In Vivo Analyses of Transcriptional Coactivator Domains

CREB结合蛋白(CBP，CREBBP)和密切相关的E1a结合蛋白p300(EP300)是转录共激活因子，与大约2000种哺乳动物转录调节蛋白中的10%发生物理或功能上的相互作用。这两个基因都是正常发育和生理所必需的，而且在癌症中会发生功能改变和失活的突变，这表明CBP和p300既可以作为癌蛋白发挥作用，也可以作为肿瘤抑制因子发挥作用。关于CBP和p300的生物学和转录作用，仍有几个主要的悬而未决的问题。首先，CBP和p300的多个不同的转录因子结合域在发育和发育过程中扮演什么角色在很大程度上是未知的。 体内的肿瘤发生。其次，许多体外研究表明，CBP和p300在生物化学上是无法区分的，但还不确定它们在体内的功能有多多余。第三，大量的体外转录研究表明，CBP和p300是重要的共激活因子，但对它们在内源基因表达中的作用知之甚少。第四，不同类别的协同激活剂是否发挥冗余作用还不确定。这些问题构成了该应用程序广泛的、长期的目标的核心，将通过测试CBP和p300共同的两个特定蛋白结合域的体内功能来探讨这些目标的各个方面。已经产生了四个品系的敲入突变小鼠。两个人身上有点突变 CBP和p300的KIX结构域的表面，抑制cAMP反应因子CREB和造血因子c-Myb的结合，以及两株在CBP和p300的CH1结构域具有缺失突变的菌株，该缺失突变取消了缺氧反应因子HIF-1的结合。使用这些小鼠将实现三个特定的目标。目的1阐明CBP和p300的KIX结构域在体内的作用。目的2确定CBP和p300的CH1结构域在体内的作用。目标3是确定CBP和p300在体内是否具有生化冗余。

CREB-binding protein (CBP, CREBBP) and the closely related E1A-binding protein p300 (EP300) are transcriptional coactivators that interact physically or functionally with about 10 percent of the estimated 2,000 mammalian transcriptional regulatory proteins. Both genes are required for normal development and physiology, and altered-function- and inactivating-mutations occur in cancer, indicating that CBP and p300 can function both as oncoproteins and as tumor suppressors. Several major unresolved questions remain with regard to the biological and transcriptional roles of CBP and p300. First, it is largely unknown what roles the multiple different transcription factor-binding domains of CBP and p300 play in development and tumorigenesis in vivo. Second, many in vitro studies indicate that CBP and p300 are biochemically indistinguishable, but it is uncertain how redundant their functions are in vivo. Third, numerous in vitro transcription studies have shown that CBP and p300 are crucial coactivators, yet little is known regarding their roles in endogenous gene expression. Fourth, it is uncertain if different classes of coactivators function redundantly. These questions form the core of the application's broad, long-term, objectives, aspects of which will be approached by testing the in vivo functions of two specific protein-binding domains common to CBP and p300. Four strains of knock-in mutant mice have been generated. Two have point mutations on the surface of the KIX domain of CBP and p300 that inhibits the binding of the cAMP-responsive factor CREB and the hematopoietic factor c-Myb, and two strains that have a deletion mutation in the CH1 domain of CBP and p300 that abrogates the binding of the hypoxia-responsive factor HIF-1. Three specific aims will be pursued using these mice. Aim 1 is to elucidate the roles of the KIX domain of CBP and p300 in vivo. Aim 2 is to determine the roles of the CH1 domain of CBP and p300 in vivo. Aim 3 is to establish if CBP and p300 are biochemically redundant in vivo.