Regulation of Bile Acid Synthesis by the Nuclear Receptor Rev-erbα Hélène Duez, Jelske N. van der Veen, Christian Duhem, Benoît Pourcet, Thierry Touvier, Coralie Fontaine, Bruno Derudas, Eric Baugé, Rick Havinga, Vincent W. Bloks, Henk Wolters, Fjodor H. van der Sluijs, Björn Vennström, Folkert Kuipers, Bart Staels  Gastroenterology  Volume 135, Issue 2, Pages 689-698.e5 (August 2008) DOI: 10.1053/j.gastro.2008.05.035 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Rev-erbα regulates bile acid metabolism in mice. Bile flow (A), biliary bile acid output rate (B), biliary cholesterol/bile acid and phospholipid/bile acid ratios (C), biliary bile acid composition (D), fecal total bile acid (E), and fecal cholic and deoxycholic acids excretion rates (F) in Rev-erbα-deficient mice (n = 6) and their wild-type littermates (n = 6). C, cholic acid; DC, deoxycholic acid. Results are mean ± SD. Statistically significant differences between groups are indicated by asterisks (Student t test: *P < .05, **P < .01). Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Rev-erbα regulates CYP7A1 gene expression in mouse liver. (A–C) Hepatic CYP7A1 mRNA and microsomal protein levels (A) and CYP8B1 (B) and CYP27A1 (C) mRNA levels in Rev-erbα-deficient mice and their wild-type littermates at 4 pm. Inset in panel A shows a representative Western blot analysis and quantification as mean ± SEM. mRNA levels were measured by quantitative polymerase chain reaction, normalized to an internal control, and are mean ± SEM (n = 6). (D–F) Wild-type mice were infected with a Rev-erbα-expressing adenovirus or a GFP-expressing control vector. Liver CYP7A1 (D), CYP8B1 (E), and CYP27A1 (F) mRNA levels are shown. Results (mean ± SEM) are expressed as percent of the GFP-infected mice. Student t test: ***P < .001; **P < .01. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Bile acid-induced repression of CYP7A1 expression is enhanced in Rev-erbα-deficient mice. Hepatic CYP7A1 mRNA (A) and protein (B) levels in Rev-erbα-deficient mice (control, n = 6; TCA, n = 4) and their wild-type littermates (control, n = 6; TCA, n = 4). Mice received a control or a 0.5% wt/wt TCA-supplemented diet for 5 days as described under the Materials and Methods section. The results (mean ± SEM) are expressed as percentage of untreated wild-type mice. Student t test: *,#P < .05, ##P < .01, ***P < .001; asterisk indicates difference between genotype, numbers symbol between chow and TCA fed mice. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Circadian variations of CYP7A1, SHP, and E4BP4 in Rev-erbα-deficient mice. Liver CYP7A1 (A), Rev-erbα (B), E4BP4 (E), and SHP (F) mRNA levels were measured in Rev-erbα-deficient and wild-type mice killed every 6 hours. Results (mean ± SEM, n = 3 or 4/group) are expressed with the control wild-type mice at Zeitgeber (ZT) 7 set as 1. Hepatic E4BP4 (C) and SHP (D) mRNA levels in Rev-erbα-deficient mice and their wild-type littermates (n = 6) at 4 pm. Student t test: *P < .05, ***P < .001. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Rev-erbα binds to a RevRE located in SHP and E4BP4 promoters. (A) Sequence of the RevRE site located in the mouse and human SHP promoter. The depicted sequence corresponds to the previously described IR-1 FXRE (see the 2 arrows below the sequence depicting the 2 half site of the IR-1), and the arrow above the sequence indicates the monomeric RevRE site. Mutations tested in EMSA and transfection experiments are underlined. (B) EMSA were performed with end-labelled consensus RevRE (lanes 1 and 2) and wild-type mouse (mSHPRevREwt) (lanes 3 and 4, 7–11, 13–15) or human (hSHPRevREwt) (lanes 5 and 6) or mutated mouse (mSHPRevREmut) (lane 12). Competition experiments were performed by adding increasing molar excess of