Functional analysis of the connexin43 gene promoter in vivo and in vitro.
Connexin43 is the principal gap junction protein found in the mammalian heart. The regulation of connexin43 gene expression may be an important determinant of normal and altered cardiac development and electrophysiology. We constructed a series of chimeric luciferase reporter genes containing nested deletions from the human connexin43 gene (-2400 to -50 basepairs, relative to transcription initiation). The transcriptional activity of the chimeric genes was assayed in several cell types and systems, including rat cardiocytes in vitro and adult rat heart in vivo. In both systems, high levels of luciferase activity required at least 175 base-pairs of 5'-flanking sequence. Constructs including 2400 base-pairs of upstream sequence increased activity two-fold in vivo, but not in vitro. Chimeric genes were also assayed in cultured cardiac non-myocytes. This population of cells accumulates significant levels of connexin43 mRNA when propagated in vitro and promoter constructs were correspondingly active. Elements within the proximal promoter may also confer tissue-specificity. Transfectional analysis of the SKHep1 cell line, which does not express connexin43, demonstrated reduced reporter gene activity, especially for the longest constructs. These studies begin to define the cis-acting elements of the connexin43 gene which regulate its strength and specificity of transcription.
De Leon
J R
JR
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.
Buttrick
P M
PM
Fishman
G I
GI
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018031:Connexin 43 / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D006321:Heart / Q000196:embryology; D007365:Intercellular Junctions; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic
D018031:Connexin 43; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins