Structure of the human 5-HT7 receptor gene and characterization of its promoter region.
The molecular mechanism regulating serotonin 5-HT(7) receptor expression is still unclear. In this study we provide evidence that transcription of the 5-HT(7) gene is at least partly regulated by Sp1 and Sp3. We isolated and sequenced >3000 bp of the upstream sequences and identified by RACE a number of transcriptional initiation sites over a region of 300 bp upstream of the coding region. This region has a high GC content, but contains no obvious TATA or a CAAT box. Besides a Sp1/Sp3 consensus motif, regulatory elements for AP2, Egr-1 and MAZ are also present. Transient transfection assays using deletion variants indicated that the GC-rich region is essential for full promoter activity. The role of Sp1 in this was confirmed by transient overexpression of both wild type Sp1 or dominant-negative forms. By gel shift and supershift analyses, targeting the Sp1 consensus sequence and the GC-rich region just upstream of the transcription initiation sites, binding of Sp1 and Sp3 was demonstrated. Both in vitro as well as in vivo experiments, using a cell line which endogenously expresses the 5-HT(7) receptor, indicated that mithramycin A, an inhibitor of Sp1/3 transcription factor binding, was able to inhibit 5-HT(7) promoter activity. Taken together these results support the essential role of Sp factors in regulating 5-HT(7) promoter activity.
Laenen
Koen
K
Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Department of Molecular Biology, Ghent University, UGent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium.
Haegeman
Guy
G
Vanhoenacker
Peter
P
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D020862:GC Rich Sequence; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008926:Plicamycin / Q000031:analogs & derivatives / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011985:Receptors, Serotonin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection
D011985:Receptors, Serotonin; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; C104425:serotonin 7 receptor; D051705:Sp3 Transcription Factor; C066851:mithramycin A; D008156:Luciferases; D008926:Plicamycin