Six putative transcription factor sites were identified by sequence homology, and their roles were tested by EMSA and site directed mutagenesis. FOXA and HNF-1α proteins are liver-enriched factors that regulate many liver-specific genes (Schrem et al., 2002). FOXA family members FOXA1, FOXA2 and FOXA3 share a highly conserved forkhead DNA binding domain and play essential roles during development and in adult animals (Friedman and Kaestner, 2006). HNF-1α is a homeodomain containing protein that regulates expression of several genes in liver, pancreatic islets and kidneys (Armendariz and Krauss, 2009). Sites 1, 2, 3, 4, and 6 were confirmed to be FOXA binding sites by EMSA with competitor oligonucleotides and by supershift assays. Site 5, which overlaps with the site 4 by two nucleotides, was predicted to be a HNF-1α site. Oligo 3, which spans sites 3, 4, and 5, produced multiple high molecular weight bands, but the FOXA specific complex was the most prominent. While none of the bands were disrupted by HNF-1α competitor, a stronger FOXA specific complex was detected (data not shown). We speculate that HNF-1 binds weakly to site 5 and this binding interferes with the binding of FOXA to the overlapping site 4.
