Expression of the human CD8a gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis. As an initial step towards understanding the molecular basis for tissue-specific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites, three of which were T cell restricted. By using a reporter-based expression approach, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8a gene. Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element. DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH proteins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat which 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 became apparent when combinations of mutations were analyzed. Taken together, these results suggest that the human CD8ae gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene.Comparison of the CD8ax enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes.The T-lymphocyte differentiation marker CD8 identifies a distinct T-cell subset which can be triggered to express cytotoxic and/or suppressor activity upon recognition of antigenic peptides presented in the context of class I major histocompatibility complex (MHC) molecules (46,62,75). Expressed on the T-cell surface as either an a/ot homodimer or a/1 heterodimer, CD8 is capable of direct extracellular interaction with the a3 domain of MHC class I molecules (12,60,63,68) and intracellular association with the tyrosine kinase p56lck (3,8), assisting both the T-cell receptor (TCR) in antigen recognition and the CD3 complex in signal transduction. Thus, CD8 serves as an integral part of a complex recognition/response apparatus which functions to initiate antigen-driven activation of mature effector T cells in the periphery as well as differentiation of immature T cells in the thymus.Tissue-specific expression of CD8 is under precise developmental control. When bone marrow-derived precursor cells begin the process of thymopoiesis, a panel of T-cellspecific genes (TCR, CD2, CD3, and CD4) including CD8a and CD83 are coordinately expressed in a defined temporal sequence (10,32,45

Expression of the human CD8 alpha gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis. As an initial step towards understanding the molecular basis for tissue-specific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites, three of which were T cell restricted. By using a reporter-based expression approach, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8 alpha gene. Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element. DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH proteins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat which 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 became apparent when combinations of mutations were analyzed. Taken together, these results suggest that the human CD8 alpha gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene. Comparison of the CD8 alpha enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes.