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

Objectives: The gap junction protein connexin40 (Cx40) is differentially ex pressed during embryonic development and in adult tissues, for which the mo lecular basis is unknown. In order to elucidate the molecular mechanisms co ntrolling Cx40 expression, we set out to map and characterize its promoter. Methods: The transcriptional activity of individual rat Cx40 (rCx40)-deriv ed 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 card iomyocytes) 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 su pershift assays. Results: Quantification of luciferase activity in cell lys ates revealed that a 235-base-pair fragment, in between map positions -150 and +85 relative to the transcription initiation site, is able to provide f or a significant level of transcription in both Cx40-expressing (A7r5, BWEM ) and -nonexpressing (N2A) cells. These results indicate that this region c ontains the basal promoter but is not sufficient to completely determine th e 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 te sted for transcriptional activity. These studies established that the regio ns in between map positions (-96, -71) and (+58, +85) contribute to promote r activity. EMSA with these regions revealed that predominantly two DNA-pro tein complexes are formed upon incubation with either A7r5, BWEM or N2A nuc lear extracts, which could be both inhibited by including excess oligonucle otide containing the Sp1 consensus binding site in the binding reaction. Pu rified recombinant human Spl provided also for a shift in the EMSA using th ese promoter regions as target fragments. When the DNA-protein complexes fo rmed with nuclear extract were subsequently incubated with either an anti-S pl or an anti-Sp3 antibody clear supershifts in the EMSA were obtained, ind icating 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. Con clusion: The results indicate that at least two Sp1/Sp3 binding sites in th e rCx40 promoter contribute to the transcriptional activation of its gene i n cultured cells. (C) 2000 Elsevier Science B.V. All rights reserved.