REGULATION OF MYOD POST TRANSCRIPTIONAL MODIFICATIONS

本研究旨在了解调节肌源性 bHLH 蛋白转录活性的分子和细胞机制。更具体地说，我们感兴趣的是研究一旦未分化的成肌细胞转变为分化的肌管时，MyoD 变得转录活跃的开关。 MyoD 活性是卫星细胞增殖所必需的，并且在横纹肌肉瘤（一种儿童实体瘤）中失调。 骨骼肌细胞再生和肿瘤转化的分子机制仍不清楚。乙酰化是一个动态过程，涉及乙酰转移酶和脱乙酰酶的作用。 MyoD 是 p300 和 PCAF 乙酰化的直接目标。尽管如此，PCAF（而非 p300）介导的乙酰化对于协助 MyoD 依赖性转录和肌肉分化是必需的。 PCAF 介导的 MyoD 乙酰化导致 DNA 结合能力增加，并改变与 DNA 结合的 MyoD 构象。我们之前分析了 p300 和 PCAF 乙酰转移酶对肌肉分化的贡献。 我们建议继续进行这一调查。具体目标是： 1．研究 HDAC 是否与 MyoD 有物理关联并阻碍肌肉分化。 2.分析MyoD、pRb和HDAC1在调节肌肉特异性转录中的功能相互作用。3．研究乙酰化在骨骼肌细胞再生和肿瘤转化中的作用。去年，我们开始表征含有 MyoD 和 HDAC1 的细胞复合物的组成，并启动了肌肉细胞中 HDAC1 过表达的实验。我们的初步结果表明，HDAC1 的强制表达会拮抗 MyoD 依赖性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.