CHARACTERIZATION OF HUMAN TCR V-BETA GENE PROMOTER - ROLE OF THE DODECAMER MOTIF IN PROMOTER ACTIVITY

During T-lymphocyte development, the T-cell antigen receptor (TCR) gen e expression is controlled by its promoter and enhancer elements and r egulated in tissue and development stage-specific manner. To uncover t he promoter function and to define positive and negative regulatory el ements in TCR gene promoters, the promoter activities from 13 human TC R V beta genes were determined by the transient transfection system an d luciferase reporter assay. Although most of the TCR V beta gene prom oters that we tested are inactive by themselves, some promoters were f ound to be constitutively strong. Among them, V beta 6.7 is the strong est. 5'-Deletion and fragmentation experiments have narrowed the full promoter activity of V beta 6.7 to a fragment of 147 base pairs immedi ately 5' to the transcription initiation site. A decanucleotide motif with the consensus sequence AGTGAYRTCA has been found to be conserved in most TCR V beta gene promoters. There are three such decamer motifs in the promoter region of V beta 6.7, and the contribution of each su ch motif to the promoter activity has been examined. Further site-dire cted mutagenesis analyses showed that: 1) when two Ts in the decamer w ere mutated, the promoter activity was totally abolished; 2) when two additional nucleotides 3' to the end of decamer were mutated, the prom oter activity was decreased to two-thirds of the full level; and 3) wh en the element with the sequence AGTGATGTCACT was inserted into other promoters, the original weak promoters become very strong. Taken toget her, our data suggest that the positive regulatory element in V beta 6 .7 should be considered a dodecamer rather than a decamer and that it confers strong basal transcriptional activity on TCR VP genes.