CBP/p300统称）是转录共激活因子，与约2，000种哺乳动物转录调节蛋白中的约15%发生物理或功能性相互作用。CBP的单倍不足（一个无效等位基因）以及较小程度的p300导致Rubinstein-Taybi综合征（RTS），其特征为颅面异常、宽拇指和脚趾以及智力和生长迟缓。CBP/p300中的几个独特结构域与许多转录因子的激活结构域结合，但突变CBP（和p300）导致RTS的功能不足尚不清楚。CBP中这些结构域之一CH 1缺陷的小鼠具有令人想起CBP单倍不足小鼠的颅面异常，后者是RTS的模型。因此，CH 1结构域是颅面发育所需的CBP的重要结构域之一。广泛的长期目标是解决CH 1结构域如何介导正常颅面发育。目前的建议的目的是确定CBP/p300 CH 1结构域在介导颅面关键转录中的作用。核心假设是，CH 1结构域是一个主要的“枢纽”，连接转录因子和靶基因的调控网络，是必不可少的颅面发育。这些网络的发现将有助于确定参与非综合征性口面裂的候选基因和途径，例如，对潜在的多基因机制知之甚少。具体目标1是确定已知的颅面决定转录因子，需要的CH 1结构域的反式激活功能和CBP/p300的相互作用，使用小鼠细胞在瞬时转染试验。具体目标2是使用CH 1结构域缺陷的原代细胞，通过使用微阵列和实时RT-PCR测量内源性基因表达，以及使用染色质免疫沉淀的CBP/p300募集来发现需要CH 1结构域的颅面决定基因。CH 1/颅面网络的意义将是阐明调节颅面基因转录的信号传导和转录机制，并揭示颅面发育的遗传相互作用。此外，一个高度连接的颅面网络的演示表明，CH 1结构域，更普遍的CBP和p300，很可能作为遗传修饰剂在多基因非综合征口面异常。从长远来看，该网络将有助于识别与人类颅面异常有关的基因和途径，并改善诊断和治疗。公共卫生相关性：许多颅面畸形的例子，如唇腭裂，与多个基因异常有关，这种复杂性阻碍了对根本原因的理解。基因相互作用网络的建立将有助于识别对头骨和面部形成至关重要的类似调控基因。这些知识将导致对这一复杂的发育过程的新看法，并可能改善颅骨和面部异常的诊断和治疗。

DESCRIPTION (provided by applicant): Craniofacial anomalies are common birth defects in the U.S., with orofacial clefts alone affecting 6,800 infants annually. In mice, mutations in more than 600 genes, many encoding transcriptional regulators, are associated with craniofacial anomalies. CREB-binding protein (CBP) and the closely related p300 (CBP/p300 collectively) are transcriptional coactivators that interact physically or functionally with ~15% of the estimated 2,000 mammalian transcriptional regulatory proteins. Haploinsufficiency (one null allele) of CBP, and to a lesser degree p300, causes Rubinstein-Taybi Syndrome (RTS), characterized by craniofacial anomalies, broad thumbs and toes, and mental and growth retardation. Several unique domains in CBP/p300 bind to the activation domains of numerous transcription factors, but the insufficient functions of mutant CBP (and p300) that lead to RTS are unclear. Mice deficient for one of these domains in CBP, CH1, have craniofacial anomalies reminiscent of CBP haploinsufficient mice, the latter being a model for RTS. The CH1 domain is therefore one of the essential domains of CBP needed for craniofacial development. The broad, long-term goal is to address how the CH1 domain mediates normal craniofacial development. The objective of the current proposal is to determine the role of the CBP/p300 CH1 domain in mediating craniofacial-critical transcription. The central hypothesis is that the CH1 domain is a major "hub" that connects transcription factors and target genes in a regulatory network that is essential for craniofacial development. Discovery of such networks will help identify candidate genes and pathways involved in nonsyndromic orofacial clefting, for example, where the underlying multigenic mechanisms are poorly understood. Specific aim 1 is to identify the known craniofacial-determining transcription factors that require the CH1 domain for transactivation function and CBP/p300-interaction using mouse cells in transient transfection assays. Specific aim 2 is to use CH1 domain-deficient primary cells to discover the craniofacial-determining genes that require the CH1 domain by measuring endogenous gene expression using microarrays and real-time RT-PCR, and CBP/p300 recruitment using chromatin immunoprecipitation. The significance of a CH1/craniofacial network will be to illuminate the signaling and transcriptional mechanisms that regulate craniofacial gene transcription, and to reveal genetic interactions that underlie craniofacial development. Moreover, demonstration of a highly connected craniofacial network would suggest that the CH1 domain, and more generally CBP and p300, are likely to act as genetic modifiers in multigenic nonsyndromic orofacial anomalies. In the long-term, the network will help identify genes and pathways involved in human craniofacial anomalies and lead to improved diagnosis and treatment. PUBLIC HEALTH RELEVANCE: Many instances of craniofacial anomalies, such as cleft lip and palate, are associated with multiple gene abnormalities, and this complexity hampers understanding the fundamental causes. The creation of a gene interaction network will help identify similarly regulated genes that are important for the formation of the skull and face. Such knowledge will lead to a new view of this complicated developmental process, and possibly to improved diagnosis and treatment of skull and facial anomalies.