title: Transcriptional regulation of N-acetylglutamate synthase.
abstract: The urea cycle converts toxic ammonia to urea within the liver of mammals. At least 6 enzymes are required for ureagenesis, which correlates with dietary protein intake. The transcription of urea cycle genes is, at least in part, regulated by glucocorticoid and glucagon hormone signaling pathways. N-acetylglutamate synthase (NAGS) produces a unique cofactor, N-acetylglutamate (NAG), that is essential for the catalytic function of the first and rate-limiting enzyme of ureagenesis, carbamyl phosphate synthetase 1 (CPS1). However, despite the important role of NAGS in ammonia removal, little is known about the mechanisms of its regulation. We identified two regions of high conservation upstream of the translation start of the NAGS gene. Reporter assays confirmed that these regions represent promoter and enhancer and that the enhancer is tissue specific. Within the promoter, we identified multiple transcription start sites that differed between liver and small intestine. Several transcription factor binding motifs were conserved within the promoter and enhancer regions while a TATA-box motif was absent. DNA-protein pull-down assays and chromatin immunoprecipitation confirmed binding of Sp1 and CREB, but not C/EBP in the promoter and HNF-1 and NF-Y, but not SMAD3 or AP-2 in the enhancer. The functional importance of these motifs was demonstrated by decreased transcription of reporter constructs following mutagenesis of each motif. The presented data strongly suggest that Sp1, CREB, HNF-1, and NF-Y, that are known to be responsive to hormones and diet, regulate NAGS transcription. This provides molecular mechanism of regulation of ureagenesis in response to hormonal and dietary changes.
authors.affiliation: Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC, United States of America.
authors.forename: Sandra Kirsch
authors.initials: SK
authors.lastname: Heibel
authors.forename: Giselle Yvette
authors.initials: GY
authors.lastname: Lopez
authors.forename: Maria
authors.initials: M
authors.lastname: Panglao
authors.forename: Sonal
authors.initials: S
authors.lastname: Sodha
authors.forename: Leonardo
authors.initials: L
authors.lastname: Mariño-Ramírez
authors.forename: Mendel
authors.initials: M
authors.lastname: Tuchman
authors.forename: Ljubica
authors.initials: L
authors.lastname: Caldovic
mesh_terms: D051046:Amino-Acid N-Acetyltransferase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000096:biosynthesis* / Q000235:genetics*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D051900:Smad3 Protein / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013045:Species Specificity; D050656:Transcription Factor AP-2 / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D023081:CCAAT-Binding Factor; D022762:CCAAT-Enhancer-Binding Proteins; C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; C496487:HNF1A protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D051046:Amino-Acid N-Acetyltransferase; D002222:Carbamoyl-Phosphate Synthase (Ammonia)