Transcriptional networks in the liver: hepatocyte nuclear factor 6 function is largely independent of Foxa2

Abstract

A complex network of hepatocyte nuclear transcription factors, including HNF6 and Foxa2, regulates the expression of liver-specific genes. The current model, based on in vitro studies, suggests that HNF6 and Foxa2 interact physically. This interaction is thought to synergistically stimulate Foxa2-dependent transcription through the recruitment of p300/CBP by HNF6 and to inhibit HNF6-mediated transcription due to the interference of Foxa2 with DNA binding by HNF6. To test this model in vivo, we utilized hepatocyte-specific gene ablation to study the binding of HNF6 to its targets in the absence of Foxa2. Chromatin immunoprecipitation using anti-HNF6 antibodies was performed on chromatin isolated from Foxa2(loxP/loxP) Alfp.Cre and control mouse livers, and HNF6 binding to its target, Glut2, was determined by quantitative PCR. In contrast to the current model, we found no significant difference in HNF6 occupancy at the Glut2 promoter between Foxa2-deficient and control livers. In order to evaluate the Foxa2/HNF6 interaction model on a global scale, we performed a location analysis using a microarray with 7,000 mouse promoter fragments. Again, we found no evidence that HNF6 binding to its targets in chromatin is reduced in the presence of Foxa2. We also examined the mRNA levels of HNF6 targets in the liver using a cDNA array and found that their expression was similar in Foxa2-deficient and control mice. Overall, our studies demonstrate that HNF6 binds to and regulates its target promoters in vivo in the presence and absence of Foxa2.

FIG. 1.

HNF6 binding to the Glut2 promoter in hepatic chromatin in the presence and absence of Foxa2. (a) Schematic representation of the model suggesting that the interaction between Foxa2 and HNF6 can synergistically stimulate or repress transcription. On a Foxa2 target promoter, HNF6 recruitment of p300/CBP activates transcription, whereas on an HNF6 target promoter, an interaction with Foxa2 inhibits HNF6 binding (modified with permission from reference 20). Liver chromatin isolated from wild-type and HNF6^−/− mice (b and c) or from wild-type and Foxa2^loxP/loxP Alfp.Cre mice (d and e) was immunoprecipitated with an anti-HNF6 antibody or control IgG. Input chromatin and precipitated DNA were amplified with primers surrounding the HNF6 binding site in the Glut2 promoter. Occupancy of the HNF6 site in the Glut2 promoter is detectable by this qualitative assay in wild-type and Foxa2-deficient liver chromatin (d), but not in HNF6^−/− chromatin (b), confirming the specificity of the anti-HNF6 antibody. In addition, immunoprecipitation with control IgG demonstrates the specificity of the assay. (c and e) Quantitative real-time PCR analysis of ChIP assays. Enrichment of the target gene Glut2 was calculated by using the 28S rRNA locus as a control for nonspecific DNA and is shown relative to the input chromatin. (c) HNF6 binding to the Glut2 promoter is 13-fold higher in wild-type than in HNF6^−/− chromatin, confirming the specificity of the anti-HNF6 antibody. (e) HNF6 binds to the Glut2 promoter in both wild-type and Foxa2-deficient liver chromatin, with a trend towards less binding in the absence of Foxa2. Black bars, control mice; white bars, HNF6^−/− mice (c) or Foxa2^loxP/loxP Alfp.Cre mice (e). Values are represented as means plus standard errors (n = 6 for each group [c] and n = 3 for each group [e]). P values were determined by Student's t test. ***, P < 0.0005. N.S., not statistically significant. (f) Glut2 mRNA levels are similar in Foxa2-deficient and control livers. Quantitative real-time PCR was performed on RNAs isolated from livers of wild-type and Foxa2^loxP/loxP Alfp.Cre mice with primers specific for Glut2, TBP, and HPRT mRNA sequences. Glut2 mRNA levels were normalized to either TBP or HPRT. Black bars, control mice; white bars, Foxa2^loxP/loxP Alfp.Cre mice. Values are represented as means plus standard errors (n = 6 for each group) N.S., not statistically significant.

