The “initiator” as a transcription control element

The enzymatic machinery that carries out RNA synthesis provides the cell with the means to adjust the patterns of transcription in response to environmental and developmental signals. In eukaryotes, this regulation is mediated in part by promoter-specific transcription factors, which are DNA-binding proteins with the ability to discriminate between distinctive DNA sequence elements found in the promoter regions of different genes. The presence of these factors bound to DNA enables other components of the transcriptional machinery, including the RNA polymerase, to initiate transcription with selectivity and accuracy.

Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins

Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II

Cytosine methylation within CpG dinucleotides has been implicated in the regulation of gene expression in vertebrates and, in some cases, has been shown to be causative in repression of transcription . We have examined whether methylation of CpG dinucleotides located within the binding site for a specific transcription factor, MLTF or USF, affects its binding to DNA. This HeLa cell factor binds to the adenovirus major late promoter (AdMLP), as well as endogenous cellular genes, and stimulates transcription in an in vitro assay. Synthetic oligonucleotides in which 5-methylcytosine replaces cytosine at specific sites were used to generate duplex DNAs, and the formation of complexes of these oligomers with MLTF was studied using a gel retardation assay. Methylation at a CpG site centrally located within the binding site strongly inhibited complex formation, whereas methylation at a site 6 bases away had no demonstrable effect. Methylation at the central site was also shown to inhibit specific transcription in vitro from the AdMLP. Methylation at the central site on only one strand caused a partial inhibition of binding, the effect being greater when the noncoding strand was methylated. The results indicate that in some cases, site-specific methylation may inhibit gene expression directly by blocking binding to DNA of factors required for optimal transcription. Along with other recent findings, they suggest an interplay between DNA methylation and transcription factors in the regulation of gene expression.

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Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter

Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes

We show that sequences located upstream from the T-A-T-A box, between positions -97 and -34, are necessary for efficient in vivo transcription from the adenovirus serotype 2 major late promoter. The effect of these upstream sequences was also investigated in vitro using a whole cell or an S100 extract and circular or linear templates. With the whole cell extract, the in vivo effect of the upstream sequences was reproduced in vitro. With the S100 extract, some effect of the upstream sequences was observed with circular, but not with linear, templates.

Sequences upstream from the T-A-T-A box are required in vivo and in vitro for efficient transcription from the adenovirus serotype 2 major late promoter

We have analysed in vivo and in vitro the sequences required for specific transcription from the Adenovirus-2 major late promoter (Ad2MLP). Our results demonstrate that, in addition to the TATA box, sequences located further upstream between positions −97 and −34 (position +1 refering to the capsite), are necessary for efficient in vivo transcription. Recombinant plasmids containing wild-type or deleted Ad2MLP, linked to the SV40 T-antigen coding sequence, were introduced by microinjection or calcium-phosphate transfection into cells in culture. T-antigen expression was assayed by immunofluorescence and specific transcription was assayed by quantitative S1 nuclease mapping. Deletion of sequences located between positions −97 and −34 resulted in a strong reduction of T-antigen production and in a 30-fold decrease in specific RNA production. The effect of these upstream sequences was also investigated in vitro using a whole cell extract or an S100 transcription system and circular or linear templates. In the whole cell extract, the in vivo effect was faithfully reproduced when circular templates were used, but the effect was also seen with linear templates. With the S100 extract, some effect of the upstream sequences on specific in vitro transcription was seen with circular, but not with linear templates.

M.-P. Gaub

Papers

Sequences upstream from the T-A-T-A box are required in vivo and in vitro for efficient transcription from the adenovirus serotype 2 major late promoter

Adenovirus-2 major late promoter : sequences upstream from the tata box are essential for efficient in vivo and in vitro transcription