VIMENTINS REGULATORY FACTORS INVOLVED IN DEVELOPMENT

肌肉发生需要肌肉特有基因的协调表达 以及肌肉不需要的其他功能的失活 表型。 尽管有大量的信息表明 骨骼基因的表达，较少知道是什么支配心脏 表达，几乎没有关于非肌肉特异性基因是如何表达的。 在肌生成过程中“关闭”，尽管基因失活 对维持肌肉表型同样重要。 细胞骨架 蛋白质，波形蛋白和结蛋白，提供了一个很好的模型系统， 确定基因在发育过程中的差异表达。 波形蛋白的合成首先在中胚层的轮廓处被检测到。 许多细胞类型从这一谱系分化，并继续 合成波形蛋白。 其他的，比如肌肉， 和“开启”结蛋白。 本提案的目标是阐明 波形蛋白基因在肌肉发生过程中特异性失活， 异常表达可能影响发育程序。 很显然， 这一决定的任何偏差都可能导致发展受到损害。 在分析波形蛋白基因表达时，我们发现了独特的阳性和阴性表达。 负调节因子，控制下调的 波形蛋白基因 在鸡骨骼肌成肌细胞中， 消极因素（沉默者）低，积极因素（抗沉默者） 蛋白质含量高，波形蛋白mRNA丰富。 随着肌生成的进行 沉默因子的活性显著增加，而 抗沉默剂活性几乎消失，波形蛋白mRNA水平 急剧下降。 因此，这两个因素的相互作用 是决定的生理正确表达的关键， 波形蛋白基因 在心脏（第14天）中，我们发现 沉默因子比骨骼肌（乳房）。 因此得出结论 在心肌发生过程中一定也有类似的程序 在本提案中，我们旨在进一步界定该机制。 我们将 继续描述和克隆这两种独特的 沉默因子和抗沉默因子。 我们把这种蛋白质称为 反消音器，因为它只与消音器协同工作 元件，并不有助于基因表达本身，不像 典型的增强子蛋白。 随着核苷酸和蛋白质的获得 序列信息，我们计划改变这些细胞的内容， 正常细胞和生肌细胞中的因子。 我们将确定 这些基因改变对波形蛋白基因表达的影响， 重要的是骨骼和心脏细胞中的生肌程序。 一 基因组数据库的屏幕显示了几个基因（从小鸡到人） 具有同源的沉默者和反沉默者元件。 如抗体或 当cRNA探针可用时，我们将检测这些关键基因的表达。 胚胎发生过程中的调节因子。 最后，我们将确定 这些调节因子控制表达的机制 波形蛋白以及其他基因。 希望通过了解如何 这些基因控制有助于发展，我们可以确定如何 心脏可能会出现异常。

Myogenesis requires the coordinate expression of genes unique to muscle and the inactivation of others which are not required for the muscle phenotype. Although considerable information exists about what controls skeletal gene expression, less is known about what governs cardiac expression, and virtually nothing about how non-muscle-specific genes are "turned-off" during myogenesis, despite the fact that gene inactivations equally important to maintaining the muscle phenotype. The cytoskeletal proteins, vimentin and desmin, provide an excellent model system for determining how genes are differentially expressed during development. Vimentin synthesis is first detected at the delineation of the mesoderm. A number of cell types differentiate from this lineage and continue to synthesize vimentin. Others, like muscle, inactivate the vimentin gene and "turn-on" desmin. The goal of this proposal is to delineate how the vimentin gene is specifically inactivated during myogenesis and how aberrant expression could affect the developmental program. Obviously, any deviation in this decision could result in compromised development. In analyzing vimentin gene expression, we have found unique positive and negative regulatory factors which control the downregulation of the vimentin gene. In chick skeletal myoblasts, the DNA binding activity of the negative (silencer) factor is low, the positive (antisilencer) protein is high, and vimentin mRNA is abundant. As myogenesis proceeds the activity of the silencer factor increases dramatically, whereas antisilencer activity virtually disappears and the level of vimentin mRNA decreases dramatically. Therefore, the interplay of these two factors is crucial for determining the physiologically correct expression of the vimentin gene. In heart (day 14) we find a 3-fold higher level of the silencer factor than in skeletal (breast) muscle. Therefore, we conclude that a similar program must be occurring during cardiac myogenesis. In this proposal we aim to further define the mechanism. We will continue with the characterization and cloning of both the unique silencer and antisilencer factor. We refer to this protein as an antisilencer, because it only functions in concert with the silencer element and does not contribute to gene expression on its own unlike the typical enhancer protein. With the acquisition of nucleotide and protein sequence information, we plan to alter the cellular content of these factors in both normal and myogenic cells. We will determine the effect of these genetic alterations on vimentin gene expression and more importantly the myogenic program in both skeletal and cardiac cells. A screen of the genomic data base reveals several genes (from chick to man) with homologous silencer and antisilencer elements. As antibodies or cRNA probes become available, we will examine the expression of these key regulatory factors during embryogenesis. Finally, we will determine the mechanism by which these regulatory factors control expression of vimentin as well as other genes. It is hoped that by understanding how these genetic controls contribute to development, we can determine how abnormalities may develop in the heart.