Farnesoid X receptor (FXR, NR1H4) belongs to

Transcription

1 Genomic Analysis of Hepatic Farnesoid X Receptor Binding Sites Reveals Altered Binding in Obesity and Direct Gene Repression by Farnesoid X Receptor in Mice Jiyoung Lee, 1 * Sunmi Seok, 1 * Pengfei Yu, 2 Kyungsu Kim, 2 Zachary Smith, 1 Marcelo Rivas-Astroza, 2 Sheng Zhong, 2 and Jongsook Kim Kemper 1 The nuclear bile acid receptor, farnesoid X receptor (FXR), is an important transcriptional regulator of liver metabolism. Despite recent advances in understanding its functions, how FXR regulates genomic targets and whether the transcriptional regulation by FXR is altered in obesity remain largely unknown. Here, we analyzed hepatic genome-wide binding sites of FXR in healthy and dietary obese mice by chromatin immunoprecipitation sequencing (ChIP-seq) analysis. A total of 15,263 and 5,272 FXR binding sites were identified in livers of healthy and obese mice, respectively, after a short 1-hour treatment with the synthetic FXR agonist, GW4064. Of these sites, 7,440 and 2,344 were detected uniquely in healthy and obese mice. FXR-binding sites were localized mostly in intergenic and intron regions at an inverted repeat 1 motif in both groups, but also clustered within 1 kilobase of transcription start sites. FXR-binding sites were detected near previously unknown target genes with novel functions, including diverse cellular signaling pathways, apoptosis, autophagy, hypoxia, inflammation, RNA processing, metabolism of amino acids, and transcriptional regulators. Further analyses of randomly selected genes from both healthy and obese mice suggested that more FXR-binding sites are likely functionally inactive in obesity. Surprisingly, occupancies of FXR, retinoid X receptor alpha, RNA polymerase II, and epigenetic gene activation and repression histone marks, and messenger RNA levels of genes examined, suggested that direct gene repression by agonist-activated FXR is common. Conclusion: Comparison of genomic FXR-binding sites in healthy and obese mice suggested that FXR transcriptional signaling is altered in dietary obese mice, which may underlie aberrant metabolism and liver function in obesity. (HEPATOLOGY 2012;56: ) Farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily. 1-3 As the primary biosensor for endogenous bile acids, FXR plays a crucial role in maintaining bile acid homeostasis by regulating the expression of numerous genes involved in bile acid metabolic pathways, including biosynthesis and transport of bile acids mainly in the liver and intestine. 2-4 In addition, recent studies have revealed new functions of FXR in triglyceride/glucose metabolism, liver regeneration, tumor suppression, and antiinflammation. 3,5-10 Despite these recent advances in understanding functions of FXR, how FXR activates or represses its genomic targets is still poorly understood. Ligand-activated FXR binds FXR response elements (FXREs) in DNA as a heterodimer with retinoid X receptor alpha (RXRa) and activates the transcription of Abbreviations: Aldh1/2, aldehyde dehydrogenase 1 and 2; Cdca4, cell division cycle-associated 4; ChIP, chromatin immunoprecipitation; ChIP-seq, ChIP sequencing; DAVID, National Institutes of Health Database for Annotation, Visualization, and Integrated Discovery; FDR, false discovery rate; FXR, farnesoid X receptor; FXRE, FXR response element; gdna, genomic DNA; GO, gene ontology; IgG, immunoglobulin G; IR1, inverted repeat 1; kb, kilobase; LRH-1, liver receptor homolog 1; MEME, Multiple Em for Motif Elicitation; mrna, messenger RNA; Oxct2b, 3-oxoacid coenzyme A transferase 2B; qpcr, quantitative polymerase chain reaction; qrt-pcr, quantitative reverse-transcription polymerase chain reaction; RNAPII, RNA polymerase II; RXRa, retinoid X receptor alpha; SHP, small heterodimer partner; TSS, transcription start site; UTR, untranslated region. From the 1 Department of Molecular and Integrative Physiology and 2 Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL. Received December 1, 2011; accepted January 17, This study was supported by the National Institutes of Health (grant no.: DK062777) and a Basic Science Award from the American Diabetes Association (to J.K.K.). *These authors contributed equally to this study. 108

