Regulation of Tissue Specific Gene Expression

描述（由申请人提供）：绝缘子是保守的基因组元件，其将染色体组织成独立的功能结构域以维持转录保真度。这些元件通过阻断增强子、沉默子和启动子之间的相互作用来保护基因表达免受正调控和负调控的影响。我们的研究目标是了解绝缘子用于组织真核生物基因组进入转录域的分子机制。 在本文所述的研究中，果蝇吉普赛绝缘子将被用作阐明绝缘子作用机制的模型。吉普赛绝缘子是一种研究充分的绝缘子，它是从吉普赛逆转录病毒中分离出来的。这种绝缘子是已知的唯一一种需要单个DNA结合蛋白的绝缘子，即毛翅抑制子[Su（Hw）]。一旦与染色体结合，Su（Hw）蛋白招募BTB/POZ结构域蛋白，修饰剂67.2，以产生建立绝缘子活性的蛋白质复合物。编码吉普赛绝缘子蛋白的基因突变是可用的，使该系统易于遗传操纵。吉普赛绝缘子蛋白与不是吉普赛逆转录病毒插入位点的数百个基因组位点相关联。这些观察结果表明，Su（Hw）和Modifier67.2在果蝇基因组中建立独立的调控结构域中具有全局性作用。我们将通过确定内源性Su（Hw）位点的DNA和蛋白质需求来测试这一假设，并定义它们在整个染色体的调控结构域组织中的作用。 这些数据将提供深入了解用于划定真核基因组内的转录调控域的机制，从而提供深入了解增强子和沉默子功能的控制的基本问题。了解绝缘子作用的机制对于基因治疗具有治疗效用，因为在基因转移载体内包含绝缘子可以改善转基因的表达，同时防止基因转移载体对内源基因表达的不适当的调节作用。

DESCRIPTION (provided by applicant): Insulators are conserved genomic elements that organize chromosomes into independent functional domains to maintain transcriptional fidelity. These elements protect gene expression from positive and negative regulatory effects by blocking interactions between enhancers, silencers, and promoters. The goal of our studies is to understand molecular mechanisms used by insulators to organize eukaryotic genomes into transcriptional domains. In studies described herein, the Drosophila gypsy insulator will be used as model to elucidate mechanisms of insulator action. The gypsy insulator is a well-studied insulator that was isolated from the gypsy retrovirus. This insulator is the only one known that requires a single DNA binding protein, the Suppressor of Hairy-wing [Su(Hw)]. Once bound to chromosomes, the Su(Hw) protein recruits the BTB/POZ domain protein, Modifier67.2, to generate a protein complex that establishes insulator activity. Mutations in the genes encoding the gypsy insulator proteins are available, making the system amenable to genetic manipulation. The gypsy insulator proteins associate with hundreds of genomic sites that are not sites of gypsy retroviral insertion. These observation suggest that Su(Hw) and Modifier67.2 have global roles in establishing independent regulatory domains in the Drosophila genome. We will test this hypothesis by determining the DNA and proteins requirements of endogenous Su(Hw) sites and define their role in organization of regulatory domains throughout chromosomes. These data will provide insights into mechanisms used to delimit transcriptional regulatory domains within eukaryotic genomes, thereby providing insights into fundamental questions of control of enhancer and silencer function. Understanding mechanisms of insulators action has therapeutic utility for gene therapy, as inclusion of insulators within gene transfer vectors can improve the expression of transgenes, while preventing inappropriate regulatory effects of gene transfer vectors on endogenous gene expression.