GENETIC ANALYSIS OF CARDIAC TERMINAL DIFFERENTIATION

终末分化是高度特化的细胞不可逆转地承担细胞命运并经历生长停滞的过程。最近对心肌的研究发现，参与心脏末端分化的两个主要蛋白质家族是Pocket蛋白(视网膜母细胞瘤基因产物(Rb)，p107，p130)和转录辅助激活因子p300/CBP。对正常分化依赖Pocket蛋白的一种解释是，某些组织特异性转录因子要求Rb共表达以实现转录活性。这导致我们和其他人假设Rb可能是心肌细胞终末分化的关键。然而，这种依赖的机制或与RB相互作用的因素尚不清楚。因此，为了确定心肌中与Rb相互作用的因素，我们利用酵母双杂交系统来鉴定心脏Rb结合蛋白。一个新的蛋白质，命名为p300和Rb结合的分化抑制因子-1(PRI-1)是主要的Rb结合克隆。PRI-1以发育调控的方式在心肌和骨骼肌中优先表达。在骨骼肌中的初步研究表明，PRI-1的过表达抑制了肌肉特异性转录。骨骼肌限制性基因的抑制是通过阻断MyoD的反式激活而介导的，与G1出口无关，令人惊讶的是，阻止PrI-1与Rb结合的突变增强了这种抑制。抑制MyoD的作用可能与PrI-1的S蛋白一样，与腺病毒蛋白E1a一样，与MyoD必需的辅活化子p300结合。因此，PrI-1与Rb和p300结合，在分化的肌肉中被Rb抑制，是MyoD功能的一种新的抑制因子。在心肌中的研究表明，PrI-1也抑制心脏特异基因的表达，尽管PrI-1‘S抑制作用的机制或潜在靶点尚不清楚。因此，PrI-1是最早被鉴定的内源性哺乳动物心脏分化抑制物之一。这第一个RO1建议进一步确定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.