2 HEPATOLOGY, Vol. 56, No. 1, 2012 LEE ET AL. 109 target genes. 1-3 FXR represses a group of target genes indirectly through the induction of an orphan nuclear receptor, small heterodimer partner (SHP) The FXR/SHP pathway has been shown to play an important role in the negative feedback regulation of bile acid biosynthesis and in hepatic triglyceride metabolism, 11-13,15 although the importance of FXR-independent bile-acid activated signaling pathways in the regulation of hepatic metabolism has been demonstrated In addition to indirect gene repression through SHP, whether FXR can directly inhibit genomic targets is largely unknown. In recent studies, we have shown the importance of post-translational modifications in modulating the levels and activities of transcriptional factors and cofactors in the regulation of hepatic metabolism In particular, we have shown that acetylation of FXR is dynamically regulated by p300 acetylase and sirtuin 1 deacetylase (SIRT1) under physiological conditions. 20,21 Though FXR acetylation increased its stability, it dampened FXR s transactivation ability by inhibiting the binding of the FXR/RXRa heterodimer to DNA. 20 Remarkably, FXR acetylation is highly elevated in the fatty livers of genetic and dietary obese mice. 20 These findings raised an important question of whether genomic targets regulated by FXR are altered in obesity, which might be associated with abnormal metabolism and aberrant liver functions. To determine whether the hepatic transcriptional regulation of FXR target genes is changed in obesity, in this study, we identified and compared the genomic binding of FXR in healthy and dietary obese mice by chromatin immunoprecipitation sequencing (ChIPseq) analysis and further investigated whether ligandactivated FXR can either directly activate or repress potential target genes. Materials and Methods Animals. Six-week-old male BALB/c mice were fed normal chow or a high-fat diet (42% fat, TD88137; Harlan Teklad, Indianapolis, IN) for 20 weeks. Increased body weights were monitored, and development of fatty liver was also confirmed by Oil Red staining and by measuring messenger RNA (mrna) levels of lipogenic genes (Supporting, Fig. 1). Hepatic expression of FXR in healthy and obese mice were also examined (Supporting Fig. 2). Mice were intraperitoneally injected with vehicle or GW4064 (30 mg/kg in corn oil) at 9:00 a.m., and 1 hour later, livers were collected for ChIP-seq analysis. Chromatin Immunoprecipitation Assays and Genomic Sequencing (ChIP-seq). Detailed procedures for ChIP-seq analysis are described in Supporting Fig. 3. Briefly, genomic samples from 4 mice per each group were immunoprecipitated by antibodies for FXR (mixture of sc-1204 and sc-13063) or control immunoglobulin G (IgG). Twenty nanograms of DNA from the immunoprecipitated chromatin pooled from four independent chromatin immunoprecipitation (ChIP) assays was subjected to deep genomic sequencing using the Illumina/Solexa Genome Analyzer II (Biotechnology Center, University of Illinois at Urbana-Champaign, Urbana, IL). Location of Binding Peaks and Gene Ontology Analysis. FXR-binding peaks were subjected to analysis with CisGenome, and the false discovery rate (FDR) (<0.001) and ratio of FXR binding to control IgG peaks (>5) were used to detect binding sites. FXR-binding sites were analyzed to identify the gene locations of the sites in the mouse genome. A list of all genes with FXR peaks within 610 kilobase (kb) of the genes was generated using CisGenome. Gene ontology (GO) analysis of potential FXR target genes was conducted by using the National Institutes of Health program, Database for Annotation, Visualization, and Integrated Discovery (DAVID), for the functional grouping of binding genes. Motif Analysis. The consensus motifs within the 250 top-scoring FXR-binding peaks were determined using the program, Multiple Em for Motif Elicitation (MEME). The coordinates of each peak were set to collect motif lengths of 6-20 base pairs. Comparison of motifs against a database of known FXREs was done in TOMTOM, generating P values of the similarity score, scoring details, and a logo alignment for each match. Re-ChIP, ChIP, and Quantitative Reverse-Transcription Polymerase Chain Reaction Analyses. Re-ChIP assays were performed as previously described. 21,23 Address reprint requests to: Jongsook Kim Kemper, Ph.D., Department of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall, MC-114, 407 South Goodwin Avenue, Urbana, IL jongsook@illinois.edu; fax: Copyright VC 2012 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article.

