Mapping and characterization of the basal promoter of the human connexin26gene

Connexin26 (Cx26) is a major gap junction protein expressed in mammary and endometrial epithelial cells. Previously, we have cloned the genomic upstre am sequence of the human connexin26 gene. In this paper, we studied the str ucture and function of its basal promoter. Various 5'-flanking regions of t he human Cx26 gene were inserted upstream of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and transfected into human immortaliz ed mammary MCF-10A and MCF-12A cell lines and endometrial RL95-2 cancer cel l line. Through CAT reporter gene analysis, we identified the basal promote r of human Cx26 gene in the proximal 5'-flanking region from -128 to +2 (re lative to the transcription initiation site). Further deletion analyses sug gested that the critical regulatory area was located within a 29 bp region (from -97 to -69), where two GC consensus boxes (CCGCCC) resided, one at -9 3 and the other at -81. Labeled oligonucleotides encompassing these two GC box DNA sequences could bind the nuclear extracts from MCF-12A and RL95-2 c ells in the electrophoretic mobility shift assay. These binding complexes c ould be competitively reduced by nonlabeled self or Spl consensus oligonucl eotide, and supershifted by antibodies against either Spl or Sp3. Mutations in the core sequence of these two GC boxes from CCGCCC to CCG<(AA)under ba r>C caused a loss of competitive ability and also produced a drastic reduct ion of basal promoter activity when integrated into promoter/reporter const ructs. Furthermore, co-transfection of Spl and/or Sp3 expressing plasmids c ould trans-activate the expression of human Cx26 promoter/reporter construc ts in Drosophila Schneider line 2 (SL2) cells. Taken together, these data i ndicated that the two GC boxes in the proximal promoter region play an impo rtant role in the control of human Cx26 gene expression. (C) 1998 Elsevier Science B.V. All rights reserved.