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.

R21AI053050

2003-09-01

2004-08-31