Identification and Functional Characterization of the Human T-Cell Receptor β Gene Transcriptional Enhancer: Common Nuclear Proteins Interact with the Transcriptional Regulatory Elements of the T-Cell Receptor α and β Genes

Abstract

A transcriptional enhancer has been mapped to a region 5.5 kilobases 3′ of the Cβ2 gene in the human T-cell receptor (TCR) β-chain locus. Transient transfections allowed localization of enhancer activity to a 480-base- pair HincII-XbaI restriction enzyme fragment. The TCR β enhancer was active on both the minimal simian virus 40 promoter and a TCR β variable gene promoter in both TCR α/β⁺ and TCR γ/δ⁺ T cells. It displayed significantly less activity in Epstein-Barr virus-transformed B cells and K562 chronic myelogenous leukemia cells and no activity in HeLa fibroblasts. DNA sequence analysis revealed that the enhancer contains a consensus immunoglobulin κE2 motif, as well as an AP-1-binding site and a cyclic AMP response element. DNase I footprint analyses using Jurkat T-cell nuclear extracts allowed the identification of five nuclear protein-binding sites, Tβ1 to Tβ5, within the enhancer element. Deletion and in vitro mutagenesis studies demonstrated that the Tβ2- Tβ3- and Tβ4-binding sites are each required for full transcriptional enhancer activity. In contrast, deletion of the Tβ1- and Tβ5-binding sites had essentially no effect on enhancer function. Electrophoretic mobility shift assays demonstrated that TCR α/β⁺ and TCR γ/δ⁺ T cells expressed Tβ2-, Tβ3-, and Tp4-binding activities. In contrast, non-T-cell lines, in which the enhancer was inactive, each lacked expression of at least one of these binding activities. TCR α and β gene expression may be regulated by a common set of T-cell nuclear proteins in that the Tβ2 element binds a set of cyclic AMP response element-binding proteins that are also bound by the Ta1 element of the human TCR a enhancer and the decamer element present in a large number of human and murine TCR β promoters. Similarly, the Tβ5 TCR β-enhancer element and the Tα2 TCR α-enhancer element bind at least one common T-cell nuclear protein. Taken together, these results suggest that TCR β gene expression is regulated by the interaction of multiple T cell nuclear proteins with a transcriptional enhancer element located 3′ of the Cβ2 gene and that some of these proteins may be involved in the coordinate regulation of TCR α and β gene expression.