3 110 LEE ET AL. HEPATOLOGY, July 2012 Briefly, liver chromatin was immunoprecipitated with FXR antibody (sc-1204, goat polyclonal) first and then washed, eluted, and reprecipitated using rabbit polyclonal antibodies for FXR (sc-13063), RXRa (sc-553), RNA polymerase II (RNAPII) (sc-9001), histone H3K9/K14 acetylation (06-599; Upstate Biotech/Millipore, Billerica, MA), and control IgG. Standard ChIP assays were also performed using antibodies for H3K9 dimethylation (07-521; Upstate Biotech/Millipore) and H3K27 trimethylation (6002; Abcam, Cambridge, MA). Then, genomic DNA (gdna) was subjected to quantitative polymerase chain reaction (qpcr) using primer sets (Supporting Fig. 4A). For gene-expression quantitative reverse-transcription polymerase chain reaction (qrt-pcr) studies, total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA), and qrt-pcr was performed using primer sets (Supporting Fig. 4B). Results Genome-wide Profiling of Hepatic FXR Binding Sites in Healthy and Obese Mice. To identify genome-wide hepatic FXR-binding sites in healthy and obese mice, mice were fed normal or high-fat chow for 20 weeks and then treated for a short time (1 hour) with a synthetic FXR agonist, GW4064, to activate FXR signaling. ChIP assays from liver chromatin were performed with FXR antibody or control IgG. The quality of the ChIP assay was confirmed by the increased binding of FXR to known FXR targets, Shp and organic solute transporter beta, and increased levels of Shp mrna also confirmed the effectiveness of the GW4064 treatment (Supporting Fig. 5). The ChIP-seq analysis generated 2.98 and 3.97 million reads for GW4064-treated healthy and obese mice, respectively (Supporting Fig. 6). FXR-binding peak analysis, with stringent FDR cutoffs of <0.001 and the elimination of peaks observed also with control IgG, identified a total of 15,263 and 5,272 FXR-binding sites in GW4064-treated healthy and obese mice, respectively (Fig. 1A, top). Of these sites, 7,440 or 2,344 were uniquely detected in healthy or high-fat dietary obese mice (Fig. 1A, bottom). The number of overlapping sites in the healthy mice was greater than that in the obese mice, because some of the FXR-binding sites in the obese group overlapped with two or more binding sites in the healthy group. Validation of ChIP-seq. To validate the ChIP-seq data, we randomly selected FXR-binding sites. Neither the size nor position of FXR-binding peaks was considered to select binding sites for validation and follow-up studies. ChIP assays revealed that binding to 24 of 27 sites in healthy mice and 20 of 21 in obese mice was enriched by at least 1.8-fold relative to vehicle-treated mice (Supporting Figs. 7 and 8), confirming binding to approximately 90% of these sites and validating the accuracy of the ChIP-seq analysis. Unique FXR-Binding Sites in Healthy or Obese Mice Suggest Altered Transcriptional Signaling in Obesity. The central question of this study was to determine whether FXR regulation might be altered in obesity, which could underlie abnormal liver function and metabolism. Therefore, we focused on the differences in FXR binding between GW4064-treated healthy and obese mice. Notably, 7,440 of the total 15,632 FXR-binding sites in healthy mice were unique in these mice, whereas 2,344 of the total 5,272 sites in obese mice were unique (Fig. 1A). Potential FXR target genes were identified based on the criteria that an FXR-binding site was within 10 kb of the gene. FXRbinding sites corresponded to 2,583 or 1,566 potential target genes unique in healthy or obese mice (Fig. 1A). These results indicate that nearly half of the total FXR-binding sites are unique in healthy or obese mice, suggesting that transcriptional regulation patterns by FXR are likely altered in obesity. Distribution of FXR-Binding Sites and Motif Analysis. Binding sites of FXR were predominantly distributed in intron (38%) and intergenic (40%) regions in both groups of mice (Fig. 1B). There was a marked reduction of FXR-binding sites to 5 UTR (untranslated region) and 3 UTR regions in obese mice, and the genes with binding sites uniquely in the UTR regions of healthy or obese mice are listed in Supporting Fig. 