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. 2009 May;58(5):1245-53.
doi: 10.2337/db08-0812. Epub 2009 Feb 2.

Functional targets of the monogenic diabetes transcription factors HNF-1alpha and HNF-4alpha are highly conserved between mice and humans

Affiliations

Functional targets of the monogenic diabetes transcription factors HNF-1alpha and HNF-4alpha are highly conserved between mice and humans

Sylvia F Boj et al. Diabetes. 2009 May.

Abstract

Objective: The evolutionary conservation of transcriptional mechanisms has been widely exploited to understand human biology and disease. Recent findings, however, unexpectedly showed that the transcriptional regulators hepatocyte nuclear factor (HNF)-1alpha and -4alpha rarely bind to the same genes in mice and humans, leading to the proposal that tissue-specific transcriptional regulation has undergone extensive divergence in the two species. Such observations have major implications for the use of mouse models to understand HNF-1alpha- and HNF-4alpha-deficient diabetes. However, the significance of studies that assess binding without considering regulatory function is poorly understood.

Research design and methods: We compared previously reported mouse and human HNF-1alpha and HNF-4alpha binding studies with independent binding experiments. We also integrated binding studies with mouse and human loss-of-function gene expression datasets.

Results: First, we confirmed the existence of species-specific HNF-1alpha and -4alpha binding, yet observed incomplete detection of binding in the different datasets, causing an underestimation of binding conservation. Second, only a minor fraction of HNF-1alpha- and HNF-4alpha-bound genes were downregulated in the absence of these regulators. This subset of functional targets did not show evidence for evolutionary divergence of binding or binding sequence motifs. Finally, we observed differences between conserved and species-specific binding properties. For example, conserved binding was more frequently located near transcriptional start sites and was more likely to involve multiple binding events in the same gene.

Conclusions: Despite evolutionary changes in binding, essential direct transcriptional functions of HNF-1alpha and -4alpha are largely conserved between mice and humans.