cold mSHPRevREwt wild-type (lanes 9–11) or mSHPRevREmut mutated (lanes 13–15) oligonucleotides. (C) Sequence of the RevRE site located in the mouse E4BP4 promoter. The mutation is underlined. (D) EMSA were performed with end-labelled consensus RevRE (lanes 1 and 2) or wild-type mouse (mE4BP4RevREwt) (lanes 3–11) oligonucleotides. Competition experiments were performed by adding increasing excess of cold mE4BP4RevREwt wild-type (lanes 5–7) or mE4BP4RevREmut mutated (lanes 8–10) oligonucleotides. Lane 11: supershifted complex (arrow) using a specific Rev-erbα antibody. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Rev-erbα binds to SHP and E4BP4 in vivo. Chromatin immunoprecipitation (ChIP) assay performed on mouse liver for recruitment of Rev-erbα to E4BP4, SHP, and Bmal1 promoters using an anti-Rev-Erbα antibody. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Rev-erbα represses SHP and E4BP4 promoter activity. RK13 (A and C) and HepG2 (B) cells were transfected with either the mouse wild-type (mSHPp wt) or mutated (mSHPp mut) SHP promoter (A) or with the human SHP promoter (B) in the presence of either hRev-erbα expression vector or empty vector. (C and D) The mouse wild-type or mutated RevRE sites located in SHP (C) and E4BP4 (D) were cloned in 3 copies and transfected in the presence of either the empty vector or the hRev-erbα expression vectors. Values were normalized to an internal control and are mean ± SD. Student t test: ***P < .001, **P < .01, *P < .05. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Scheme summarizing the role of Rev-erbα in the regulation of CYP7A1 expression and bile acid metabolism. CYP7A1 is regulated by numerous factors, some of them being depicted on the cartoon. The present report identifies Rev-erbα as one of these transcriptional regulators. During the late light phase (4 pm), Rev-erbα expression levels are high, leading to E4BP4 and SHP inhibition and ensuing derepression of CYP7A1 and bile acid synthesis. This mechanism may serve to adequately and timely maintain circadian bile acid and cholesterol homeostasis. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure S1 Rev-erbα regulates bile acid metabolism in mice. Hepatic Bmal1, BSEP, ABCB4 (mdr-2), NTCP, ABCG5, and ABCG8 mRNA levels in Rev-erbα-deficient mice (n = 6) and their wild-type littermates (n = 6) at 4 pm. mRNA levels were measured by quantitative PCR and normalized to an internal control. Results are mean ± SEM. ***P < .001 by Student t test. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure S2 Adenovirus-mediated Rev-erbα overexpression in vivo. Wild-type mice were infected with a Rev-erbα-expressing adenovirus or a GFP-expressing control vector. Liver Rev-erbα mRNA levels (mean ± SEM) are expressed as percent of the GFP-infected mice. Student t test: *P < .05. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure S3 Diurnal variations of Bmal1 mRNA levels in liver from Rev-erbα-deficient and wild-type mice. Hepatic mRNA levels were measured in Rev-erbα-deficient and wild-type mice around the clock. mRNA levels were measured by quantitative PCR and normalized to an internal control. Results are fold induction compared with wild-type mice at ZT7 and are mean ± SEM. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions

Figure S4 Expression of clock-regulated genes and nuclear receptors involved in CYP7A1 regulation in Rev-erbα-deficient mice. Hepatic DBP, Dec2, PPARα, LXRα, and HNF4α mRNA levels in Rev-erbα-deficient mice (n = 6) and their wild-type littermates (n = 6) at 4 pm. mRNA levels were measured by quantitative PCR and normalized to an internal control. Results are mean ± SEM. Gastroenterology 2008 135, 689-698.e5DOI: (10.1053/j.gastro.2008.05.035) Copyright © 2008 AGA Institute Terms and Conditions