Identification of protein-binding sequences mediating constitutive and 12-O-tetradecanoylphorbol-13-acetate-induced VL30 transcription in cultured mouse and human keratinocytes.
A retrovirus-like 30S (VL30) gene induced in mouse epidermis after a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) was used as a model gene to define mechanisms of transcriptional regulation in keratinocytes. Sequences important for TPA and epidermal growth factor-induced transcription were found to be separated from eacho other within the long terminal repeat. Deletion mapping of the long terminal repeat region and linking short sequences to a heterologous promoter made it possible to identify a 28-base pair VL30 TPA-responsive element. VL30 TPA-responsive element mediated both basal and TPA-induced transcription in the mouse keratinocyte cell line Balb/MK and in normal human keratinocytes. Gel-retardation and transient transfection experiments indicated that two nuclear factors (VLX and VLY) bind independently to the VL30 TPA-responsive element in juxtaposed positions and that the binding sites collaborate functionally in constitutive and TPA-induced transcription. The sequence involved in VLY binding shows no homology to previously identified binding motifs. Two different sequences involved in mediating TPA-induced transcription of the urokinase plasminogen activator and of the c-jun gene, respectively, competed for proteins with affinity toward the VLX binding site. No competition was found with sequences containing the consensus AP-1 binding site.
Bohm
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Karolinska Institute, Center for BioTechnology, NOVUM, Huddinge, Sweden.
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA; D004815:Epidermal Growth Factor / Q000378:metabolism; D005786:Gene Expression Regulation; D005814:Genes, Viral; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D009687:Nuclear Proteins; D004815:Epidermal Growth Factor; D012313:RNA; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate