9. Although a relatively small fraction of the total binding sites, a peak of total or unique FXR-binding sites was observed within 1 kb of the transcription start site (TSS) in both groups (Fig. 1C; Supporting Fig. 10). The highest scored binding motif for the 250 top-scoring FXR-binding sites was an inverted repeat 1 (IR1) motif with similar
preferred sequences in both healthy this website and obese mice (Fig. 1D). To identify possible biological functions regulated by FXR, potential FXR target genes were assigned to functional groups by GO analysis. Many potential FXR target genes represent previously unknown functions, such as cellular signaling, hypoxia, autophagy, apoptosis, RNA processing, and many transcriptional regulators. Notably, genes encoding components of diverse cellular signaling pathways, such as G-protein signaling, Wnt signaling, mitogen-activated protein kinase signaling, numerous kinases, and phosphatases, were identified (Fig. 1E). These results suggest that previously unknown functions of FXR, particularly in the regulation of cellular signaling pathways, are different in healthy and obese Wnt pathway mice, which could underlie abnormal regulation in obesity. Overall, these GO studies, together with the analysis of genome-wide FXR binding, reveal novel potential FXR target genes, suggesting that FXR may have much broader biological functions than previously appreciated.
Examples of FXR-binding peaks detected near selected genes unique in either healthy or obese mice are shown in Fig. 2 and Supporting Fig. 11. These analyses reveal previously unrecognized genomic targets of FXR in liver with novel biological functions, suggesting that transcriptional patterns and biological pathways regulated by ligand-activated FXR are likely altered in obesity. To initially examine whether differences
in FXR-binding correlate with relative gene expression, ChIP and qRT-PCR studies were performed for randomly selected potential target genes. FXR binding was detected Glycogen branching enzyme by ChIP analysis in 86% (13 of 15 genes) or 100% (5 of 5 genes) of these target genes unique to healthy or obese mice, which validated the accuracy of the ChIP-seq analysis (Fig. 3A,B). For 15 genes with FXR binding unique to healthy mice, mRNA levels of nearly all of the genes were changed, compared to obese mice (Fig. 3C), whereas only 5 of 14 genes with FXR binding unique in obese mice showed significant changes in mRNA levels (Fig. 3D). These results suggest either that FXR-binding sites are likely not functional for a large fraction of the genes in obese mice, or that factors other than FXR may contribute to the overall difference in expression of these genes in obesity. To correlate the binding of FXR at a gene with its expression, mRNA levels of randomly selected genes with FXR-binding sites were measured.
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