ANALYSIS OF THE IMPERFECT OCTAMER-CONTAINING HUMAN-IMMUNOGLOBULIN V(H)6 GENE PROMOTER

The octamer sequence ATGCAAAT is highly conserved in the promoter of i mmunoglobulin heavy and light chain genes and is one of the sequence m otifs involved in the control of transcription of these genes. The pro moter region of an human immunoglobulin heavy chain variable gene, the sole member of the V(H)6 gene family, was found to differ from other V(H) gene promoters: it contains neither the conserved octamer motif n or a heptamer sequence, and generally bears little resemblance to othe r V(H) gene transcriptional control regions. An imperfect octamer sequ ence with a single nucleotide substitution (AgGCAAAT) is located 108 b p upstream of the ATG translation start site, and 81 bp upstream of th e transcription initiation site. We sought to determine which sequence elements within the V(H)6 promoter were responsible for transcription initiation by creating progressive deletions of a 1 kb fragment from this region and testing their ability to function as promoter elements in B and non-B cells (HeLa). The minimum fragment required for full p romoter function was 110 bp, but a fragment with only 65 bp retained 3 0 - 50% activity in B cells. Similar levels of transcription were seen when the -146 bp promoter containing two point mutations in the imper fect octamer was tested. Mutation of a possible pyrimidine box sequenc e located downstream of the TATA box was shown to have only a minor ef fect (10-30%) on transcription when three nucleotides were changed. Su rprisingly, CAT activity was not B cell-specific, as all constructs ha d virtually the same activity in several B cell lines and in HeLa cell s. Removal of the TATA box led to a 50% reduction in CAT activity, and the region upstream of the TATA box functioned as a promoter in both orientations. The transcriptional activity of the VH6 promoter was vir tually enhancer independent: only a minor increase was observed when t he immunoglobulin or SV40 enhancer was added to the promoter construct . Electrophoretic mobility shift assays of transcription factor bindin g to the region around the imperfect octamer indicated that binding wa s weak when nuclear extracts from either B cells or HeLa cells were us ed. The amount of complex shifted was increased by mutating the imperf ect octamer to a perfect one. Chimeras produced between the VH6 promot er and a B cell-specific promoter from a member of the human V(H)2 gen e family demonstrated that the lack of tissue specificity was due to t he absence of a repressor of non-B cell transcription in the VH6 promo ter. These results indicate that the VH6 promoter is relatively simple , requiring little more than the TATA element and the imperfect octame r, and transcription from this promoter lacks B cell specificity and i s not dependent on the enhancer element.