Molecular cloning and characterization of alpha1-soluble guanylyl cyclase gene promoter in rat pituitary cells.
Soluble guanylyl cyclase is a cytosolic enzyme which catalyzes conversion of GTP to the second messenger cyclic GMP. The transcriptional regulation at the promoter levels of four soluble guanylyl cyclase subunits, termed alpha1, alpha2, beta1, and beta2, is largely unknown. In this study, we identified the transcription start site of alpha1-soluble guanylyl cyclase gene in rat 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 pituitary cells with constructs of variable lengths confirmed the relevance of different promoter regions in the control of transcriptional activity. Among them, the -49 to + 156 region was critical for basal transcriptional activity. Electrophoretic mobility shift assay using nuclear proteins extracted from normal and immortalized pituitary cells indicated that the CCAAT/Sp1 site within the -49 to + 156 region was able to specifically interact with CCAAT-binding factor and Sp1. These two sites were partly overlapped and both of them conferred stimulatory effects. The in vivo recruitment of CCAAT-binding factor and Sp1 was confirmed by chromatin immunoprecipitation. These results indicate that the composite CCAAT/Sp1 cis-element contributes to the expression of alpha1-sGC subunit in resting pituitary cells.
Jiang
Yonghua
Y
Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-4510, USA.
Stojilkovic
Stanko S
SS
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005260:Female; D017353:Gene Deletion; D006162:Guanylate Cyclase / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010902:Pituitary Gland / Q000166:cytology* / Q000201:enzymology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D000071756:Soluble Guanylyl Cyclase; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D023081:CCAAT-Binding Factor; D018160:Receptors, Cytoplasmic and Nuclear; D016329:Sp1 Transcription Factor; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase