MOLECULAR STUDIES OF EUKARYOTIC GENE REGULATION

真核基因调控的分子研究

• 批准号: 5200925
• 负责人: B M PATERSON
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 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/5200925
• 关键词: Caenorhabditis elegans; Drosophilidae; antisense nucleic acid; cell differentiation; gene induction /repression; glutamates; larva; myogenesis; phosphoproteins; point mutation; tissue /cell culture; transcription factor; transfection /expression vector; virus RNA

We have continued our studies on the role of the MyoD family of basic helix-loop-helix muscle-specific gene regulatory factors during myogenic cell commitment and differentiation, analyzing vertebrate muscle formation in vitro or larval muscle formation in developing Drosophila embryos. Vertebrates express four different myogenic factors in a developmentally regulated pattern whereas Drosophila has only a single related factor. In vertebrates, MyoD and myogenin are the two factors expressed in cultured muscle. We have shown that the putative auto regulation of the avian MyoD(CMD1) promoter is indirect and does not involve the direct binding of the bHLH myogenic factors or MEF2. The myogenic bHLH proteins are nuclear phosphoproteins. We have identified a regulatory serine residue in the carboxy terminal portion of CMD1 that is a potential casein kinase II site and plays a role in the homo-heterodimer equilibrium of CMD1 and the ubiquitous E- protein, E12. The same site is conserved in Drosophila MyoD. The myogenic factors bind DNA predominately as heterodimers with the E2A- related proteins, E12 and E47 but little is known about the in vivo complex. We have shown that MyoD and myogenin are complexed in muscle cultures with E2A proteins and that myogenesis can be regulated depending upon the level of the E2A gene products using combinations of sense and antisense E12 RNA expression vectors. We have previously shown, by comparison with Drosophila MyoD, that dimerization specificity within the vertebrate myogenic factors and E proteins is determined, to a large extent, by the nonconserved nonhydrophobic residues in the HLH domain. We have extended this study to further demonstrate conserved glutamate residues in helix 2 of MyoD are required to overcome the inhibitory domain of E12 and that this inhibitory domain blocks E12 homodimerization and not DNA binding, as previously proposed. The role of dimerization specificity in the regulation of the myogenic factors has been demonstrated directly in vivo and in vitro: we have been able to rescue the lethal C. elegans MyoD point mutation, HLH1, with Drosophila MyoD and, to a lesser extent, with avian myogenin; conversion of mouse 10T1/2 fibroblasts to muscle only works with Drosophila MyoD when it is paired with daughterless, the Drosophila homolog to the E-proteins, or the C. elegans E-protein homolog. The Drosophila MyoD alone will not activate myogenesis in mouse cells which demonstrates the homodimer is not functional and is consistent with the results of the E12 antisense experiments.

我们继续研究Myod基本家族的作用 螺旋环螺旋特异性基因调节因素 肌原细胞的承诺和分化，分析脊椎动物 肌肉形成在体外或幼虫肌肉形成中 果蝇胚胎。 脊椎动物表达四种不同的肌原性 在发育中受调节的模式中的因素，而果蝇具有 只有一个相关因素。 在脊椎动物中，myod和肌根蛋白是 在培养的肌肉中表达的两个因素。 我们已经证明了 假定的自动调节鸟类Myod（CMD1）启动子是间接的 并且不涉及BHLH肌原因子的直接结合 或MEF2。 肌源性BHLH蛋白是核磷蛋白。 我们 已经确定了羧基终端中的调节性丝氨酸残留物 CMD1的一部分是一个潜在的酪蛋白激酶II位点，并且播放了 CMD1和无处不在的E-中的均型异二聚体平衡中的作用 蛋白质，E12。 同一地点在果蝇Myod中是保守的。 这 肌源性因子主要以E2A-的异二二聚体结合DNA 相关蛋白质E12和E47，但对体内知之甚少 复杂的。 我们已经表明，肌蛋白和肌根蛋白在肌肉中复杂化 具有E2A蛋白的培养物，可以调节肌发生 根据使用组合的E2A基因产物的水平 感官和反义E12 RNA表达载体。 我们以前有 与果蝇Myod相比，显示了二聚体 脊椎动物肌原因素和E蛋白的特异性是 在很大程度上，通过非保守的非嗜恐惧症确定 HLH域中的残留物。 我们已经扩展了这项研究以进一步 在MYOD的螺旋2中证明保守的谷氨酸残基是 克服E12的抑制域所需的 抑制结构域阻止E12同构化而不是DNA结合，为 先前提出的。 二聚体特异性在 肌原因子的调节已直接证明 体内和体外：我们已经能够营救致命的秀丽隐杆线虫 Myod点突变HLH1，果蝇Myod，较小 程度，与鸟类肌蛋白；鼠标10T1/2的成纤维细胞转换为 肌肉仅与果蝇肌一起配对时 女儿，果蝇与e蛋白的同源物，或C。 秀丽隐杆线虫电子同源物。 果蝇myod不会 激活显示同型二聚体的小鼠细胞中的肌发生 不起作用，与E12反义的结果一致 实验。