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Figures

FIG. 1.
FIG. 1.
HNF-1α and -4α are only essential for transcription in a subset of the genes to which they bind. Dark lines depict the distribution of liver gene expression ratios for all genes in the experimental models described in the title of the horizontal axis. Colored lines depict expression ratios for the subset of genes that are bound in liver by either HNF-1α or -4α using different platforms indicated in the upper legends. KO, knockout; WT, wild type. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Conservation of HNF-1α function. A and E: HNF-1α binding in mice (M) and humans (H) in the study by Odom et al. (8). The larger Venn diagrams represent binding in all studied genes; smaller diagrams below represent the subset of genes that were downregulated in Hnf1a−/− liver (A) or HNF1A-deficient hepatocellular adenomas (E) (17). Only genes represented in both binding and expression arrays were analyzed. B and F: Binding conservation was 3-fold higher in genes that were significantly downregulated in Hnf1a−/− liver (B) and 2.7-fold higher in genes downregulated in HNF1A-deficient adenomas (F) (17), in comparison with nonregulated genes. C and G: HNF-1α binding was enriched in mouse genes that were downregulated in Hnf1a−/− liver (C) and human genes downregulated in HNF1A-deficient tumors (G). In contrast to the expectation if HNF-1α function is divergent, HNF-1α binding enrichment was comparable in the orthologs of such HNF-1α–dependent genes. D and H: Genes downregulated in Hnf1a−/− mice or HNF1A-deficient adenomas showed a marked enrichment of conserved binding events (mice and humans). Species-specific binding (mice only, human only) was also moderately enriched, but this was not selective for the species where gene regulation is experimentally verified. *P < 0.01, **P < 0.001, ***P < 0.0001, Fisher's exact test. NS, nonsignificant effect of species on binding enrichment in downregulated genes using logistic regression analysis.
FIG. 3.
FIG. 3.
Comparison of HNF-1α occupancy in different platforms. A: Venn diagrams depicting HNF-1α–bound genes in mouse BCBC arrays versus human and mouse Agilent arrays from Odom et al. (8). We analyzed 2,150 genes with data in both platforms. Note that Agilent arrays cover 10-Kb surrounding transcription start sites, whereas BCBC arrays cover 1- to 2-Kb 5′ flanking regions, and thus complete binding overlap is not expected. B: Concordance of HNF-1α occupancy in mouse BCBC arrays at different Log2 binding ratio thresholds (M >1, >0.8, and >0.6; P < 0.001) with that in Agilent arrays expressed as the fold increase over the random expectation. Statistical significance was calculated with the hypergeometric distribution. ***P < 0.0001, #P < 0.05, only values for overrepresented classes are shown. C: Volcano plot of HNF1 binding ratios for all probes in mouse BCBC arrays (formula image) and for genes classified as human-specific binding events using either default or stringent criteria in the report by Odom et al. (8) (● and ○). The results show that 15–37% of genes classified as human-specific HNF1α targets are bound in mouse BCBC arrays using low or moderate stringency criteria. Dashed and dotted lines depict lenient and stringent binding criteria in the BCBC arrays. IP, immunoprecipitate.
FIG. 4.
FIG. 4.
Conservation of high-affinity HNF1 motifs in HNF-1α–dependent genes. A and C: We identified HNF1 motifs with scores >0.9 in the immediate (500 bp) 5′ flanking regions of all mouse and human genes. Motifs were strongly enriched in mouse and human genes that are experimentally determined to be HNF-1α dependent in Hnf1a−/− liver (A) and HNF1A-deficient tumors (C) (17). In contrast to the expectation if HNF-1α function is divergent, high-affinity HNF1 motifs were also enriched in the orthologs of such HNF-1α–dependent genes. B and D: Genes downregulated in Hnf1a−/− mice or HNF1A-deficient adenomas showed a marked enrichment of conserved HNF1 motifs (mouse and human). Species-specific binding (mouse only, human only) was also moderately enriched, but this was not selective for the species where gene regulation is experimentally verified. The effect of species on HNF1 motif enrichment in downregulated genes was studied with logistic regression analysis. ***P < 0.0001, Fisher's exact test. H, human; M, mouse.
FIG. 5.
FIG. 5.
Conservation of HNF-4α binding among HNF-4α–dependent genes. A: Venn diagrams depict HNF-4α–bound genes in mice (M) and humans (H) from the study by Odom et al. (8) in all genes and in the subset that is downregulated in liver-specific Hnf4a-deficient mice. Only genes represented in both binding and expression arrays were analyzed. Note that the overall binding frequency of HNF-4α is twofold higher in human chromatin, and therefore binding enrichment comparisons in ortholog pairs are uninformative because even if there is 100% conservation, the enrichment will be twofold higher in mouse genes. HNF-4α binding was nevertheless significantly enriched in mouse Hnf4α-dependent genes and their human orthologs (3.7- and 1.8-fold, respectively). B: Fraction of HNF-4α–bound mouse genes that exhibit conserved binding in human orthologs, according to their expression changes in Hnf4a-deficient liver. Statistical significance was calculated with Fisher's exact test. #P < 0.05. C: Venn diagrams depicting HNF-4α–bound genes in mouse BCBC arrays versus human and mouse Agilent arrays from the study by Odom et al. (8). We analyzed 2,495 genes with data in both platforms. Note that Agilent arrays cover 10-Kb surrounding transcription start sites, whereas BCBC arrays cover 1- to 2-Kb 5′ flanking regions, and thus complete binding overlap is not expected.
FIG. 6.
FIG. 6.
Distinct binding properties of species-specific versus conserved binding. A: Fraction of genes with two or more binding peaks among mouse-specific versus conserved HNF-1α and -4α targets. B: Fraction of downregulated genes in Hnf4a-deficient mouse liver according to the number of HNF-4α peaks in human or mouse orthologs. A similar analysis is not shown for HNF-1α because the frequency of multiple binding events is low. The results show that HNF-4α peak multiplicity correlates with both binding conservation and regulation in Hnf4a-deficient cells. C and D: Spatial distribution of mouse HNF-1α and -4α binding events that are either species-specific (formula image) or conserved (●). Circles represent the fraction of peaks that are located within 200-bp intervals relative to the transcriptional start site (TSS). Results show that proximal binding is more frequently conserved. *P < 0.01; **P < 0.001; ***P < 0.0001; #P < 0.05.

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