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 and obese mice (Fig. 1D). Functional GO Analysis. 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

4 HEPATOLOGY, Vol. 56, No. 1, 2012 LEE ET AL. 111 Fig. 1. Unique FXR-binding sites in healthy and obese mice. (A) Venn diagrams showing the number of FXR-binding sites in healthy or dietary obese mice (top) and the number of sites uniquely detected in either healthy (ND) or obese (HFD) mice treated with GW4064 for 1 hour (bottom). The numbers of genes near the unique FXR-binding sites in healthy and obese mice are shown in parenthesis. (B) Distribution of total or unique FXR-binding sites in GW4064-treated healthy and obese mice. FXR sites located >10 kb from genes were assigned to intergenic regions. (C) Distance from the center of each FXR-binding peak to the TSS. (D) Logo showing the 13 nucleotide consensus IR1 motif from the top 250 FXR peaks in GW4064-treated healthy and obese mice. The size of the letters is proportional to the frequency of occurrence. (E) Gene ontology categories of the genes nearest to unique FXR binding sites in GW4064-treated normal or obese mice. Genes located within 10 kb of FXR sites were identified using the program DAVID. functions of FXR, particularly in the regulation of cellular signaling pathways, are different in healthy and obese 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. Correlation of FXR Binding With Relative Gene Expression. 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 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

5 112 LEE ET AL. HEPATOLOGY, July 2012 Fig. 2. Effects of GW4064 on FXR binding in healthy or dietary obese mice by ChIP-seq. (A-F) ChIP-seq data showing FXR-binding sites in vehicle- or GW4064-treated healthy and obese mice are displayed using the University of California Santa Cruz genome browser. Chromosomal locations are shown at the top of each display. The Y-axis shows the numbers of mapped sequence tags, with the maximum ranging from 160 to 485. Gene positions are indicated at the bottom of each display, with the arrows indicating the TSS. 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. Effects of GW4064 Treatment on Potential FXR Target Gene Expression. To correlate the binding of FXR at a gene with its expression, mrna levels of randomly selected genes with FXR-binding sites were measured. For genes with FXR binding restricted to healthy mice, GW4064 treatment overnight significantly increased mrna in only 2 genes of 16 examined, whereas unexpectedly, mrna levels were markedly reduced in eight genes (Fig. 4A). Similar trends were observed from the samples treated with GW4064 for a short time only (1 hour) (Supporting Fig. 12), which is consistent with a direct effect of FXR on gene expression, rather than delayed indirect responses. For the genes unique to obese mice, mrna levels were increased significantly for 5 of 13 genes and decreased significantly in one (Fig. 4B). These results suggest that a large fraction of potential FXR target genes examined are likely repressed by agonist-activated FXR.

