Localization of regulatory protein binding sites in the proximal region ofhuman myometrial connexin 43 gene

Parturition is preceded by a large increase in gap junctions between myomet rial smooth muscle cells. Connexin 43 is the major structural protein of my ometrial gap junctions. To explore transcriptional regulation of the myomet rial Cx43 gene, we used DNase I footprinting, electrophoretic mobility shif t and transient transfection assays to examine a 312 bp promoter region (-1 64 to +148) of the gene, utilizing human myometrial cell cultures and nucle ar extracts. The DNase I studies showed four regions of nucleoprotein inter actions. Protection of region 1 (-80 to -31) encompassed an Activator Prote in 1 (AP1) (-44 to -36) and two Specificity Protein 1 (Sp1) (-77 to -69 and -59 to -48) consensus sequences. Regions 2 to 4 included the transcription initiation site (-10 to +25), an Ets/NF-kB consensus sequence (+47 to +74) and a TA-rich region (+81 to +101) respectively. Gel mobility shift and su pershift assays demonstrated c-Jun and Sp1 binding at the AP1 and Sp1 sites respectively. Promoter mutagenesis and transient transfection analyses com bined with Sp1 and c-Jun/c-Fos over-expression studies indicate that both S p1 and c-Jun are required for maximal promoter activity and, therefore, may positively regulate transcription of myometrial Cx43 during the initiation of labour.