The promoter of the Xwnt-5C gene contains octamer and AP-2 motifs functional in Xenopus embryos.
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ABSTRACT:
SUBMITTER: Kuiken GA
PROVIDER: S-EPMC308048 | biostudies-other | 1994 May
REPOSITORIES: biostudies-other
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Project description:We have identified in an intron of an X. laevis alpha-tubulin gene a member of a novel family of short (226-431 bp) interspersed repetitive elements. We have isolated other members of this family, which we term Ocr, from ovary cDNA and genome libraries and have identified another two in the published sequences of an H1B histone gene cluster and an actin gene intron. The termini of the Ocr elements are formed by a 19 bp inverted repeat that has clear sequence homologies to those of certain large transposable elements, such as 1723 (Xenopus) and Ac (maize). However, the Ocr elements do not appear to be deletion derivatives of larger transposons. The internal regions of the Ocr elements contain multiple copies of the octamer motif (ATTTGCAT) arranged as divergently-orientated dyads. We have shown by a gel mobility shift assay that these octamer dyads specifically bind what is presumably an OTF-type activator protein in oocyte nuclear extracts. We speculate that short interspersed repetitive families of this type may be generated by a mechanism of replicative transposition that uses a DNA intermediate and involves the interaction of DNA-binding proteins also utilised in other cellular processes.
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Project description:Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.
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Project description: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 nucleotide 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. Electrophoretic 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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