LINEAGE COMMITMENT, GENE SILENCING, AND EPIGENETIC REGULATION IN THYMOCYTES
The distinct stages of T cell development in the thymus can be readily followed by the pattern of expression of various cell surface markers, particularly the CD4 and CD8 glycoproteins. These molecules are co-receptors that interact with membrane-proximal domains of MHC class II and class I, respectively, and participate in TCR-mediated recognition of MHC/antigen complexes and the initiation of signal transduction. The most immature thymocytes are double negative (DN) cells, which express neither CD4 nor CD8. Thymocytes with productive rearrangement at the TCRb locus and expression of a pre-TCR (composed of pre-Ta and TCRb) are selected to differentiate to the double positive (DP, CD4+CD8+) stage, after which there is rearrangement at the TCRa locus and cell surface expression of clonally-restricted TCRab heterodimers. The DP thymocytes then undergo selection, such that strongly self-reactive cells are deleted (negative selection) while cells that will form a useful repertoire are selected for migration to secondary lymphoid organs (positive selection), and differentiate into CD4+CD8- T helper cells and CD4-CD8+ cytotoxic T cells. The tight coupling of CD4 and CD8 expression with the functional specification of T cells suggests that these functions may be regulated by shared transcriptional signaling pathways. Our approach has been to characterize the factors that regulate CD4 and CD8 gene expression at different stages of T cell development. We expect that this will provide insight into the mechanism of thymocyte lineage commitment, which has been an area of considerable interest and controversy.
By using a combination of transfection, transgenic, and gene targeting analyses, we have shown that CD4 gene expression is regulated by a stage-specific silencer element. In the absence of the silencer, CD4 is de-repressed in both DN and CD8-lineage thymocytes and T cells. By using Cre-Lox-mediated deletion of the silencer, we have shown that it is required for establishment of silencing, but not for maintenance of silencing in CD8+ cytotoxic T cells. We have evidence that a different silencing mechanism operates in DN thymocytes, in which silencing needs to be reversed. For example, we have shown that members of the Runx family of transcription factors are differentially required for silencing in DN versus mature CD4-CD8+ thymocytes. The roles of these factors in recruitment of silencing factors and in remodeling of chromatin, as well as in lineage specification, are being studied in a series of recently-generated mutant mice.
The closely-linked CD8a and CD8b genes are regulated not by silencing, but by a series of enhancers that act in concert at distinct stages of T cell development. We have identified an enhancer that functions exclusively in DP thymocytes, but is down-regulated as cells undergo positive selection. Another enhancer directs expression of reporter genes only at the most mature stage of CD8 lineage differentiation, after cells have shut off CD4, yet collaborates with a third enhancer to activate the CD8 genes in DP thymocytes. The different mode of regulation of the CD4 and CD8 loci suggests that these molecules evolved independently, perhaps to serve distinct functions during positive selection of thymocytes. We continue to characterize the factors involved in this regulation and examine the mechanisms by which these factors are controlled by signals from the TCR and co-receptors.
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