Chromatin based gene regulation in T lymphocytes

描述(申请人提供)：现在很明显，经典转录控制序列(启动子、增强子、抑制子)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。对“基于染色质的”基因调控的研究有望完成我们对这些过程的理解。这些机制已被提出在免疫细胞的发育和功能中发挥重要作用。然而，基于染色质的基因调控的分子本质及其与体内T细胞基因活性的相关性仍然不清楚。转基因小鼠转录调控的研究已经确定了一类新的具有染色质活性的基因调控序列。一个这样的元素是轨迹控制区(LCR)。我们正在研究小鼠T细胞受体(TCR)-α/Dad1基因座上的LCR。利用转基因小鼠，我们已经鉴定出具有染色质活性的LCR的一个离散的亚元件，称为DNA酶超敏位点(HS)-6。我们假设，我们对该元件的研究将揭示T细胞中基于染色质的基因调控背后的分子和机制。在这项建议中，我们的具体目标是： 1.确定hs6中与体内LCR活性有关的功能序列 2.用标准的和新颖的方法表征功能性hs6序列的作用机制 3.确定与hs6功能序列相互作用的转录因子在LCR活性中的作用 4.检测hs6载体在转基因小鼠体内的治疗作用 我们的初步数据已经通过体内足迹确定了hs6中的三个因子结合位点。LCR中这些位点的突变会损害其基于染色质的活性。因此，这些将是目标1和2的重点。AML-1和Elf-1蛋白被发现与这些位点相互作用，并将成为目标3的重点。通过提供关于LCR活性和基于染色质的基因调控的分子基础的清晰图景，这些研究将有助于缩小我们对T细胞特异性基因表达如何实现的了解的差距。这些信息可能有助于改进先天性免疫疾病、白血病和注意力缺陷多动障碍的基因治疗策略。

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. The study of "chromatin based" gene regulation holds the promise of completing our understanding of these processes. Such mechanisms have been proposed to play an important role in immune cell development and function. However, the molecular nature of chromatin based gene regulation and its relevance to T cell gene activity in vivo is still poorly defined. The study of transcriptional regulation in transgenic mice has identified a novel class of gene regulatory sequences with chromatin based activity. 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. Using transgenic mice, we have identified a discrete sub-element of the LCR with chromatin based activity, named DNase hypersensitive site (HS)-6. We hypothesize that our study of this element will reveal the molecules and mechanisms behind chromatin based gene regulation in T cells. In this proposal, we specifically aim to: 1. Determine the functional sequences within HS6 contributing to LCR activity in vivo 2. Characterize the mechanism of action of functional HS6 sequences using standard and novel methods 3. Determine the role of transcription factors interacting with HS6 functional sequences in LCR activity 4. Test the therapeutic utility of HS6 containing vectors in transgenic mice Our preliminary data have identified 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 Aims 1 and 2. The AML-1 and Elf-1 proteins were found to interact with these sites and will be the focus of Aim 3. 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 for congenital immune disorders, leukemia and A.I.D.S.