IDENTIFICATION AND CHARACTERIZATION OF AN ALU-CONTAINING, T-CELL-SPECIFIC ENHANCER LOCATED IN THE LAST INTRON OF THE HUMAN CD8-ALPHA GENE

Expression of the human CD8alpha gene is restricted to cells of the ly mphoid lineage and developmentally regulated during thymopoiesis. As a n initial step towards understanding the molecular basis for tissue-sp ecific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hyper sensitivity mapping and found four hypersensitive sites, three of whic h were T cell restricted. By using a reporter-based expression approac h, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last in tron of the CD8alpha gene. Deletion studies demonstrated that the mini mal enhancer is adjacent to a negative regulatory element. DNA sequenc e analysis of the minimal enhancer revealed a striking cluster of cons ensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH pro teins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat wh ich contained the GATA-3, bHLH, and LyF-1 binding sites. Site-directed mutation of the Ets-1 and GATA-3 sites dramatically reduced enhancer activity. The functional importance of the other binding sites only be came apparent when combinations of mutations were analyzed. Taken toge ther, these results suggest that the human CD8alpha gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcri ptional enhancer located in the last intron of the gene. Comparison of the CD8alpha enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factor s regulates several T-cell genes.