Functional interaction of NF-Y and Sp1 is required for type a natriuretic peptide receptor gene transcription.
The vasorelaxant and anti-mitogenic activities of the atrial and brain natriuretic peptides depend upon their binding to the type A natriuretic peptide receptor (NPR-A) expressed on the surface of vascular cells. Intervention strategies aimed at controlling NPR-A expression are limited by the paucity of studies in this area. Here we identify a sequence CCAAT between -141 and -137 of the NPR-A promoter that, when mutated, reduces promoter activity by 90% in rat aortic smooth muscle (RASM) cells. Protein/DNA cross-linking and immunoperturbation of electrophoretically shifted complexes formed between RASM nuclear extracts and an oligonucleotide surrounding the CCAAT sequence indicates that the heterotrimeric transcription factor NF-Y binds specifically to the wild-type, but not mutated, CCAAT element. Cotransfection of a dominant negative mutant of the NF-YA subunit results in a concentration-dependent decrease in the activity of the NPR-A promoter in RASM cells confirming that endogenous NF-Y is an activator of the promoter. Mutation of the CCAAT element, in conjunction with mutation of all three Sp1 sites previously shown to be involved in NPR-A promoter regulation, virtually eliminates NPR-A promoter activity in RASM cells. Coexpression of all three NF-Y subunits together with Sp1 in Drosophila cells deficient in these factors indicates that NF-Y and Sp1 act synergistically to reconstitute NPR-A promoter activity. A direct physical association between NF-Y and Sp1 can be demonstrated both in vitro by glutathione S-transferase pull-down assay and in the intact cell by coimmunoprecipitation and functional studies. Together, these studies show that NPR-A promoter activity is dominantly regulated through functional, and possibly physical, interactions of NF-Y and Sp1.
Liang
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Metabolic Research Unit and Department of Medicine, University of California, San Francisco, California 94143-0540, USA.
Schaufele
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Gardner
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DG
D000818:Animals; D001011:Aorta / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004330:Drosophila; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006162:Guanylate Cyclase / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D023081:CCAAT-Binding Factor; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D001616:beta-Galactosidase; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A