In Vivo Analysis of a Transcriptional Coactivator Domain

描述：(从申请者的摘要中浏览)许多发育和 生理过程涉及到通过特定的基因表达调节 DNA结合转录因子(TF)。对cAMP和钙的反应 CREB因子和造血细胞决定因子c-Myb与它们的 分别针对启动子和增强子，并通过以下方式刺激转录 结合转录共激活物CREB结合蛋白(CBP)及其 旁路P300。在人类中，CBP突变与急性髓系疾病有关 白血病和Rubinstein-Taybi综合征(RTS)以精神症状为特征 发育迟缓，头面部缺陷，大拇指和拇指宽大，以及异常 肿瘤发病率)。CBP和p300至少有四个不同的Tf结合 作为不同细胞内信号的核焦点的结构域 小路。其中一个结构域，KIX，在体外被广泛研究为一种 CAMP-和钙-信号的汇聚点通过CREB，但也作为一个 C-Myb组织特异性活性的介体。为了了解TFS是如何 与转录共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结构域将 通过以下方式将具有靶向MX突变的基因导入小鼠体内进行测试 同源重组。CBP中KIX的突变导致亚型蛋白 在转录方面具有与野生型CBP不同的特征 化验。这些突变小鼠将被用来阐明CBP和p300-KIX依赖 在体内发育、生理和基因表达方面的功能。胚胎 来自KIX突变小鼠的成纤维细胞将用于研究MX 利用体外转录实验定位CBP/p300结构域的功能 对于挽救依赖KIX的转铁蛋白活性是必要的和充分的，并 对突变的共活化子进行生化表征。如果成功，这些 研究将开发新的体外和体内研究模型系统 CBP和p300在组织特异性和信号依赖转录中的作用。 我们将深入了解MX领域在以前的 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突变小鼠也将被 研究这些共激活子在生理学中作用的有价值的模型 与人类健康有关的过程，如记忆、免疫、生殖、 生长、衰老和新陈代谢。

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.