Identification of proximal and distal regulatory elements of the rat connexin32 gene

We have investigated the genetic basis of the transcriptional regulation of the rat connexin32 gene which encodes the major gap junction protein in rat liver. Primer extension analysis of RNA isolated from adult rat liver identified multiple initiation sites clustered between -110 bp and -50 bp upstream from the translation start codon. An approx. 760 bp genomic DNA fragment upstream of the first exon which included the mRNA start sites was cloned 5′ to the luciferase reporter cassette in p19LUC to yield pCx 32-800/-33-LUC. Transfection of pCx 32-800/-33-LUC resulted in a 200-fold increase in luciferase activity above p19LUC in the human hepatoma cell line HuH-7. Using a series of vectors containing 5′ deletions of the 760 bp fragment, a basal promoter was localized between -179 bp and -134 bp. Three DNA:protein complexes were identified with the basal promoter fragment by DNA mobility shift assay using nuclear extracts from HuH7 cells. Two of the DNA-binding complexes appeared to be related to the transcription factor Sp1. In addition, three DNase hypersensitive (HS) sites were identified within the genomic locus of connexin32 in adult rat liver. Two of the DNase HS regions behaved as silencer elements with both the native promoter and a heterologous promoter in HuH-7 cells. These data demonstrate that (1) the active promoter responsible for rat connexin32 mRNA transcription is located upstream of the first exon, (2) a basal promoter region was localized to a 50 bp region which formed multiple DNA:protein complexes, and (3) multiple proximal and distant regulatory elements are involved in the expression of connexin32.