Nucleic Acids Research, 1994, Vol. 22, No. 5 850-860
© 1994
MOLECULAR BIOLOGY |
Analysis of the imperfect octamer-containing human immunoglobulin VH6 gene promoter
Department of Virology and Molecular Biology, St Jude Children’s Research Hospital 332 N Lauderdale Street, Memphis, TN 38101-0318, USA
*To whom correspondence should be addressed
Received August 17, 1993. Revised December 17, 1993. Accepted December 17, 1993.
The octamer sequence ATGCAAAT is highly conserved in the promoter of immunoglobulin heavy and light chain genes and is one of the sequence motifs involved in the control of transcription of these genes. The promoter region of an human immunoglobulin heavy chain variable gene, the sole member of the VH6 gene family, was found to differ from other VH gene promoters: It contains neither the conserved octamer motif nor a heptamer sequence, and generally bears little resemblance to other VH gene transcriptional control regions. An imperfect octamer sequence with a single nucleotlde substitution (AgGCAAAT) is located 108 bp upstream of the ATG translation start site, and 81 bp upstream of the transcription initiation site. We sought to determine which sequence elements within the VH6 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 promoter function was 110 bp, but a fragment with only 65 bp retained 30 - 50% activity in B cells. Similar levels of transcription were seen when the -146 bp promoter containing two point mutations in the Imperfect octamer was tested. Mutation of a possible pyrimidine box sequence located downstream of the TATA box was shown to have only a minor effect (10-30%) on transcription when three nucleotides were changed. Surprisingly, CAT activity was not B cell-specific, as all constructs had virtually the same activity in several B cell lines and in HeLa cells. 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 virtually enhancer Independent: only a minor Increase was observed when the immunoglobulin or SV40 enhancer was added to the promoter construct. Electrophoretlc mobility shift assays of transcription factor binding to the region around the imperfect octamer Indicated that binding was weak when nuclear extracts from either B cells or HeLa cells were used. The amount of complex shifted was Increased by mutating the Imperfect octamer to a perfect one. Chimeras produced between the VH6 promoter and a B cell-specific promoter from a member of the human VH2 gene family demonstrated that the lack of tissue specificity was due to the absence of a repressor of non-B cell transcription in the VH6 promoter. These results indicate that the VH6 promoter is relatively simple, requiring little more than the TATA element and the imperfect octamer, and transcription from this promoter lacks B cell specificity and is not dependent on the enhancer element.
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