This project seeks to identify a novel class of human gene regulatory elements that are different from conventional enhancers, but cooperate with a conventional enhancer to cause powerful gene activation in developing T-cells. Transfection, biochemical, and transgenic analyses of cis-elements in the human adenosine deaminase (ADA) gene first intron have uncovered crucial control sequences that have been termed """"""""facilitators"""""""" by this investigator. The facilitators are presently defined by two l kb segments that bilaterally flank a conventional 300 bp T-cell enhancer. Their presence can drastically increase gene expression by the enhancer, but their effects are only detectable in transgenic mice. Facilitators prevent insertion site-dependent gene suppression and they cause thymocyte gene expression to increase strictly in proportion to gene copy number. Both facilitators must be present for their function: with either segment deleted, cell-type specificity of expression is maintained, but the transgenes are subject to positional effects that cause poor expression. A potentially important clue for their mechanism is that facilitator deletion abolishes DNase I hypersensitive site formation over the adjacent enhancer domain and also completely prevents any increase in gene expression from increased gene copy number. Hundreds of unfacilitated gene copies are expressed at a level equal to or less than that of only one or two facilitated copies. Similar size human DNA fragments could not substitute for a missing facilitator segment indicating that specific sequences were responsible. Facilitator sequences are hypothesized to function during T-cell development by interacting with nuclear factors responsible for organizing chromatin structure: they may be representative of a novel clams of regulatory elements that enable the formation of regulatory domains within chromosomes. This application will test the hypothesis that facilitators are composed of specific definable elements that cooperate with the ADA gene enhancer.
The specific aims are (1) to identify specific sequences responsible for facilitator activity, and (2) to determine the rules or constraints that govern how these control sequences cooperate with the enhancer. The strong effect of each facilitator segment in transgenic analyses provides a clear means to identify its individual elements and properties using mutational techniques. Long range goals are to determine the potential ability of faciIitators to interact with other enhancers and transferable genes, and to determine facilitator mechanisms by identifying nuclear components that interact with the specific facilitator elements. Detailed study of the facilitators will provide insight into a crucial regulatory region that controls purine catabolism necessary for T-cell development, and should increase our understanding of how chromatin accessibility is regulated. Our ability to use facilitator elements in the future may greatly improve the design of genes capable of correcting inherited illnesses or combating viral or neoplastic diseases.
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