6 HEPATOLOGY, Vol. 56, No. 1, 2012 LEE ET AL. 113 Fig. 3. Correlation of FXR binding with expression of the nearest genes. (A and B) To validate FXRbinding sites unique to GW4064-treated healthy or obese mice, healthy or dietary obese mice were treated with GW4064 for 1 hour and livers were collected. Occupancies of FXR were examined at randomly selected genes by ChIP assays in healthy (ND) (A) or high-fat-diet induced obese (HFD) (B) mice. gdna from three independently performed ChIP assays from 3 mice were pooled and used for the qpcr analysis. (C and D) mrna levels of genes near the unique FXR-binding sites in healthy (A) or high-fat-diet induced obese (B) mice treated with GW4064 overnight were measured by qrt-pcr. Statistical significance was determined by the Student s t test from nine qrt-pcr readings of three replicate assays each for 3 mice (standard error of the mean; n ¼ 9). *P < 0.05; **P < 0.01; ***P < 0.001; and statistically not significant. Direct Gene Repression by Agonist-Activated FXR Is Likely Common. Direct gene repression by agonistactivated FXR was expected to be rare, because nearly all of the direct FXR target genes have been reported to be activated. 2,3,19 We therefore further examined epigenetic histone markers of gene activation and repression, as well as occupancy by RNAPII. 21,24,25 Genes with increased expression, as well as representative genes that were repressed or not significantly affected (Fig. 4A), were examined. Two-step re-chip and qrt-pcr analyses were performed, and the known FXR target gene, Shp, was first analyzed as a control. Co-occupancy of RNAPII with FXR and acetylated histone H3K9/K14 at the Shp promoter was increased, whereas co-occupancy of RXRa with FXR was not changed after GW4064 treatment (Fig. 5A). As expected, mrna levels for Shp were increased after a 1-hour treatment with GW4064 (Fig. 5B). For selected potential FXR genomic targets, occupancy of FXR was increased in nearly all of the genes examined after GW4064 treatment (Fig. 5C). For the three genes with increased mrna levels after GW4064 treatment (Fig. 4A), aldehyde dehydrogenase 1 and 2 (Aldh1/2), 3-oxoacid coenzyme A transferase 2B (Oxct2b), and cell division cycle-associated 4 (Cdca4), co-occupancy of RXRa and RNAPII with FXR was increased and acetylated histone H3K9/K14 levels were increased, as expected, for activated genes (Fig. 5D-F). For the remaining genes, RNAPII occupancy and acetylated histone H3 levels were decreased or unchanged, which would be consistent with either no induction or suppression of these genes, as observed from the mrna levels.

7 114 LEE ET AL. HEPATOLOGY, July 2012 Fig. 4. Effects of GW4064 treatment on mrna levels of potential FXR target genes. Mice were treated with vehicle or GW4064 overnight (16 hours), and qrt-pcr analysis was done to measure mrna levels of genes nearest to unique FXR-binding sites in healthy (A) or high-fat-diet induced obese (B) mice. mrna levels of genes nearest to FXR-binding sites that were unique to healthy mice were determined by qrt-pcr. Statistical significance was determined by the Student s t test from 11 qrt-pcr readings from 4 mice (standard error of the mean; n ¼ 11). *P < 0.05; **P < Finally, to directly determine whether binding of agonist-activated FXR leads to the repression of genes examined, ChIP assays were performed to measure the levels of known epigenetic histone marks for gene repression, such as H3K9 di- and H3K27 tri-methylation. 24,25 After treatment with GW4064, occupancies of FXR and RNAPII were increased and tri-methylated H3K9 and H3K27 levels were decreased at the control Shp gene promoter (Fig. 6A). For the potential FXR target genes with increased expression, such as Ald1/2, Oxc2b, and Cdca4, levels of repressed histone marks (H3K27 and H3K9) were unchanged or decreased after GW4064 treatment (Fig. 6B). In contrast, levels of gene-repression histone marks, either H3K9 dimethylation, H3K27 trimethylation, or both, were markedly increased for the FXR target genes with decreased expression after either a 1- or 3-hour treatment with GW4064 (Fig. 6C). These results are consistent with decreased mrna levels (Fig. 4A) and decreased occupancy of RNAPII and acetylated H3 levels at those genes (Fig. 5C-F). Collectively, these studies suggest that a large fraction of the agonist-activated FXR target genes examined is directly repressed. Because FXR was shown to increase its target genes in nearly all previous studies and to repress some target genes