Chromatin based gene regulation in T lymphocytes

描述（由申请人提供）：现在很明显，经典转录控制序列（启动子、增强子、沉默子）直接影响RNA聚合酶活性的能力并不能完全解释整个动物染色质中基因表达的组织特异性模式。然而，这些知识对于理解淋巴细胞分化和在染色质环境中有效的基因治疗载体的发展是必不可少的。转基因小鼠的转录调控研究发现了一类新的基因调控序列，其基于染色质的活性明显不同于经典的转录增强子。其中一个元素是基因座控制区（LCR）。我们正在研究小鼠T细胞受体(TCR)- α /Dad1基因位点的LCR。我们目前的长期目标是解释LCR活性的分子基础及其在复杂位点调控中的作用。通过这一研究，我们旨在进一步了解T细胞分化的基本原理，并帮助开发基因治疗载体。我们已经确定了LCR基于染色质的活性的一个关键组成部分，命名为dna酶敏感位点(HS)-6。我们假设诸如HS6之类的元件参与了最近描述的针对其基因位点的表观遗传控制机制。

DESCRIPTION (provided by applicant): It is now apparent that the ability of classical transcriptional control sequences (promoters, enhancers, silencers) to directly affect RNA polymerase activity does not fully explain the tissue-specific patterns of gene expression in chromatin of the whole animal. Yet this knowledge is essential to the understanding of lymphocyte differentiation and the development of gene therapy vectors that are effective in a chromatin environment. The study of transcriptional regulation in transgenic mice has identified a novel class of gene regulatory sequences with apparent chromatin-based activities distinct from those of classical transcriptional enhancers. One such element is the Locus Control Region (LCR). We are studying the LCR in the mouse T cell receptor (TCR)-alpha/Dad1 gene locus. Our current long-term goal is to explain the molecular basis for this LCR's activity and its role in the regulation of its complex locus. Through this, we aim to further the basic understanding of T cell differentiation and aid the development of gene therapy vectors. We have identified a key component of this LCR's chromatin-based activity, named DNase hypersensitive site (HS)-6. We hypothesize that elements such as HS6 are involved in targeting recently described epigenetic control mechanisms to its gene locus. In this project, using well characterized reporter transgenes in mice, we aim to determine the functional sequences and associated factors within HS6 contributing to LCR activity in vivo. We also aim to identify the mechanism of action of functional HS6 sequences. Using a combination of standard and novel methods developed in our laboratory, we will examine the effect of HS6 mutations on reporter transgene chromatin structure, in vivo factor occupancy, DNA methylation, histone modification and intra-nuclear localization patterns. Our preliminary data have identified two functional regions and three factor-binding sites in HS6 by in vivo footprinting. Mutation of these sites in the LCR impairs its chromatin based activity. Thus, these will be the focus of the proposed experiments. By providing a clear picture of the molecular bases of LCR activity and chromatin based gene regulation, these investigations will help close the gap in our knowledge of how T cell specific gene expression is achieved. This information may help improve gene therapy strategies against congenital immune disorders, leukemia and A.I.D.S.