Dr. Sen's long term goal is to understand at a molecular level the sequential steps of T-cell differentiation. Dr. Sen proposes that identification of key transcription factors that regulate stage-specific gene expression provides an important mechanism to examine the differentiation process. The regulation of the T-cell receptor beta chain gene and the CD8 alpha chain gene will be studied as probes of gene expression in double negative and double positive stages of T-cell development, respectively.
The Specific Aims of this application are: 1) To study the biological function and regulation of a promoter identified in the J beta 2-C beta 2 intron. It is proposed that this promoter may regulate TCR beta rearrangements, which will be investigated by deleting the promoter in embryonic stem cells. Critical promoter factors will be identified by transfection, mutagenesis and in vitro binding studies. 2) To identify the factors that activate the TCR beta enhancer in double negative T- cells. A core enhancer will be defined and differentiation stage specific factors that interact with these sequences will be identified by comparing extracts from mutant mice and staged fetal thymocytes. 3) To define and characterize CD8 alpha gene regulatory elements. Transgenic mice will be used to identify combinations of DNAase I hypersensitive sites that function as a locus control region and confer developmentally appropriate regulation. The combined genetic and biochemical experiments are expected to identify factors that respond to developmental cues in the thymus and, thereby, provide targets for the manipulation of T- cell differentiation.