indirectly through the induction of SHP, 3,4,11,14 our finding that direct gene repression by FXR is common is unexpected. Discussion In this study, ChIP-seq analysis of hepatic genomic binding of agonist-activated FXR in healthy and obese mice resulted in two major findings. First, of the total hepatic FXR-binding sites, nearly half of the sites were unique to healthy or obese mice, implying altered FXR transcriptional signaling in obesity. Second, further analyses utilizing ChIP and qrt-pcr assays suggested that a large fraction of FXR target genes examined are directly repressed by ligand-activated FXR. Approximately 80% of identified FXR-binding sites are localized in intergenic and intron regions, at a

8 HEPATOLOGY, Vol. 56, No. 1, 2012 LEE ET AL. 115 Fig. 5. Effects of GW4064 treatment on co-occupancy of FXR, RXRa, RNAPII, and acetylated histone H3K9/K14 at potential FXR target genes. Mice were treated with vehicle or GW4064 for 1 hour, and livers were collected for two-step re-chip (A and C-F) and qrt-pcr (B) analyses. Initial ChIP assays were done using the FXR antibody (goat polyclonal), and then immunoprecipitated chromatin was eluted and second ChIP assays were performed using rabbit polyclonal antibodies to FXR, RXRa, RNAPII, and acetylated histone H3K9/K14. Occupancy of these proteins and acetylated histone H3K9/K14 levels at the potential FXR target genes (C-F), as well as the Shp gene promoter as a positive control (A), were examined by qpcr and semiquantitative PCR of the immunoprecipitated chromatin DNA, respectively. In (A), band intensities were determined using ImageJ, and the intensities relative to vehicletreated mice are indicated below the bands. (C-F) Values of the samples from mice treated with GW4064 are normalized relative to those from mice treated with vehicle. Consistent results were observed for qpcr readings from triplicate re-chip assays each from 2 mice. Statistical significance was determined by the Student s t test (standard error of the mean; n ¼ 6). *P < 0.05; **P < 0.01; and statistically not significant). consensus IR1 motif, in healthy and obese mice. These findings are consistent with recently reported ChIP-seq analysis of FXR binding in healthy mice Thomas et al., for the first time, identified and compared genomic FXR-binding sites in liver and intestine in mice treated with GW4064. Interestingly, only 11% of total FXR-binding sites were shared between liver and intestine, demonstrating tissue-specific FXR target genes. 26 Chong et al. identified FXR-binding sites in mouse hepatic chromatin, and showed that binding sites for liver receptor homolog 1 (LRH-1) were enriched near the asymmetric IR1 FXR site and that LRH-1 and FXR can coactivate gene expression. 27 Lee et al. also identified functional FXR sites within promoters, introns, or intragenic regions of selected genes involved in xenobiotic metabolism, suggesting a role for FXR in liver protection against toxic substances, such as acetaminophen. 28 This current study reveals numerous previously unknown potential FXR target genes unique in healthy and obese mice and categorization of these genes identifies new functions, suggesting that biological pathways potentially regulated by FXR are altered in obesity. We have shown that acetylation of FXR inhibits DNA binding of the FXR/RXRa heterodimer, and that FXR acetylation levels are highly elevated in obese mice. 20 The present studies suggest that potential FXR genomic targets and biological pathways are altered in fatty liver of dietary obese mice, which raises the intriguing possibility that aberrantly elevated acetylation of FXR might be associated with altered transcriptional regulation in obesity in such a way as to contribute to fatty liver and, subsequently, detrimental metabolic outcomes. Furthermore, reduced DNA binding of FXR/ RXRa caused by FXR hyperacetylation may contribute to the decreased FXR-binding sites observed in obesity (5,272, compared to 15,263 sites in healthy mice). An important, unexpected finding in these studies is that binding of agonist-activated FXR was often associated with repression of gene expression. In a large fraction (8 of 16) of genes examined, binding of ligandactivated FXR was associated with decreased mrna levels, which was confirmed by decreased RNAPII occupancy and reduced acetylated histone H3K9/K14 levels. More important, levels of known histone generepression marks as well as H3K9 and H3K27

