In Vivo Analysis of a Transcriptional Coactivator Domain

转录辅激活因子结构域的体内分析

• 批准号: 6330900
• 负责人: PAUL K BRINDLE
• 金额: $ 26.82万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6330900
• 关键词: binding proteins; cAMP response element binding protein; developmental genetics; genetically modified animals; immunoprecipitation; laboratory mouse; microarray technology; oligonucleotides; oncoproteins; point mutation; protein structure function; transcription factor

DESCRIPTION: (Scanned from the applicant's abstract) Many developmental and physiological processes involve the regulation of gene expression by specific DNA-binding transcription factors (TFs). The cAMP- and calcium-responsive factor CREB, and the hematopoietic cell-determining factor c-Myb, bind to their respective target promoters and enhancers, and stimulate transcription by binding the transcriptional coactivators CREB-binding protein (CBP) and its paralog p300. In humans, CBP mutations are associated with acute myeloid leukemia and Rubinstein-Taybi Syndrome (RTS is characterized by mental retardation, craniofacial defects, broad big toes and thumbs, and an abnormal incidence of neoplasms). CBP and p300 have at least four distinct TF-binding domains that act as nuclear foci for different intracellular signaling pathways. One of these domains, KIX, has been extensively studied in vitro as a convergence point for cAMP- and calcium-signals through CREB, but also as a mediator of c-Myb tissue specific activity. In order to understand how TFs interact with transcriptional co-activators to drive tissue- and signal-specific gene expression in mammals, the CBP and p300 KIX domains will be tested in vivo by introducing genes with targeted MX mutations into mice by homologous recombination. Mutating KIX in CBP results in a hypomorphic protein that has characteristics unique from those of wild-type CBP in transcription assays. These mutant mice will be used to elucidate CBP- and p300-KIX-dependent functions in development, physiology and gene expression in vivo. Embryonic fibroblast cells derived form KIX mutant mice will be used to study MX functions using transcription assays in vitro, to map CBP/p300 domains necessary and sufficient to rescue KIX-dependent TF activity, and to biochemically characterize the mutant co-activators. If successful, these studies will develop new model systems for studying in vitro and in vivo the roles of CBP and p300 in tissue-specific and signal-dependent transcription. Additional insight will be gained about the role of the MX domain in previously described target tissues of the CREB and Myb families of proteins, including the brain, blood, mammary gland, and testes. KIX mutant mice will also be valuable models for studying the roles of these co-activators in physiological processes relevant to human health such as memory, immunity, reproduction, growth, aging, and metabolism.

描述：（从申请人的摘要扫描）许多发展和 生理过程涉及通过特异性免疫调节基因表达。 DNA结合转录因子（TF）。cAMP和钙离子反应性 CREB因子和造血细胞决定因子c-Myb与它们的 各自的靶启动子和增强子，并通过 结合转录辅激活因子CREB结合蛋白（CBP）及其 paradox p300.在人类中，CBP突变与急性骨髓性白血病相关。 白血病和Rubinstein-Taybi综合征（RTS的特征是精神分裂症， 发育迟缓，颅面缺陷，宽的大脚趾和拇指，以及异常的 肿瘤发生率）。CBP和p300具有至少四种不同的TF结合 作为不同细胞内信号传导的核焦点的结构域 途径。这些结构域之一，KIX，已经在体外被广泛研究，作为一种免疫调节剂。 通过CREB的cAMP和钙信号的会聚点，但也作为一个 c-Myb组织特异性活性的介质。为了了解TF是如何 与转录辅激活因子相互作用以驱动组织-和 在哺乳动物中的信号特异性基因表达，CBP和p300 KIX域将 通过将具有靶向MX突变的基因引入小鼠体内进行体内测试， 同源重组突变CBP中的KIX导致亚型蛋白 其在转录方面具有不同于野生型CBP的独特特征 测定。这些突变小鼠将用于阐明CBP和p300-KIX依赖性 在体内发育、生理和基因表达中起作用。胚胎 来自KIX突变小鼠的成纤维细胞将用于研究MX 使用体外转录测定功能，以定位CBP/p300结构域 必要和足够的救援KIX依赖的TF活性，并 对突变的共激活因子进行生物化学表征。如果成功，这些 研究将开发新的模型系统，用于体外和体内研究， CBP和p300在组织特异性和信号依赖性转录中的作用。 将获得有关MX域在以前的 描述了CREB和Myb蛋白家族的靶组织，包括 大脑血液乳腺和睾丸KIX突变小鼠也将 为研究这些共激活剂在生理学中的作用提供了有价值的模型。 与人类健康相关的过程，如记忆，免疫，生殖， 生长衰老和新陈代谢