The Principal Investigator's (PI's) laboratory studies transcriptional regulation of the human LAT gene, which encodes a transmembrane adaptor protein linking receptor engagement to the activation of numerous downstream signaling pathways. This gene, highly conserved among mammals, is critical for the development and/or function of T cells, mast cells, Natural Killer cells, and megakaryocytes/platelets.
The specific aims outlined in this grant proposal build upon previous findings from the PI's lab and also initiate new studies focused on the contribution of distal control regions in the transcriptional regulation of LAT.
The specific aims of this grant proposal are to: 1. Use gene knockdown experiments to explore the functional contribution of Ets1, Elf1, and Runx1 in LAT gene expression. A demonstrated role for these transcription factors will be followed by investigations that examine the underlying mechanism by which they operate and will focus on their modulation of histone acetylation levels within the promoter region. 2. Employ systematic promoter deletions, chromatin immunoprecipitation assays, gene knockdown experiments, and other laboratory techniques to delineate and characterize additional critical control regions within the proximal LAT promoter that have yet to be examined in detail. 3. Implement reporter gene assays to investigate putative distal control regions that display a number of features consistent with their role as enhancer elements for the LAT gene. Confirmation that one or more of these regions serve as an enhancer will be followed by experiments to map the important regulatory sites and characterize interacting proteins.
This specific aim will also include chromatin conformation capture experiments to determine if the enhancer element(s) interact with the LAT proximal promoter region. This project will provide important new insights into the specific molecular processes that control transcription of the human LAT gene. As the details of LAT gene regulation continue to emerge, it may be possible to use this knowledge to devise intervention strategies that alter LAT expression as a means of modulating the immune responses and/or platelet function in situations where it would be therapeutically beneficial. This research will also aid in further understanding the general principles that govern gene regulation in eukaryotes. Finally, this research will provide an opportunity to integrate research and learning at Agnes Scott College, a small liberal arts college for women. Undergraduate students will contribute to each of the specific aims outlined in this proposal. Aspects of this research will also be incorporated into courses that the PI teaches.
This research will significantly enhance our understanding of the mechanisms that regulate expression of the human LAT gene, which is essential for the development and/or function of various cells of the immune system as well as those from which platelets are derived. Cells that express LAT contribute to numerous pathological conditions. Understanding how this critical gene is regulated will provide novel insights into the regulation of these cells under normal and altered states, and may identify potential targets for the development of new therapeutic agents.