Organization and regulation of the rat Cx31 gene - Implication for a crucial role of the intron region

The connexin31 (Cx31) gene, a member of the connexin multigene family, is e xpressed in a characteristic spatiotemporal pattern during placental develo pment in rodents. To elucidate the trophoblast-specific regulation of Cx31, we have isolated the rat Cx31 gene and performed structural and functional promoter analysis. The isolated Cx31 gene contains two exons separated by an intron of 2.6 kb. The first exon of the Cx31 gene is preceded by a TATA- less promoter region. Transcription is initiated in exon 1 from two transcr iption start sites producting transcripts of 105 and 139 bp. The 935 bp of the 5' flanking region of exon 1 comprises five putative binding sites for the GATA transcription factors as well as a NF-kappaB element, a CAAT-box a nd E-box/E-box-related sequences. For functional promoter analysis, the rat choriocarcinoma cell line Rcho-1 and the mouse keratinocyte cell line Hel3 7, which both express Cx31, were chosen. Only constructs including exon 1 a nd the complete intron showed high activity in transient transfection exper iments in both cell lines. All deletion fragments of the putative promoter region, but which contain the entire intron sequence, did not reveal any ob vious changes in luciferase activity. However, deletion of 1.1 kb of the in tron sequence downstream of the splice donor site resulted in the loss of p romoter activity. The intron exhibits no enhancer activity for the gene; ho wever, the mRNA stability was increased in the presence of the intron seque nce. These results indicate that parts of the intron sequence are critical for basic promoter function of the Cx31 gene.