One of the key features of thymocyte differentiation is the expression of the T-cell receptor (TCR) for antigen. The molecular mechanisms by which the TCR-alpha and -beta genes are activated and regulated during thymocyte maturation is not well understood. This research plan exploits a series or related T- lymphoma cell clones derived from a common source to examine TCR gene regulation. The SL12 clones differ in the expression of transcripts encoding TCR-alpha, TCR-beta, and the noncovalently associated invariant T3-delta and -epsilon subuints. Some of the SL12 cell clones can be induced to express TCR-alpha and -beta mRNAs in response to specific reagents and physiological factors. Preliminary studies indicate that repressor molecules, protein kinase C, cytoplasmic free Ca++ and cyclosporin A all participate in regulating TCR/T3 gene expression. The accumulation of TCR/T3 mRNA is regulated by transcriptional and post- transcriptional mechanisms in SL12 cell clones. The proposed studies will assess whether post-transcriptional regulation results from differences in RNA stability, processing or transport. To gain information on the precise mode of regulation, a combination of methodologies will be used, including the nuclear run-off assay, Northern analysis of precursor RNAs in the nucleus and labeling experiments to determine RNA half-life. Cis- and trans-acting regulatory elements involved in post-transcriptional regulation of selected TCR/T3 genes will be characterized by in vitro assays. Normal lymphoid cells will be assessd to determine whether they use the same post-transcriptional regulatory mechanisms as do SL12 cell clones. The transcriptional regulation of TCR-beta mRNA expression by a putative labile repressor protein(s) will be examined in more detail. In a collaborative effort, mutated TCR- beta promoter and J-C intron constructs will be introduced into SL12 cell clones and assayed for transcriptional activity to identify cis-acting regulatory elements which the TCR-beta gene possesses. Novel cDNA clones corresponding to genes which are regulated in parallel with TCR/T3 genes have been isolated by """"""""subtraction hybridization"""""""" methodologies. The expression of the novel genes will be assessed in normal lymphoid cells, including thymocyte subsets corresponding to distinct stages of thymocyte maturation. One of the novel genes will be characterized in detail, including DNA sequencing. The transcriptional and post- transcriptional regulation of the novel gene will be compared with regulatory mechanisms which control the expression of TCR/T3 genes. A molecular analysis of the unique and common modes of regulation used by a subset of T-cell specific genes may provide some insight into the orchestration of gene activation during lymphocyte differentiation.
