MOLECULAR STUDIES OF EUKARYOTIC GENE REGULATION

真核基因调控的分子研究

• 批准号: 6100780
• 负责人: B M PATERSON
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6100780
• 关键词: Caenorhabditis elegans; Drosophilidae; embryology; gene induction /repression; genetic promoter element; mesoderm; myogenesis; phosphoproteins; tissue /cell culture; transcription factor

The determination, proliferation and differentiation of muscle cells during development in both vertebrates and invertebrates depend upon the complex regulated expression, dimerization and function of the MyoD family of basic helix-loop-helix proteins. We have continued to study the role of the MyoD related proteins during vertebrate and Drosophila myogenesis. In the vertebrates, the MyoD family of muscle gene regulatory proteins or MRFs, were thought to directly auto regulate their expression levels through MRF binding sites in the promoters of these genes. However, we have demonstrated that this pathway is indirect, at least for avian MyoD (CMD1), and does not involve any presently known muscle-specific transcription factors. In Drosophila we have shown that the single MyoD gene, nau is not auto regulated but expression depends upon an earlier mesodermal transcription factor, DMEF2. The single DMEF2 gene encodes a MADS domain containing SRF homologue essential for muscle formation in Drosophila. DMEF2 is expressed just after the onset of twist expression, the key mesodermal determination gene in flies. Our studies indicate that, in addition to DMEF2, twist also regulates nau in cultured Drosophila cells, suggesting twist is epistatic to nau and is a possible regulator of nau during embryogenesis at the earliest point of mesoderm formation. The MyoD family of proteins are phosphoproteins. Two adjacent serine residues outside the HLH domain in the carboxy terminal portion of vertebrate MyoD regulate the formation of the homodimer in vitro and in vivo. This site and its function are conserved in nau. Phosphorylation of this site also regulates a transcriptional activation domain and may be responsive to signal transduction pathways that modulate MyoD function. We have demonstrated the importance of dimerization specificity among the MyoD/E-protein family members by using a newly developed competitive EMSA, demonstrating the heterodimer dependent myogenic conversion of nonmuscle cells from both vertebrates and Drosophila, and conducting the rescue of a MyoD lethal mutation in C. elegans with Drosophila MyoD, a predicted heterodimer partner from our studies on dimerization specificity. MyoD is not only a transcription factor but also appears to play a role in the decision to exit the cell cycle during differentiation. Preliminary experiments indicate that MyoD can regulate CDK function in a concentration dependent manner, inhibiting the phosphorylation of Rb. The fragment of MyoD containing the CDK binding site can itself inhibit cell growth.

肌肉细胞的测定，增殖和分化 在发育过程中，脊椎动物和无脊椎动物都取决于 MYOD的复杂调节表达，二聚化和功能 基本螺旋环螺旋蛋白的家族。我们继续学习 脊椎动物和果蝇中与MYOD相关蛋白的作用 肌发生。在脊椎动物中，肌肉基因家族 被认为是直接自动调节的调节蛋白或MRF 它们通过MRF结合位点的启动子中的MRF结合位点的表达水平 这些基因。但是，我们已经证明了这条路是 间接，至少对于Avian Myod（CMD1），并且不涉及任何 目前已知的肌肉特异性转录因子。在果蝇中 已经表明单个myod基因Nau不是自动调节的，而是 表达取决于早期的中胚层转录因子， DMEF2。单个DMEF2基因编码包含SRF的MADS域 果蝇中肌肉形成必不可少的同源物。 DMEF2是 在扭曲表达开始后，钥匙中胚层 果蝇中的测定基因。我们的研究表明，除了 DMEF2，Twist还调节培养的果蝇细胞中的NAU，表明 扭曲对NAU是上毒的，是NAU的可能调节器 胚胎发生在中胚层形成的最早。 myod 蛋白质家族是磷蛋白。两个相邻的丝氨酸残基 脊椎动物的羧基终端部分的HLH域外 Myod在体外和体内调节同型二聚体的形成。这 位点及其功能在NAU中是保守的。该地点的磷酸化 还调节转录激活域，并且可能具有响应性 信号调节MYOD函数的转导途径。我们有 证明了二聚化特异性的重要性 Myod/e蛋白家庭成员使用新发展的竞争力 EMSA，证明了异二聚体依赖性的肌原性转化 来自脊椎动物和果蝇的非肌肉细胞，并进行 用果蝇Myod拯救了秀丽隐杆线虫中的Myod致命突变，一个 从我们的二聚化研究中预测的异二聚体伙伴 特异性。 Myod不仅是转录因素，而且还出现 在决定退出细胞周期的决定中发挥作用 分化。初步实验表明MYOD可以调节 CDK以浓度依赖的方式功能，抑制 RB的磷酸化。包含CDK结合的Myod的片段 部位本身可以抑制细胞生长。