Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor α in mouse liver reveals extensive sharing of binding sites - PubMed (original) (raw)

doi: 10.1128/MCB.06175-11. Epub 2011 Dec 12.

Thomas Åskov Pedersen, Barbara Gross, Simon J van Heeringen, Dik Hagenbeek, Christian Bindesbøll, Sandrine Caron, Fanny Lalloyer, Knut R Steffensen, Hilde I Nebb, Jan-Åke Gustafsson, Hendrik G Stunnenberg, Bart Staels, Susanne Mandrup

Affiliations

Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor α in mouse liver reveals extensive sharing of binding sites

Michael Boergesen et al. Mol Cell Biol. 2012 Feb.

Abstract

The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

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Figures

Fig 1

Fig 1

Genome-wide mapping of LXR and RXR binding sites in mouse liver. (A) LXR ChIP-qPCR on the livers from wild-type (WT) and LXRα/β-deficient (LXR double knockout [LXRdKO]) mice gavaged with the LXR agonist T0901317 (30 mg/kg body weight [mpk]) once daily for 14 days. Sterol regulatory element-binding protein 1c (SREBP-1c) (61) and carbohydrate response element-binding protein (ChREBP) (8) LXREs were used as positive controls for LXR binding, whereas “No gene” is a negative control (see Materials and Methods for details). Bars represent the means plus standard deviations (SD) (error bars) (n = 3). (B) Venn diagrams representing the number of “LXR only” (blue), “RXR only” (yellow), and shared LXR-RXR binding sites (green) in mouse liver detected by ChIP-seq. Mice were gavaged with vehicle (1% carboxymethyl cellulose), the RXR agonist bexarotene (100 mpk), or the LXR agonist T0901317 (30 mpk) once daily for 14 days. Peaks were called using FindPeaks (FDR > 0.001). (C) Scatterplots illustrating the intensity of LXR and RXR binding in vehicle-treated mouse liver (log2 number of reads per peak) at sites that are defined as LXR specific (blue), RXR specific (red), and shared (green). The percentages indicate the fractions of sites that are above or below the broken line. (D) Genomic positions of shared LXR-RXR binding sites in T0901317-treated mouse liver relative to the nearest gene (PinkThing) are shown as follows: Distant, distance to the TSS > 25 kb; 5′far, 25 to 5 kb upstream of the TSS; Promoter, <5 kb upstream of the TSS; Intragenic, intragenic peaks downstream of intron 1; 3′near, <5 kb downstream of the 3′ end; 3′far, 5 to 25 kb downstream of the 3′ end.

Fig 2

Fig 2

Assessment of genomic LXR and RXR binding in mouse liver during treatment with LXR and RXR agonists. Mice were gavaged with vehicle (Veh) (1% carboxymethyl cellulose), the RXR agonist bexarotene (Bexa) (100 mpk), or the LXR agonist T0901317 (T09) (30 mpk) once daily for 14 days. (A and C) Venn diagrams representing the number of genome-wide binding sites of LXR (A) and RXR (C) in livers from mice treated with vehicle (yellow), bexarotene (red), and T0901317 (blue). (B and D) Box plots illustrating the number of tags per LXR peak (B) and RXR peak (D) at sites that are conserved between the different treatments (gray area in panels A and C). (E and F) ChIP-qPCR validation of ligand-dependent RXR binding (E) and LXR binding (F) to selected loci identified by ChIP-seq (see Fig. S3 in the supplemental material) (n = 4). Values that are significantly different from the vehicle by one-way analysis of variance (ANOVA) are indicated as follows: ∧, P < 0.01; ∗, P < 0.05. (G) Venn diagram representing the number of LXR-RXR binding sites in vehicle- and T0901317-treated mice and overlap with ENCODE DNase I-hypersensitive (DHS) sites. (H and I) Box plots illustrating the number of DHS (H) and LXR (I) tags per peak in overlap with T0901317-independent and T0901317-dependent LXR binding sites. For all box plots, the rectangles show the interquartile ranges (IQR) from the first quartile to the third quartile and the lines in the middle of the boxes represent the medians. The whiskers are drawn to the nearest value not exceeding 1.5 times the IQR, and outliers are not shown. Wilcoxon test with Bonferroni's correction was used.

Fig 3

Fig 3

LXR, RXR, and RNA polymerase II (RNAP2) binding to the Scd1 (A) and Ehhadh (B) gene loci in mouse liver. UCSC Genome Browser tracks derived from LXR, RXR, and RNAPII ChIP-seq data are shown. The black arrow shows the position of a previously reported LXRE (11).

Fig 4

Fig 4

LXR is not required for binding of RXR to the majority of LXR-RXR binding sites. (A) Venn diagram showing the number of RXR binding sites in the livers of LXR dKO mice treated with vehicle (veh) (yellow), bexarotene (bexa) (red), and T0901317 (T09) (blue). (B) Boxplot illustrating the number of tags per RXR peak at sites that are conserved between the different treatments in panel A. Values that are significantly different (P < 2.2e−16 by Wilcoxon test with Bonferroni's correction) from those of vehicle-treated mice are indicated by an asterisk. (C) Venn diagram showing the number of genome-wide RXR binding sites in the livers of T0901317-treated WT (red) and LXR dKO mice (blue) and overlap with LXR binding sites in T0901317-treated WT mice (yellow). (D) RXR occupancy at LXR-RXR sites in WT mice (shared between vehicle-, bexarotene-, and T0901317-treated animals) that are present (LXR independent) or absent (LXR dependent) in LXRdKO mice. (E) Relative overrepresentation (fold enrichment compared to a random background) of direct, inverted, and everted repeats with the core sequence RGKTCA under LXR-RXR peaks where RXR binding is either LXR independent (gray) or LXR dependent (orange). For all box plots, the rectangles show the IQR from the first quartile to the third quartile and the lines in the middle of the boxes represent the medians. The whiskers are drawn to the nearest value not exceeding 1.5 times the IQR, and outliers are not shown.

