EKLF transactivates the mouse PIT1 promoter. (A) Luciferase assay on PIT1 5000-bp wild-type promoter. Results are expressed as the fold induction normalized with the empty pGL3-vector. Data indicate the means ± SEM of at least 3 independent experiments. Significant differences from Sport6 vector are indicated. ***P < .001. (B) Luciferase assay on PIT1 5000-bp wild-type promoter, EKLF binding site mutants, and wild-type promoter shorter constructs. Results are normalized with empty sport6 expression vector for each construct and are expressed as the fold induction. Data indicate the means ± SEM of 4 independent experiments. Significant differences from pGL3 (*P < .05; **P < .01; ***P < .001), from the mouse PIT1 wild-type promoter (#P < .05; ###P < .001), and from the −119-bp construct ($P < .05; $$P < .01) are indicated. (C) Phylogenetic alignment of the 600 proximal bp of the PIT1 promoter. Underlined sequence indicates EKLF2 consensus binding site, bold sequence indicates −119-bp construct, and highlighted sequence indicates the beginning of the PIT1 gene. Asterisks indicate nucleotide identities between the 3 species. (D) EKLF occupancy in the mouse PIT1 promoter using the UCSC browser as described by Bodin et al.³³  (E) Quantitative PCR analysis of EKLF ChIP experiments. EKLF-immunoprecipitated DNA from G1E-ER-Gata1 cells treated or not with β-estradiol for 24 hours were analyzed by quantitative PCR using primers located along the mouse PIT1 promoter. Negative controls were performed using DNA incubated with beads but without anti-EKLF Ab. Means ± SEM of 4 experiments are presented. Significant differences from negative control are indicated. *P < .05.