In Vivo Analysis of a Transcriptional Coactivator Domain

描述：（扫描自申请人的摘要）许多发展和

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.