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. 2008 Aug;36(14):4549-64.
doi: 10.1093/nar/gkn382. Epub 2008 Jul 8.

Global analysis of in vivo Foxa2-binding sites in mouse adult liver using massively parallel sequencing

Elizabeth D Wederell  1 , Mikhail Bilenky, Rebecca Cullum, Nina Thiessen, Melis Dagpinar, Allen Delaney, Richard Varhol, YongJun Zhao, Thomas Zeng, Bridget Bernier, Matthew Ingham, Martin Hirst, Gordon Robertson, Marco A Marra, Steven Jones, Pamela A Hoodless
Affiliations

Global analysis of in vivo Foxa2-binding sites in mouse adult liver using massively parallel sequencing

Elizabeth D Wederell et al. Nucleic Acids Res. 2008 Aug.

Abstract

Foxa2 (HNF3 beta) is a one of three, closely related transcription factors that are critical to the development and function of the mouse liver. We have used chromatin immunoprecipitation and massively parallel Illumina 1G sequencing (ChIP-Seq) to create a genome-wide profile of in vivo Foxa2-binding sites in the adult liver. More than 65% of the approximately 11.5 k genomic sites associated with Foxa2 binding, mapped to extended gene regions of annotated genes, while more than 30% of intragenic sites were located within first introns. 20.5% of all sites were further than 50 kb from any annotated gene, suggesting an association with novel gene regions. QPCR analysis demonstrated a strong positive correlation between peak height and fold enrichment for Foxa2-binding sites. We measured the relationship between Foxa2 and liver gene expression by overlapping Foxa2-binding sites with a SAGE transcriptome profile, and found that 43.5% of genes expressed in the liver were also associated with Foxa2 binding. We also identified potential Foxa2-interacting transcription factors whose motifs were enriched near Foxa2-binding sites. Our comprehensive results for in vivo Foxa2-binding sites in the mouse liver will contribute to resolving transcriptional regulatory networks that are important for adult liver function.

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Figures

Figure 1.
Figure 1.
(A) Distances from sites similar to Foxa2-binding sequences to the peak maximum, normalized to peak width. This graph shows that Foxa2-binding sequence sites tended to cluster around peak maxima. (B) Peak enrichment in Foxa2-like sequences, as assessed with PWM scan scores. The Foxa2 PWM used was created from the aligned sequences used to generate the sequence logo shown in Supplementary Figure 2 (see Methods section). The graph indicates that peak genomic sequences contained higher scoring Foxa2-binding sequences than randomized peak sequences; that is, peaks were enriched relative to a random expectation.
Figure 2.
Figure 2.
Characterized target Foxa2-binding sites. (A) Table of six characterized target sites in five target genes showing peak presence or absence, peak height and fold enrichment by qPCR. All three sites that contained a peak also showed enrichment above 2-fold. (B) UCSC genome browser mm8 screenshots of four target sites at two of the target genes. Of the HNF4α enhancer and promoter sites, only the enhancer was covered by a peak in our adult liver dataset. The Ttr gene has two closely related promoter sites from different publications, both of which were overlapped by a single peak in our dataset. An additional track displaying the Foxa2-like binding sequence sites from scanning our 10-mer model is also shown (‘Foxa2 logo sites’).
Figure 3.
Figure 3.
Characterization of novel target sites associated with known Foxa2-target genes. (A) UCSC genome browser screenshots of peaks associated with the Alb and Afp genes. The Alb novel site is an additional location to the characterized target site; both sites were overlapped by peaks, indicating binding by Foxa2 in the liver. The Afp novel site is an alternate location 5′ to the site characterized in embryonic liver. (B) QCPR fold enrichment for four novel target sites in characterized Foxa2-target genes averaged over three biological replicate Foxa2 ChIP experiments. Both the fold enrichment and the peak height are shown; the red line indicates the minimum peak height threshold for our dataset (i.e. 10 XSETs). All enrichments were calculated using IgG enrichment as a control in all graphs showing qPCR results. All four novel target sites had fold enrichments above 13.
Figure 4.
Figure 4.
QPCR fold enrichment of annotated genes containing Foxa2-binding site peaks. All genes shown contained SAGE tag(s) indicating that they were expressed in the adult liver. QPCR fold enrichment and peak height are shown for each target; the red line indicates the peak height threshold of 10 XSETs. As previously, IgG enrichment served as the control. (A) Annotated genes that have characterized roles in liver function. Peaks are divided into those that contained a Foxa2-binding sequence and those that did not. All eight targets showed fold enrichments above 4. (B) Annotated genes that have not previously been shown to be expressed in the liver, with peaks divided into those that contained a Foxa2-binding sequence and those that did not. Dock8 and Cask, which did not show enrichment, were the only targets out of 41 peaks tested that were not enriched. The peak height of these two targets was close to the peak height threshold. The remaining six targets all showed fold enrichments above 6. (C) Transcription factors that have a characterized role in liver function in literature. All six targets showed fold enrichments above 5. (D) Transcription factors with uncharacterized roles in the liver. Expression of these transcription factors in the liver is a novel finding. All four targets showed fold enrichments above 5.
Figure 5.
Figure 5.
Examples of peaks located >50 kb away from annotated genes. (A) UCSC genome browser mm8 screenshots of two remote peaks in regions containing no known genes. The first peak (unknown #4) lies close to a mouse EST, while the second peak (unknown #5) overlaps and is close to highly conserved sequence regions. (B) Graph of qPCR results of all six peaks lying in unknown gene regions. Four peaks contained Foxa2-binding sequences and two peaks did not. As previously, both fold enrichment by qPCR and peak height are shown. Fold enrichments are relative to IgG control. All six peaks showed fold enrichments above 19.
Figure 6.
Figure 6.
Positive correlation between fold enrichment by qPCR and peak height. Each data point represents the average fold enrichment of three separate Foxa2 ChIP experiments. As indicated, taller peaks showed greater fold enrichments. For this dataset, peak height can be considered to be a predictor of expected fold enrichment.
Figure 7.
Figure 7.
Foxa2 knockdown in Hepa1-6 cells and its effect on expression of targets identified by ChIP–Seq. Foxa2 siRNA treatment results in ∼50% knockdown of Foxa2 expression compared to control siRNA. Upon Foxa2 knockdown, expression of both known (Alb, HNF4α) and novel (HNF6, Hes1, Fgf1, Vtn) decreased. Expression of HNF1β increased, while expression of Cited2 and Gtls3 remained unchanged. The expression of Foxa1 was unaffected by Foxa2 knockdown, indicating the specificity of the Foxa2 siRNA. *P < 0.05.
Figure 8.
Figure 8.
The distribution of TFBS sequence models relative to the corresponding Foxa2-binding sequence for three of the top 20 cotranscription factors in peaks containing a Foxa2-binding sequence. (A) The distribution of GATA4 TFBS models (Transfac model M00632). Motifs for this transcription factor show a uniform distribution across the 500 bp sequence. (B) The distribution of Pax6 TFBS models (Transfac model M00979). Pax6 motifs clustered at a distance of ∼40 bp away from the Foxa2-binding sequence (P = 1.1E-3). (C) The spatial distribution of HNF1 TFBS models (TRANSFAC model M00790). HNF1 motifs are spatially concentrated near the Foxa2-binding sequence (P = 5.7E-9).
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