A) An alignment of
NANOG sequences from seven vertebrates in the region -119 to -63 relative
to the human TSS. Oct4, Sox2 and the Nanog binding sites are perfectly
conserved and indicated by solid bars, whereas the Zfp281/Zic3 site by a
wavy line. The human (Hs) Zfp281/Zic3 site was mutagenized to produce
a consensus Zic3/Zfp281 mouse site (Mm) by mutating CTGGTAG to
CCTGCAG.(B) Luciferase assay was performed in CJ7 ESCs. Cells were transfected with the NANOG promoter reporter constructs (left) and analyzed for promoter activity. The Oct4 binding site was mutated GCAT to AACC and used as a positive control to show ablation of promoter activity. The Nanog binding sites were mutated from ATT to CCG at the conserved tetramer ATTA/C. Firefly luciferase expression levels were normalized to the luciferase activity of internal Renilla control. The no promoter containing plasmid (No Prom) was used as the internal control and its activity was normalized to 1. Data presented are the mean ± SEM of triplicates from one of two independent experiments.
