Transcription of urea cycle genes is in part regulated by the glucocorticoid and glucagon signaling pathways. Therefore, we postulate that there exists a nitrogen sensing mechanism that is both responsive to amino acid and hormone stimulation and that an understanding of the transcriptional regulation of NAGS could contribute to the understanding of such mechanism. In this study, we identified two regulatory regions upstream of the NAGS translation start site that contain highly conserved protein-binding DNA motifs. We subsequently confirmed that these regions function as promoter and enhancer and that the enhancer is most effective in liver cells. Avidin-agarose protein- DNA pull-down assays have been used to confirm binding of Sp1 and CREB within the NAGS promoter and Hepatic Nuclear Factor 1 and NF-Y within the enhancer regions. Chromatin immunoprecipitation and GSK1363089 quantitative realtime PCR have been used to independently verify that Sp1 and CREB bind to the promoter region, and HNF-1 and NF-Y bind to the enhancer region. We also used 59RACE analysis to identify multiple transcription start sites for NAGS that may be species and tissue specific. These findings provide new information on the regulation of the NAGS gene, and suggest possible mechanisms for coordinated regulation of the genes involved in ureagenesis. To validate our strategy for identification of conserved regions, the same analyses were conducted for CPS1, a gene in which a proximal promoter and an enhancer element located 6.3 kb upstream of rat Cps1, have been characterized. Reporter assays were used to examine the functionality of each of the following: wild type NAGS promoter, control reversed promoter, enhancer alone, promoter and enhancer, and enhancer in both orientations with the heterologous TATA-box promoter by measuring the expression of a MK-2206 luciferase reporter gene in cultured HepG2 cells. The human NAGS promoter alone, stimulated transcription of the luciferase gene while the upstream regulatory region alone, did not. When the NAGS promoter and upstream regulatory region were both present, transcription increased by 50% compared to the promoter alone confirming that the upstream conserved region can function as an enhancer of transcription. When the NAGS enhancer was paired with a heterologous promoter containing a TATA-box, in the 4.23Enh construct, the transcription of luciferase about three times higher compared to construct with minimal TATA-box. The backbone vector 4.10 did not stimulate expression of the luciferase gene. As expected, positive control vector 4.13, containing a strong promoter, activated transcription in this cell culture system. The promoter in the reverse orientation did not activate luciferase expression indicating that the NAGS promoter acts in a direction dependent manner.