Molecular cloning and characterization of α 1‐soluble guanylyl cyclase gene promoter in rat pituitary cells

The transcriptional regulation at the promoter levels of four soluble guanylyl cyclase subunits, termed α 1, α 2, β1 and β2, has been incompletely characterized. In this study, we identified the transcription start site of α 1‐soluble guanylyl cyclase gene in rat anterior pituitary cells and cloned the 3.5 kb 5′‐promoter. Sequence analysis of this TATA‐less promoter revealed the presence of several putative binding sites for transcriptional factors, including CCAAT site at −41 to −32 and Sp1 site at −34 to −24. Transfection of immortalized pituitary cells with constructs of variable lengths confirmed the relevance of different promoter regions in bidirectional control of basal transcriptional activity. Among them, a construct carrying −516 to +156 promotor region showed elevated basal transcriptional activity, which was significantly reduced by mutation of CCAAT/Sp1 site located in this region. Furthermore, the electrophoretic mobility shift assay using nuclear proteins extracted from normal and immortalized pituitary cells showed that the CCAAT/Sp1 site was able to specifically bind with CCAAT binding factor and Sp1. Finally, the in vivo recruitment of CCAAT binding factor and Sp1 was confirmed by chromatin immunoprecipitation. These results indicate that a composite CCAAT/Sp1 cis‐element contributes to the expression of α 1‐sGC subunit in resting pituitary cells. Supported by the Intramural Research Program of the NICHD, NIH.