GENETIC ANALYSIS OF CARDIAC TERMINAL DIFFERENTIATION

终末分化是高度特化的细胞不可逆地进入细胞命运并经历生长停滞的过程。 最近在心肌中的研究已经确定了参与心脏终末分化的两个主要蛋白家族，口袋蛋白（视网膜母细胞瘤基因产物（Rb），p107，p130）和转录辅激活因子，p300/CBP。正常分化依赖口袋蛋白的一种解释是，某些组织特异性转录因子需要Rb的共表达来获得转录活性。 这使我们和其他人推测Rb可能是心肌细胞终末分化的关键。 然而，这种依赖性的机制或与Rb相互作用的因素是未知的。 因此，为了确定心肌中与Rb相互作用的因子，我们利用酵母双杂交系统鉴定心肌Rb结合蛋白。 一种新的蛋白质，命名为p300-和Rb-结合分化抑制剂-1（PRI-1）是主要的Rb-结合克隆。 PRI-1以发育调节的方式优先在心肌和骨骼肌中表达。 在骨骼肌中的初步研究表明，PRI-1的过表达抑制肌肉特异性转录。骨骼肌限制性基因的抑制是通过阻断MyoD的反式激活介导的，独立于G1出口，并且令人惊讶的是，通过阻止PRI-1与Rb结合的突变来增强。 MyoD的抑制可以通过PRI-1的能力来解释，就像腺病毒蛋白E1 A一样，结合p300，一种必需的MyoD辅激活因子。 因此，PRI-1结合Rb和p300，在分化的肌肉中被Rb抑制，并且是MyoD功能的新型阻遏物。 心肌研究表明，PRI-1也抑制心脏特异性基因表达，尽管PRI-1抑制作用的机制或潜在靶点尚不清楚。 因此，PRI-1代表了第一个内源性的，哺乳动物的心脏分化抑制剂被确定。 这第一个RO 1建议进一步定义PRI-1在心肌细胞分化中的作用，并阐明其生化特性。

Terminal differentiation is a process whereby highly specialized cells commit irreversibly to a cellular fate and undergo growth arrest. Recent studies in cardiac muscle have identified two major families of proteins involved in cardiac terminal differentiation, pocket proteins (retinoblastoma gene product (Rb), p107, p130) and the transcriptional coactivators, p300/CBP. One explanation for the dependence of normal differentiation on pocket proteins, is the requirement of some tissue-specific transcription factors on the coexpression of Rb for transcriptional activity. This led us and others to hypothesize that Rb might be the key to terminal differentiation in cardiac myocytes. However, the mechanism underlying this dependence or the factors interacting with Rb are unknown. Therefore, to determine the factors interacting with Rb in cardiac muscle, we utilized the yeast two-hybrid system to identify cardiac Rb- binding proteins. One novel protein, designated p300- and Rb- binding Inhibitor of differentiation-1 (PRI-1) was the predominant Rb-binding clone. PRI-1 is preferentially expressed in cardiac and skeletal muscle in a developmentally regulated manner. Initial studies in skeletal muscle demonstrated that overexpression of PRI-1 inhibited muscle-specific transcription. Repression of skeletal muscle-restricted genes was mediated by a block to transactivation by MyoD, independent of G1 exit, and, surprisingly, was potentiated by a mutation that prevents PRI-1 binding to Rb. Inhibition of MyoD may be explained by PRI-1's ability, like adenoviral protein E1A, to bind p300, an essential MyoD coactivator. Thus, PRI-1 binds both Rb and p300, was inhibited by Rb in differentiated muscle, and was a novel repressor of MyoD function. Studies in cardiac muscle demonstrate that PRI-1 also inhibits cardiac-specific gene expression although the mechanism or potential targets of PRI-1's inhibitory effects are unknown. Thus PRI-1 represents one of the first endogenous, mammalian inhibitors of cardiac differentiation to be identified. This first RO1 proposes to further define the role of PRI-1 in cardiac myocyte differentiation and elucidate its biochemical properties.