9 116 LEE ET AL. HEPATOLOGY, July 2012 Fig. 6. Gene-repression histone marks are increased at potential FXR target genes after GW4064 treatment. Healthy mice were treated with vehicle or GW4064 for either 1 or 3 hours, and livers were collected for ChIP analysis. (A) As a control, occupancy of FXR, RXRa, RNAPII, and levels of H3K9 di- and H3K27 tri-methylation were detected at the Shp gene promoter. In (A), band intensities were determined using Image J, and intensities relative to vehicle-treated mice are indicated below the bands. (B and C) Gene-repression histone marks as well as H3K9 di- and H3K27 tri-methylation were detected at the potential FXR target genes with increased expression (B) or decreased expression (C) after GW4064 treatment. Values of the samples from GW4064-treated mice are normalized relative to those from vehicle-treated mice. Each bar represents the average of three qpcr readings from each ChIP assay. methylation, were markedly increased at those genes that were repressed in healthy mice after exposure to the FXR agonist, GW4064, for a short 1- or 3-hour treatment. Because mrna levels were measured after 1-hour treatment, in addition to overnight treatment with GW4064, direct effects of FXR on gene transcription were likely detected. Although our follow-up epigenetic and gene-expression studies have suggested that gene repression by FXR is common, direct comparison of FXR binding with a comprehensive global transcriptome analysis using RNA sequencing or microarray will be necessary to definitively determine the extent of gene repression relative to gene activation by agonist-activated FXR. FXR is well known to repress its target genes indirectly through the induction of SHP These present studies suggest that FXR may also directly repress its target genes by unknown mechanisms. FXR could directly repress by binding to the DNA as a FXR/ RXRa heterodimer or as a monomer or homodimer, as previously shown in the regulation of apolipoprotein A1, 29 which results in the inhibition of DNA binding of key transcription factors. In addition, FXR could directly inhibit genes by tethering to DNA-binding transcription factors and masking their interaction with coactivators and/or facilitating the interaction with corepressors. Sumoylation of peroxisome proliferator-activated receptor gamma and liver X receptor has been shown to be directly involved in the repression of inflammatory genes by the tethering of these nuclear receptors to DNA-binding activators, such as, nuclear factor kappa light-chain enhancer of activated B cells or activator protein We have evidence that FXR is sumoylated in mouse liver extracts (D.H.K. and J.K.K., unpublished data), and FXR was shown to inhibit inflammatory responses, 9,10 so that this is a

10 HEPATOLOGY, Vol. 56, No. 1, 2012 LEE ET AL. 117 possible mechanism for FXR gene repression. Whether FXR directly suppresses its target genes by binding to DNA or tethering to other transcription factors is an important area of future investigation. In conclusion, these studies analyze, for the first time, a genome-wide comparison of FXR-binding sites in the livers of healthy and dietary obese mice. Our studies identify numerous previously unknown potential FXR target genes that are involved in a wide range of hepatic functions, which suggests that FXR may have much broader functions than previously appreciated. Approximately half of the potential target genes in both healthy and obese mice were unique to each, suggesting that potential FXR target genes and biological pathways are altered in obesity. Moreover, a large fraction of the potential FXR target genes examined were repressed by ligand-activated FXR, suggesting that direct gene repression by FXR might be more common than previously thought. Additional studies will be required to elucidate the molecular mechanisms by which FXR directly represses these potential genomic targets. Acknowledgment: The authors are grateful to Dr. Grace L. Guo (University of Kansas Medical Center) for her helpful suggestions for the ChIP-seq analysis. The authors also thank Ms. Ting Fu for kindly performing Oil Red staining of liver sections. The authors also thank Byron Kemper for his critical comments on the manuscript for this article. References 1. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995;83: Lee FY, Lee H, Hubbert ML, Edwards PA, Zhang Y. FXR, a multipurpose nuclear receptor. Trends Biochem Sci 2006;31: Lefebvre P, Cariou B, Lien F, Kuipers F, Staels B. 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