Fig 5

Fig 5

Genome-wide overlap between LXR and PPARα binding sites in mouse liver. (A) PPARα, LXR, and RXR ChIP-qPCR on selected loci in the livers of wild-type (WT) and PPARα knockout (KO) mice. Bars represent the mean plus range of two independent experiments. (B) Venn diagram representing the overlap between PPARα, LXR, and RXR binding sites in the livers of vehicle-treated WT mice. (C) Scatterplots illustrating the intensity of LXR and PPARα binding in vehicle-treated mouse liver (log2 number of reads per peak) at sites that are defined as LXR specific (blue), PPARα specific (red), and shared (green). The percentages indicate the fractions of sites that are above or below the broken line. (D) Venn diagram representing the overlap between PPARα binding sites in vehicle-treated WT liver with LXR and RXR binding sites in T0901317-treated (T09) WT liver. (E) Boxplot representing the number of PPARα ChIP-seq tags per peak in clusters that do not overlap with RXR and LXR binding (red), overlap with only RXR (orange) and overlap with both RXR and LXR (gray). Wilcoxon test with Bonferroni's correction was used. For all box plots, the rectangles show the IQR from the first quartile to the third quartile and the lines in the middle of the boxes represent the medians. The whiskers are drawn to the nearest value not exceeding 1.5 times the IQR, and outliers are not shown.

Fig 6

Fig 6

UCSC Genome Browser tracks derived from ChIP-seq data showing LXR, PPARα, RXR, and RNAP2 binding to the Ehhadh (A), Agpat2 (B), Gbpc (C), and Sik1 (D) loci. The black arrows indicate overlapping binding of LXR, PPARα, and RXR.

Fig 7

Fig 7

LXR and PPARα bind to common binding sites in a mutually exclusive manner. (A) Direct repeat motif search (RGKTCA[1.…5]RGKTCA) on LXR and PPARα genomic binding regions in mouse liver treated with vehicle (veh) and T0901317 (T09). LXR-RXR (T09) refers to LXR-RXR binding sites in T0901317-treated liver that are not overlapping with PPARα binding sites. (B) Web logos of DR1 and DR4 sequence motifs identified by de novo motif search on PPARα-RXR (vehicle) and LXR-RXR (vehicle) binding sites (ROC_AUC = 0.632/0.744). (C) Fold enrichment over background of DR1 and DR4 de novo motifs (FDR of 1%). (D) Fraction of peaks with either a single de novo DR1 or DR4 motif or both. (E) Distance between peak centers of overlapping LXR, RXR, and PPARα peaks determined by FindPeaks. Distances were calculated for each site and are shown as 25-bp bins. (F) ChIP-reChIP on liver chromatin from wild-type mice treated with 30 mpk T0901317 24 h and again 8 h before euthanasia. Bars indicate reChIP recoveries when using an antibody against LXR in the first ChIP and against either RXR, PPARα, or IgG in the second ChIP (left y axis), as well as when using an antibody against PPARα in the first ChIP and against either RXR, LXR, or IgG in the second ChIP (right y axis). Bars represent the mean plus range of data from two mice.

Fig 8

Fig 8

Genome-wide cooccurrence of mouse hepatic transcription factors (TFs). Comparison of LXR, RXR, and PPARα genome-wide binding with published binding profiles of HNF4α and C/EBPα (68), Rev-erbα (19), FXR (80), and SREBP2 (71). The percentage of binding sites of the transcription factors on the y axis that have any overlap (≥1 bp) with binding sites for the TFs on the x axis was calculated for each pair and visualized as a heat map. The total number of binding sites for each factor is indicated in parentheses after the TF name on the y axis.

Fig 9

Fig 9

Functional cross talk between LXR and PPARα in mouse liver. Wild-type C57BL6 mice were treated with PPARα agonist fenofibrate (FF) (200 mpk) (n = 7), LXR agonist T0901317 (T09) (30 mpk) (n = 8), both FF and T09 (200 and 30 mpk, respectively) (n = 8), or vehicle (n = 7) once daily for 5 days. On the fifth day of treatment, the animals were sacrificed, and liver and plasma samples were taken. (A to C) mRNA levels of selected genes in liver samples as determined by qPCR relative to cyclophilin A. PEPCK, phosphoenolpyruvate carboxykinase. (D) Liver and plasma triglyceride (TG); (E) liver and plasma total cholesterol. Values that are significantly different by one-way ANOVA are indicated by black lines and the following symbols: ∧, P < 0.01; ∗, P < 0.05.

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References

    1. Alberti S, et al. 2001. Hepatic cholesterol metabolism and resistance to dietary cholesterol in LXRbeta-deficient mice. J. Clin. Invest. 107:565–573 - PMC - PubMed
    1. Apfel R, et al. 1994. A novel orphan receptor specific for a subset of thyroid hormone-responsive elements and its interaction with the retinoid/thyroid hormone receptor subfamily. Mol. Cell. Biol. 14:7025–7035 - PMC - PubMed
    1. Baes M, et al. 1994. A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Mol. Cell. Biol. 14:1544–1552 - PMC - PubMed
    1. Bedoucha M, Atzpodien E, Boelsterli UA. 2001. Diabetic KKAy mice exhibit increased hepatic PPARgamma1 gene expression and develop hepatic steatosis upon chronic treatment with antidiabetic thiazolidinediones. J. Hepatol. 35:17–23 - PubMed
    1. Blumberg B, et al. 1998. SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev. 12:3195–3205 - PMC - PubMed

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