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.

R01AI053050

2004-03-15

2009-02-28