Table 1.
Plasmids generated for luciferase reporter assays.
Table 2.
Mutations in Sp1 and CREB binding sites in the promoter, and HNF-1 and NF-Y in the enhancer of human NAGS.
Figure 1.
Regions upstream of the mammalian NAGS genes that are highly conserved.
Conservation of mammalian NAGS DNA by phastCons (green) and phyloP (blue) algorithms is shown with the highly-conserved regions indicated in red boxes (A). Pair-wise blast analysis of mammalian non-coding regions of NAGS identified highly conserved sequences upstream of the translational start site termed the promoter (purple) and enhancer (cyan) (B).
Figure 2.
Highly conserved regulatory regions, upstream of the NAGS gene, function as promoter and enhancer elements.
In liver derived cells the NAGS promoter (4.10Prom), promoter+enhancer (4.10PromEnh), enhancer with TATA promoter (4.23Enh), and positive control promoter vector (pGL4.13) significantly simulate transcription while the enhancer (4.10Enh), basic vector (pGL4.10) does not stimulate transcription above baseline (A). Reverse insertion of the promoter (4.10PromRev) did not stimulate transcription compared to 4.10Prom and pGL4.10 vector (B), but reverse enhancer (4.23EnhRev) significantly stimulated transcription compared to 4.23Enh and pGL4.23 vector (C). Calculated results are an average of three independent experiments that were each carried out in triplicate, normalized to Rluc expression, and expressed relative to the promoter for each experiment with error reported as ±SEM. Lowercase letters indicate statistically significant differences.
Figure 3.
Transcription start sites (TSS) are species and tissue specific.
TSS identified in the promoter of NAGS by 5′RACE analysis (blue circles), the Database of Transcriptional Start Sites (DBTSS) (green circles) and 5′ termination sites of Expressed Sequence Tags (ESTs) from Genbank (orange circles) were aligned on the DNA sequence 5′ of the human (A) and mouse (B) NAGS coding sequence. The arrow indicates the translation start site.
Figure 4.
Sequence alignment of NAGS promoters and enhancers from seven mammalian species indicate conserved motifs.
DNA sequence of the promoter (A) and enhancer (B) regions were aligned using CLUSTALW alignment software. CLOVER analysis was used to identify transcription factor binding motifs. Binding sites for C/EBP (green), Sp1 (red), CREB/ATF (pink), AP-2 (purple), HNF-1 (blue), NF-Y (olive), and SMAD 3 (cyan) were highly conserved.
Figure 5.
DNA-protein avidin-agarose pull-down assay results confirm transcription factor binding.
Two probes for the promoter (A) and one probe for the enhancer (B) encompass the highly conserved transcription factor binding motifs of NAGS. The motif colors reflect the colors used in figures 4A and B. Assays followed by immunoblot confirmed binding of Sp1 and CREB, but not C/EBP within the promoter (C) and HNF-1 and NF-Y, but not SMAD3 or AP-2 within the enhancer regions (D). Lanes 1–4 represent precipitated proteins from mouse liver nuclear extract bound to biotinylated probes of the regions of interest (Lane 1), non-biotinylated probes of the regions of interest (Lane 2), biotinylated probes of non-specific regions (Lane 3), and no probe (Lane 4). Lanes 5–8 represent supernatant fluid from overnight incubation of biotinylated probes of the region of interest (Lane 5), non-biotinylated probes of the region of interest (Lane 6), biotinylated probes of the non-specific regions (Lane 7), or no probe (Lane 8). Immunoblots are representative of at least three replicate experiments.
Figure 6.
Chromatin Immunoprecipitation (ChIP) results confirm transcription factor binding.
ChIP with transcription factor antibodies was compared to negative control IgG antibody. Real-Time PCR using promoter or enhancer specific primers was compared to primers for the negative locus MIP-2. The results confirmed that Sp1 and CREB but not C/EBP bind within the promoter (A) and HNF-1 and NF-Y but not AP-2 or SMAD2/3 bind within the enhancer region (B) of NAGS. Calculated error was from three replicate experiments and reported as ± SEM. One asterisk (*) indicates p<0.05 and two asterisks (**) indicate p<0.005.
Figure 7.
Transcription factors Sp1, CREB, HNF-1, and NF-Y are functionally important for stimulating expression of reporter gene transcription.
Mutagenesis of the putative transcription factor binding sites significantly decreases transcription by the promoter (A) and the enhancer with TATA promoter (B) in liver derived cells when compared to non-mutated sites. Addition of Sp1 with the promoter (A) and HNF-1 with the enhancer (B) increases transcription driven by non-mutated constructs. Calculated results are an average of three independent experiments that were each carried out in triplicate, normalized to Rluc expression, and expressed relative to the promoter for each experiment with error reported as ±SEM. Lowercase letters indicate statistically significant differences.
Figure 8.
The NAGS enhancer shows tissue specificity.
The enhancer with NAGS promoter (4.10PromEnh) increases transcription relative to the promoter in liver derived cells but not in intestine or lung derived cells (cyan bars) without the addition of HNF-1 protein (teal bars). Calculated results are an average of three independent experiments that were carried out in triplicate, normalized to Rluc expression, and expressed relative to the promoter for each experiment with error reported as ±SEM. Lowercase letters indicate statistically significant differences.