Foxa1-Specific Targets Are Enriched for p53 Binding Sites.
(A) A set of Foxa1-only targets was defined as those sequences that had one or fewer sequence tags Foxa2 ChIP-Seq data set (yellow circle). Motif analysis of these sequences found in a weaker forkhead consensus. In addition to the forkhead consensus, several other motifs appeared in this set of sequences. The first (ACATG and ATG repeats with a spacer in the middle) comprises parts of the positional weight matrix (PWM) for p53. The second closely resembles the PWM of Klf12, also known as repressor of AP-2alpha (Tfap2a). Orthogonal analysis of sites bound only bound by Foxa1 (yellow circle) and gene expression changes in livers of Foxa1-deficient mice (light purple circle) shows that thirty-seven are direct targets of Foxa1. (B) Verification of Foxa1-only targets by qPCR. Filled bars, ChIP of liver chromatin with an anti-Foxa1 antibody, open bars, ChIP with an anti-Foxa2 antibody. Three negative control regions (Nc1, Nc2, and Nc3) with a low amount of reads are included Binding is expressed as enrichment of the PCR amplicon relative to input DNA in liver chromatin. (C) Binding of p53 to cis-regulatory elements of its previously identified targets (positive controls), the alpha-fetoprotein (AFP) and TNF receptor superfamily Fas genes and twelve additional Foxa1-only targets, including cell-cycle associated Aatf and Zwint genes in both wildtype (black bars) and Foxa1 mutant livers (white bars) (left panel) by ChIP of liver chromatin followed by qPCR. None of Foxa2-only sites tested were bound by p53 in wiltype livers (right panel) (D) Quantitative RT- PCR analysis for mRNA of Zwint, Zwint mRNA levels are significantly downregulated in livers of Foxa1 mutant mice by 30%, Values are represented as means plus standard error. P values were determined by Student's t test. * p-value<0.05 (E) Foxa1 targets are enriched in genes involved in cell differentiation, morphogenesis, movement, and cellular cycle. (F) A regulatory feed-forward loop involving Foxa1. In the set of Foxa1-specific targets, Foxa1 binds to regulatory elements of the nuclear receptor Esr2 (estrogen receptor), its co-activator Smyd3, and target gene Pgr (progesterone receptor). (G) Foxa1 only sites are more distributed more broadly surrounding TSS (+/−100 Kb) than dual Foxa1/Foxa2 targets (compare to Figure 4E).