Molecular basis of transvection

描述(由申请人提供)：我们的实验室有兴趣了解主导增强子-启动子相互作用的机制以及绝缘体在基因表达调控中的作用。这项提议的目的是探索横行现象背后的分子机制。增强子与启动子的相互作用是导致对基因表达进行时间和组织特异性调控的精心安排的过程中的关键步骤。即使在超过100kb的距离上，增强子也可以特异性地激活启动子。值得注意的是，增强子还可以反式激活位于同源染色体上的启动子，这一现象被称为转基因。转基因依赖于染色体之间的体细胞配对，其中一个基因的表达受到来自同源染色体上相应等位基因的调控信号的影响。转基因在果蝇的遗传水平上已经得到了很好的描述，但从酵母、植物和哺乳动物等多种生物中报道的大量例子表明，反式激活(即转基因)是真核生物中一种保守的调节特征。我们认为绝缘子是分子载体，通过它，增强子可以与位于不同染色体上的启动子进行交流。以下两个目的旨在检验这一假设。答：使用转基因检测评估吉普赛绝缘子介导横行感染的能力。细胞和分子生物学分析以及突变分析将评估绝缘子在实验控制的传播环境中的参与程度。B：评估内源性绝缘体是否在横行中起普遍作用。我们将使用计算机分析、荧光显微镜、凝胶漂移分析和转基因检测来定位和表征特定的内源性绝缘子，并预测其在转基因中的作用。遗传分析还将评估绝缘体蛋白在先前报道的感染现象中所起的作用。这些研究可能会对体细胞和生殖系发育所涉及的调控过程的总体理解产生立竿见影的影响。转基因和基因组绝缘子都可能参与控制许多基因的转录，因此在包括癌症在内的人类疾病的生物学中具有广泛的影响。了解增强子如何与启动子相互作用，以及绝缘体在这一过程中的作用，可能会为遗传病的研究提供新的途径。

DESCRIPTION (provided by applicant): Our laboratory is interested in understanding the mechanisms presiding enhancer-promoter interactions and the role of insulators in gene expression regulation. The goal of this proposal is to explore the molecular mechanisms underlying the phenomenon of transvection. Interaction of enhancers with promoters is a critical step in the orchestrated process leading to time and tissue specific regulation of gene expression. Enhancers can specifically activate promoters even at distances longer than 100 kb. Remarkably, enhancers can also trans-activate promoters located in homologue chromosomes, a phenomenon known as transvection. Transvection depends on somatic pairing between chromosomes, wherein the expression of one gene is influenced by regulatory signals emerging from its corresponding allele on the homologue chromosome. Transvection has been very well characterized at the genetic level in Drosophila, but numerous examples reported from multiple organisms including yeasts, plants and mammals, suggest that trans-activation (i.e. transvection) is a conserved eukaryotic regulatory feature. We propose that insulators are the molecular vehicles by which enhancers can communicate with promoters located in separate chromosomes. The following two aims are designed to test this hypothesis. A: assess the ability of gypsy insulators to mediate transvection using transgenic assays. Cell and molecular biology assays as well as mutational analysis will asses the involvement of insulators in experimentally controlled transvection settings. B: assess whether there is a general role of endogenous insulators in transvection. We will map and characterize specific endogenous insulators with a predicted role in transvection using computer analysis, fluorescence microscopy, gel shift assays and transgenic assays. Genetic analysis will also assess the role that insulator proteins play in previously reported transvection phenomena. These studies might have an immediate repercussion in the general understanding of the regulatory processes involved in somatic and germ-line development. Both, transvection as well as genome insulators may be involved in the control of transcription of numerous genes and therefore have a broad impact in the biology of human diseases, including cancer. Understanding how enhancers interact with promoters and the role of insulators in this process may provide new avenues to approach the study of genetic diseases.