Species-specific strategies underlying conserved functions of metabolic transcription factors - PubMed (original) (raw)

Species-specific strategies underlying conserved functions of metabolic transcription factors

Raymond E Soccio et al. Mol Endocrinol. 2011 Apr.

Abstract

The winged helix protein FOXA2 and the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) are highly conserved, regionally expressed transcription factors (TFs) that regulate networks of genes controlling complex metabolic functions. Cistrome analysis for Foxa2 in mouse liver and PPARγ in mouse adipocytes has previously produced consensus-binding sites that are nearly identical to those used by the corresponding TFs in human cells. We report here that, despite the conservation of the canonical binding motif, the great majority of binding regions for FOXA2 in human liver and for PPARγ in human adipocytes are not in the orthologous locations corresponding to the mouse genome, and vice versa. Of note, TF binding can be absent in one species despite sequence conservation, including motifs that do support binding in the other species, demonstrating a major limitation of in silico binding site prediction. Whereas only approximately 10% of binding sites are conserved, gene-centric analysis reveals that about 50% of genes with nearby TF occupancy are shared across species for both hepatic FOXA2 and adipocyte PPARγ. Remarkably, for both TFs, many of the shared genes function in tissue-specific metabolic pathways, whereas species-unique genes fail to show enrichment for these pathways. Nonetheless, the species-unique genes, like the shared genes, showed the expected transcriptional regulation by the TFs in loss-of-function experiments. Thus, species-specific strategies underlie the biological functions of metabolic TFs that are highly conserved across mammalian species. Analysis of factor binding in multiple species may be necessary to distinguish apparent species-unique noise and reveal functionally relevant information.

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Figures

Fig. 1.

Fig. 1.

Conservation of TF-binding regions. The overall conservation of binding regions approximates 10% between mouse and human. All FOXA2 (A) and PPARγ (B) binding regions are represented in Venn diagrams. The darker-colored pie slices of each circle show the regions that fail to convert to the other genome, whereas the intersection of circles shows the conservation of convertible regions based on centers within 500 bp. There are different numbers of conserved regions in each species, reflecting cases in which a single GLITR region in one species overlaps multiple regions in the other species. C, Conserved regions are more likely to be located in gene promoters. Each dataset of binding regions was divided as in the Venn diagrams, and the percentage of regions localized within 5 kb of the transcription start site of the nearest gene is shown. D, The strongest regions are more likely to be conserved. The percent conservation of the 100 strongest convertible binding regions for each dataset is shown. E, Conservation is found even among the weakest binding regions. All convertible binding regions were divided into quintiles based on strength, and the percent conservation is shown. The data are clearly skewed from the null hypothesis of 20% (the P value for the overall skew in each dataset is <10−10), but even the weakest quintile accounts for more than 10% of all conserved sites. *, P < 0.05; **, P < 0.001; ***, P <10−10 vs. all sites.

Fig. 2.

Fig. 2.

Motifs in TF-binding regions. A, The complete datasets of mouse and human binding regions for FOXA2 and PPARγ were searched using de novo motif analysis, and the top scoring motif is shown for each. These motifs were compared with the TRANSFAC database to find the most similar motif. B, Conserved and species-specific binding correlates with the occurrence of motifs in binding regions. The top human de novo motif for FOXA2 (left) and PPARγ (right) was used to interrogate the convertible binding regions as well as control datasets matched for length and guanine and cytosine content. The binding regions were divided into conserved and species-specific regions, and coordinates were mapped on the mouse (mm8) and human (hg18) genomes as indicated. *, P < 0.01; **, P <10−8; ***, P <10−70 for comparisons as indicated. #, P <10−70 vs. mouse-only regions mapped on human genome, ^, P <10−70 vs. human-only regions mapped on mouse genome. C, PhastCons scores for conserved and species-specific (nonconserved) binding. PhastCons scores were calculated based on placental mammal sequences and were calculated in human binding regions. Conserved binding regions (yellow) had high conservation scores, while non-conserved regions (green) had much lower conservation scores, but were still higher than all regions (red). Non-convertible (blue) and matched control (purple) regions showed background conservation.

Fig. 3.

Fig. 3.

Shared genes nearest to TF-binding regions. A, The sharing of nearest genes approximates 50%. The nearest gene was found to each binding region, and this list was filtered to unique genes with orthologs in the other species. The intersection of the Venn diagrams represents orthologous genes with nearest binding regions in both species. B, Most shared genes have only nonconserved nearest regions. For each list of genes nearest to factor-binding regions in one species, about 4% of genes did not have an ortholog in the other species (red). When there was an ortholog, it either had no nearest region (yellow), only nonconserved nearest regions (dark blue), or a conserved nearest region (light blue). C, Shared genes have more nearest binding regions. The genes are divided as in B with the average number of nearest regions shown. *, P <10−6 by permutation t test, compared with 106 random permutations of the data.

Fig. 4.

Fig. 4.

Transcriptional regulation of shared and species-unique target genes by FOXA2 and PPARγ. A, Microarray experiments were performed comparing liver gene expression in Foxa1/a2 double-mutant mice to control mice (FOXA2) and 3T3-L1 mouse adipocyte gene expression in PPARγ siRNA knockdown cells vs. control siRNA (PPARγ). All nonredundant genes on each microarray were divided into the four classes shown based on the presence or absence of nearest factor-binding regions in the mouse and human genomes. For each class, the percentage of genes with statistically significant more than 1.5-fold microarray down-regulation upon factor knockout/knockdown was determined. *, P < 10−9; **, _P_ <10−40; NS, _P_ > 0.1 vs. negative control (black bar, genes with binding regions in neither species). B, UCSC browser tracks on the mouse genome (mm8) are shown to illustrate several classes of genes, with tracks shown for the mouse and convertible human PPARγ-binding regions. The fold down-regulation upon PPARγ siRNA knockdown from the microarray experiment in panel A is shown for each gene.

Fig. 5.

Fig. 5.

Conservation of metabolic pathways regulated by TFs. A, DAVID/PANTHER biological pathway analysis of FOXA2 nearest genes that are human unique (red), mouse-unique (blue), or shared human (purple). B, DAVID/PANTHER biological pathway analysis of PPARγ nearest genes that are human unique (green), mouse unique (yellow), or shared human (light green). All gene lists were generated by the analysis in Fig. 3A, and all pathways with P < 0.01 are shown.

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