FIG. 2.

HNF6 mRNA and protein levels are similar in Foxa2-deficient and control livers. (a) RNAs were isolated from livers of wild-type or Foxa2^loxP/loxP Alfp.Cre mice and reverse transcribed. PCRs were performed with primers specific for HNF6, TBP, and HPRT mRNA sequences, and PCR products were resolved by agarose gel electrophoresis. (b) Quantitative real-time PCR analysis shows similar HNF6 mRNA levels in Foxa2^loxP/loxP Alfp.Cre and control mice. HNF6 mRNA levels were normalized as described in the legend to Fig. 1f. Black bars, control mice; white bars, Foxa2^loxP/loxP Alfp.Cre mice. Values are represented as means plus standard errors (n = 6 for each group). The y axis is in a logarithmic scale. (c) Western blot analysis of protein nuclear extracts (10 μg) from two control (lanes 1 and 2) and two Foxa2^loxP/loxP Alfp.Cre (lanes 3 and 4) mice with antibodies against HNF6, Foxa2, and TBP (loading control). The faint bands in the two lanes for Foxa2^loxP/loxP Alfp.Cre mice detected with the anti-Foxa2 antibody are due to a partial cross-reactivity of this antibody with other Foxa family members.

FIG. 3.

Schematic representation of experimental approach combining promoter and expression microarray analysis. Chromatin and RNA were isolated from wild-type or Foxa2^loxP/loxP Alfp.Cre mice. Chromatin was cross-linked and immunoprecipitated with an anti-HNF6 antibody. The resulting material was amplified via ligation-mediated PCR, fluorescently labeled, and hybridized to the mouse promoter microarray. Isolated RNAs were reverse transcribed, and the cDNAs were fluorescently labeled and hybridized to the PancChip 5.0. Microarray data were analyzed using the SAM and PaGE software packages to determine whether there were significant differences between the expression levels or binding levels of promoters by HNF6 in the wild-type and Foxa2^loxP/loxP Alfp.Cre mice.

FIG. 4.

Global promoter occupancy by HNF6 is similar in Foxa2-deficient and control liver chromatin. (a) Plot generated by the SAM analysis package showing no differences in enrichment of HNF6-bound promoters between Foxa2^loxP/loxP Alfp.Cre and control liver chromatin. The solid line indicates y = x, where the observed differences are identical to the expected differences. The distance from the diagonal represents the likelihood of differential enrichment between the two groups. The two dashed lines on either side of the diagonal represent a false discovery rate of 10% (see the text for details). Notice that there are no points outside the area bound by the dashed lines. (b) Promoter microarray values of enrichment for 97 promoters, which were previously shown to be in vivo HNF6 targets, plotted for the Foxa2^loxP/loxP Alfp.Cre versus control mice. Enrichment was normalized relative to genomic DNA. Most promoters are enriched to a similar degree in Foxa2-deficient and control liver chromatin, and some are more highly enriched in the presence of Foxa2 (circled dots). An arrow indicates Glut2, a well-established HNF6 target.

FIG. 5.

HNF6 binds its target promoters similarly in Foxa2-deficient and control mice. Liver chromatin isolated from wild-type or Foxa2^loxP/loxP Alfp.Cre mice was immunoprecipitated with an anti-HNF6 (a) or anti-Foxa2 (b) antibody. Input and precipitated DNAs were subjected to quantitative real-time PCR with primers specific for several putative HNF6 target promoters. Enrichment of the target promoters was calculated, using the 28S rRNA locus as a reference, and is shown relative to the input chromatin. Black bars, control mice; white bars, Foxa2^loxP/loxP Alfp.Cre mice. Values are represented as means plus standard errors (n = 3 for each group) P values were determined by Student's t test. *, P < 0.05; **, P < 0.01.

FIG. 6.

Expression levels of HNF6 targets in the liver are similar for Foxa2-deficient and control mice. Mouse PancChip 5.0 microarray fluorescence intensity values of enrichment for 97 HNF6 in vivo target genes were plotted for Foxa2-deficient versus control mice.