REGULATION OF MYOD POST TRANSCRIPTIONAL MODIFICATIONS

本研究旨在了解调控肌源性bHLH蛋白转录活性的分子和细胞机制。更具体地说，我们感兴趣的是，一旦未分化的成肌细胞转变为分化的肌管，允许MyoD成为转录活性的开关。MyoD活性是卫星细胞增殖所必需的，在横纹肌肉瘤(一种儿科实体肿瘤)中则被解除调节。骨骼肌细胞再生和肿瘤转化的分子机制尚不清楚。乙酰化是一个由乙酰基转移酶和脱乙酰基酶共同作用的动态过程。MyoD是p300和PCAF乙酰化的直接靶点。然而，需要PCAF而不是p300介导的乙酰化来辅助MyoD依赖的转录和肌肉分化。PCAF介导的MyoD乙酰化导致DNA结合能力增强，并改变了MyoD与DNA结合的构象。我们之前已经分析了p300和PCAF乙酰转移酶在肌肉分化中的作用。我们建议继续进行这方面的调查。其具体目的是：1.研究HDAC是否与MyoD有物理联系，并阻碍肌肉分化。2.分析MyoD、pRb和HDAC1在肌肉特异性转录调控中的功能相互作用。探讨乙酰化在骨骼肌细胞再生和转化中的作用。在过去的一年里，我们已经开始表征含有MyoD和HDAC1的细胞复合体的组成，并开始在肌肉细胞中过度表达HDAC1的实验。我们的初步结果表明，强制表达HDAC1可以拮抗MyoD依赖的转录，并抑制肌肉分化。

The present study is aimed at the understanding of the molecular and cellular mechanisms that regulate the transcriptional activity of the myogenic bHLH proteins. More specifically, we are interested in studying the switches that allow MyoD to become transcriptionally active once undifferentiated myoblasts transition into differentiated myotubes. MyoD activity is required for proliferation of satellite cells and is deregulated in rhabdomyosarcoma, a pediatric solid tumor. The molecular mechanisms of regeneration and neoplastic transformation of skeltal muscle cells remian unclear. Acetylation is a dynamic process involving the actions of both acetyltransferases and deacetylases. MyoD is a direct target of acetylation by both p300 and PCAF. Nonetheless, acetylation mediated by PCAF, but not by p300, is required to assist MyoD-dependent transcription and muscle differentiation. PCAF-mediated acetylation of MyoD results in an increased DNA binding capability and modify the conformation of MyoD bound to DNA. We have previously analyzed the contribution of the p300 and PCAF acetyltransferases to muscle differentiation. We propose to continue this line of investigation. The specific aims are:1. To investigate whether the HDACs physically associate with MyoD and impede muscle differentiation. 2. To analyze the functional interplay of MyoD, pRb and HDAC1 in regulating muscle specific transcription.3. To investigate the role of acetylation in both regeneration and neoplastic transformation of skeletal muscle cells. In the past year, we have begun characterizing the composition of a cellular complex containing MyoD and HDAC1 and have initiated experiments of HDAC1 overexpression in muscle cells. Our preliminary results indicate that forced expression of HDAC1 antagonizes MyoD-dependent transcription and prohibits muscle differentiation.