Testing the effect of over-expression of CREB protein was hindered by its capacity to homo- and heterodimerize with multiple partners. The effect of NF-Y was not tested because this transcription factor is a heterotrimer and its LEE011 CDK inhibitor coexpression with reporter plasmids would require stable expression of NF-Y subunit proteins by in vitro cell culture before reporter plasmids can be transfected and assayed for NF-Y effect on transcription. From the data provided herein, we can speculate on the potential role these factors play in regulating NAGS transcription. First, in the absence of a canonical TATA-box, transcription initiated by Sp1 often results in multiple transcriptional start sites. Sp1 is a strong activator of transcription and when multiple Sp1 sites are present, as in NAGS, multiple Sp1 proteins can form complexes with each other and synergistically activate transcription. Because transcription is significantly increased by co-expression with Sp1 protein and decreased following mutation of the Sp1 binding sites, Sp1 may prove to be the activator of NAGS transcription, similar to its role for ASS, ASL and ARG1. Second, studies have shown that glucagon and second messenger cAMP trigger a cascade that phosphorylates CREB and allows for DNA binding and activation of transcription. In CPS1 and ASS, CREB stimulates transcription upon glucagon signaling. Decrease in transcription following CREB mutation and the close proximity of Sp1 and CREB binding sites among the TSS suggests that the transcription initiation machinery may be recruited by these factors, and future research should examine this postulate. Our experiments and other studies confirm the role of HNF-1 in NAGS expression. HNF-1 is essential for stimulation of NAGS expression by its enhancer. This factor is in part regulated by HNF-3, HNF-4, and C/EBP, each of which are known to regulate other urea cycle genes. Future research will focus on the mechanism of control between these factors, HNF-1, and NAGS. Our study has also shown that NF-Y is an activator of NAGS expression, and future studies will focus on the exact mechanism of its function in this context. The human NAGS gene on the forward strand of chromosome 17 partially overlaps with the peptide YY gene, which is on the reverse strand. This overlap was identified with a PYY cDNA isolated from a brain astrocytoma cDNA Niltubacin abmole bioscience library that has an 80 nucleotide long exon located between regions A and B of the NAGS promoter. Other full-length PYY transcripts initiate about 500 bp upstream of the PYY coding region, which is located 51 kb upstream of the NAGS translation initiation codon. Recent analysis of human transcripts revealed that many protein coding loci are associated with at least one transcript that initiates from a distal site, but the significance or function of these transcripts remains to be elucidated.