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.
Highlights
Ammonia, the toxic product of protein catabolism, is converted to urea by the urea cycle in the liver of mammals
To validate our strategy for identification of conserved regions, the same analyses were conducted for carbamylphosphate synthetase 1 (CPS1), a gene in which a proximal promoter and an enhancer element located 6.3 kb upstream of rat Cps1, have been characterized [55,56,57]. 15 kb of CPS1 genomic DNA sequence 59 of the translational start site was collected from human, chimpanzee, dog, mouse and rat and compared using pair-wise BLAST
In this study we used bioinformatic analyses to predict regulatory regions based on the hypothesis that non-coding DNA sequences that are highly conserved between species are important for gene regulation
Summary
The toxic product of protein catabolism, is converted to urea by the urea cycle in the liver of mammals. While studies in the 1980s and 1990s identified the cis-acting motifs regulating transcription of the urea cycle enzymes CPS1. [7,8,9,10], OTC [11,12,13,14], ASS [15,16,17], ASL [18,19,20], and Arg1 [21,22], the mammalian NAGS gene was not identified until 2002 [2] and we can report for the first time on its transcriptional regulation. Sp1 and nuclear factor 1 (NF-1)/CCAAT-binding transcription factor (CTF) activate ARG1 transcription while two C/EBP factors and two unidentified proteins bind within an enhancer in intron 7 to confer glucocorticoid responsiveness [22]
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