Chromatin based gene regulation in T lymphocytes

描述（由申请人提供）：现在很明显，经典的转录控制序列（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开发在染色质环境中有效的基因治疗载体至关重要。转基因小鼠转录调控的研究已经确定了一类新的基因调控序列，具有明显的基于染色质的活性，不同于经典的转录增强子。基因座控制区（Locus Control Region，LCR）。我们正在研究小鼠T细胞受体（TCR）-α/Dad 1基因位点的LCR。我们目前的长期目标是解释这种LCR活性的分子基础及其在调节其复杂位点中的作用。通过这一点，我们的目标是进一步了解T细胞分化的基本知识，并帮助基因治疗载体的发展。我们已经确定了这个LCR的染色质为基础的活动，命名为DNA酶超敏位点（HS）-6的关键组成部分。我们假设，元素，如HS 6参与靶向最近描述的表观遗传控制机制，其基因位点。 在这个项目中，使用良好的特点报告转基因小鼠，我们的目标是确定功能序列和相关因素内HS 6有助于LCR活性在体内。我们还旨在确定功能性HS 6序列的作用机制。使用我们实验室开发的标准和新方法的组合，我们将研究HS 6突变对报告基因转基因染色质结构，体内因子占有率，DNA甲基化，组蛋白修饰和核内定位模式的影响。我们的初步数据已经确定了两个功能区和三个因子结合位点的HS 6体内足迹。LCR中这些位点的突变损害其基于染色质的活性。因此，这些将是拟议实验的重点。通过提供LCR活性和基于染色质的基因调控的分子基础的清晰图像，这些研究将有助于缩小我们对如何实现T细胞特异性基因表达的知识的差距。这些信息可能有助于改善针对先天性免疫疾病、白血病和艾滋病的基因治疗策略。

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.