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Additional Supporting Information may be found in the online version of this article.

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HEP_23450_sm_SuppLegTabs.doc117KSupplementary Tables
HEP_23450_sm_SuppFig1.tif1004KSupporting Figure 1: Anti-FXR Antibody Validation. (S1a) FXR protein levels in the nuclear extraction of wild-type and FXR KO mice show that the FXR antibody used is specific for mouse FXR, revealed by Western blot assay. (S1b) Binding of FXR to the downstream IR1 in the Nr0b2 gene in wild-type and FXR KO mice by ChIP-qPCR assay. * indicates P < 0.05.
HEP_23450_sm_SuppFig2.tif909KSupporting Figure 2: Initial Validation of FXR binding to known FXRREs by ChIP-qPCR. Predicted FXRREs are found at −320 to −220 bp upstream of Nr0b2 transcriptional start site (TSS) in the liver, −1245 to −1145 bp upstream of TSS of Osta and −220 to −150 bp upstream of Ostb in the intestine. Primers were designed to amplify these specific sites. The graphs are displayed as number of binding events detected per 1000 cells (y-axis) versus a specific amplified site (x-axis). S1a illustrates FXR binding at −320 to −220 bp upstream of Nr0b2 in the liver of mice treated with vehicle or GW4064 for 2, 4, or 8 hrs. S1b illustrates FXR binding at −1245 to −1145 bp upstream of Osta and −220 to −150 bp upstream of Ostb in the intestine of mice treated with vehicle or GW4064 for 2, 4, or 8 hours. These ChIP-qPCR results show FXR binding at these sites is higher than for untranscribed regions of the genome (Untr 6). This indicates that the assay and the anti-FXR antibody used are specific to detect FXR bound regions of the genome. The greatest enrichment factor of GW4064 treated mice versus vehicle control is 1.59 (seen for Osta in the intestines for the 8 hr treatment group: S1b). These results indicate that FXR binding at these target sites is not sufficiently enhanced after treatment of animals with an FXR ligand. Therefore, only tissues from animals treated with GW4064 were used for ChIP-seq analysis.
HEP_23450_sm_SuppFig3.tif892KSupporting Figure 3: ChIP-qPCR of FXR Binding Sites Identified by ChIP-Seq Analysis. (a) FXR binding sites identified by ChIP-seq results with a peak value above 300 were confirmed by ChIP-qPCR. Slc9a8 (encoding sodium hydrogen exchanger 8: Nhe8) was bound by FXR in the liver and intestine at around -5350 to -5250 bp upstream of the gene (peak value: around 800 in liver and 1000 in intestine). The 3′ end of Nr0b2 (encoding Shp: 950 to 1050 downstream of gene) was also bound by FXR in both liver and intestine (peak value: 498 in liver and around 400 in intestine). Only the binding site in the liver is shown here. Binding of FXR to Slc9a8 in liver and intestine increased 3 and 25 fold after GW4064 treatment, and binding to Nr0b2 in liver increased 6 fold after GW4064 treatment. (b) Sites identified by ChIP-Seq results with a peak value of 100-300 were confirmed by ChIP-qPCR. Actg1 (encoding actin, gamma, cytoplasmic 1: Actg1) was bound by FXR -5260 to -5160 bp upstream of the gene in the intestine (peak value: 181). Cdk5rap2 (encoding CDK5 regulatory subunit associated protein 2: Cdk5rap2) was bound by FXR 32150 to 32250 bp within the gene in the liver (peak value: 224). Binding of FXR to Actg1 in the intestine and Cdk5rap2 in the liver and intestine increased 5 and 2.5 fold after GW4064 treatment. (c) Sites identified by ChIP-Seq results with a peak value of less than 100 were confirmed by ChIP-qPCR. Bccip (encoding BRCA2 and CDKN1A interacting protein: Bccip) was bound by FXR -380 to -280 bp upstream of the gene in the intestine (peak value: around 30). Nr1d2 (encoding nuclear receptor subfamily 1, group D, member 2: Nr1d2) was bound by FXR 2000 to 2100 bp within the gene in the liver (peak value: 81). Binding of FXR to Bccip in the intestine and Nrld2 in the liver and intestine increased 1.75 and 3 fold after GW4064 treatment. Statistical significance indicated by a p-value < 0.05.

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