Characterization of the rat connexin40 promoter: two Sp1/Sp3 binding sites contribute to transcriptional activation

Objectives: The gap junction protein connexin40 (Cx40) is differentially expressed during embryonic development and in adult tissues, for which the molecular basis is unknown. In order to elucidate the molecular mechanisms controlling Cx40 expression, we set out to map and characterize its promoter. Methods: The transcriptional activity of individual rat Cx40 (rCx40)-derived promoter fragments fused to the luciferase reporter gene was determined by transfection/reporter assays in Cx40-expressing (A7r5, rat smooth muscle embryonic thoracic aorta cells, and BWEM, v-myc transformed rat fetal cardiomyocytes) and Cx40-nonexpressing cells (N2A, mouse neuroblastoma cells). The nature of DNA-protein interactions was investigated by a combination of standard electrophoretic-mobility-shift assays (EMSA) and EMSA/antibody supershift assays. Results: Quantification of luciferase activity in cell lysates revealed that a 235-base-pair fragment, in between map positions -150 and +85 relative to the transcription initiation site, is able to provide for a significant level of transcription in both Cx40-expressing (A7r5, BWEM) and -nonexpressing (N2A) cells. These results indicate that this region contains the basal promoter but is not sufficient to completely determine the endogenous Cx40-expression pattern within these cell types. In search for the responsible transcriptional regulatory element(s), additional segments of the (-150, +85) region were deleted and the remaining fragments were tested for transcriptional activity. These studies established that the regions in between map positions (-96, -71) and (+58, +85) contribute to promoter activity. EMSA with these regions revealed that predominantly two DNA-protein complexes are formed upon incubation with either A7r5, BWEM or N2A nuclear extracts, which could be both inhibited by including excess oligonucleotide containing the Sp1 consensus binding site in the binding reaction. Purified recombinant human Spl provided also for a shift in the EMSA using these promoter regions as target fragments. When the DNA-protein complexes formed with nuclear extract were subsequently incubated with either an anti-Spl or an anti-Sp3 antibody clear supershifts in the EMSA were obtained, indicating Sp1 and Sp3 binding to both the (-98, -64) and (+53, +87) regions. The introduction of mutations within the core sequence of the putative Sp1/Sp3 binding sites present in these regulatory elements reduced the level of transcriptional activity and abrogated Sp1/Sp3 binding to these sites. Conclusion: The results indicate that at least two Sp1/Sp3 binding sites in the rCx40 promoter contribute to the transcriptional activation of its gene in cultured cells. (C) 2000 Elsevier Science B.V. All rights reserved.