The p300 Acetylase Is Critical for Ligand-activated Farnesoid X Receptor (FXR) Induction of SHP

The primary bile acid receptor farnesoid X receptor (FXR) maintains lipid and glucose homeostasis by regulating expression of numerous bile acid-responsive genes, including an orphan nuclear receptor and metabolic regulator SHP. Using SHP as a model gene, we studied how FXR activity is regulated by p300 acetylase. FXR interaction with p300 and their recruitment to the SHP promoter and acetylated histone levels at the promoter were increased by FXR agonists in mouse liver and HepG2 cells. In contrast, p300 recruitment and acetylated histones at the promoter were not detected in FXR-null mice. p300 directly interacted with and acetylated FXR in vitro. Overexpression of p300 wild type increased, whereas a catalytically inactive p300 mutant decreased, acetylated FXR levels and FXR transactivation in cells. While similar results were observed with a related acetylase, CBP, GCN5 did not enhance FXR transactivation, and its recruitment to the promoter was not increased by FXR agonists, suggesting functional specificity of acetylases in FXR signaling. Down-regulation of p300 by siRNA decreased acetylated FXR and acetylated histone levels, and occupancy of FXR at the promoter, resulting in substantial inhibition of SHP expression. These results indicate that p300 acts as a critical coactivator of FXR induction of SHP by acetylating histones at the promoter and FXR itself. Surprisingly, p300 down-regulation altered expression of other metabolic FXR target genes involved in lipoprotein and glucose metabolism, such that beneficial lipid and glucose profiles would be expected. These unexpected findings suggest that inhibition of hepatic p300 activity may be beneficial for treating metabolic diseases. The primary bile acid receptor farnesoid X receptor (FXR) maintains lipid and glucose homeostasis by regulating expression of numerous bile acid-responsive genes, including an orphan nuclear receptor and metabolic regulator SHP. Using SHP as a model gene, we studied how FXR activity is regulated by p300 acetylase. FXR interaction with p300 and their recruitment to the SHP promoter and acetylated histone levels at the promoter were increased by FXR agonists in mouse liver and HepG2 cells. In contrast, p300 recruitment and acetylated histones at the promoter were not detected in FXR-null mice. p300 directly interacted with and acetylated FXR in vitro. Overexpression of p300 wild type increased, whereas a catalytically inactive p300 mutant decreased, acetylated FXR levels and FXR transactivation in cells. While similar results were observed with a related acetylase, CBP, GCN5 did not enhance FXR transactivation, and its recruitment to the promoter was not increased by FXR agonists, suggesting functional specificity of acetylases in FXR signaling. Down-regulation of p300 by siRNA decreased acetylated FXR and acetylated histone levels, and occupancy of FXR at the promoter, resulting in substantial inhibition of SHP expression. These results indicate that p300 acts as a critical coactivator of FXR induction of SHP by acetylating histones at the promoter and FXR itself. Surprisingly, p300 down-regulation altered expression of other metabolic FXR target genes involved in lipoprotein and glucose metabolism, such that beneficial lipid and glucose profiles would be expected. These unexpected findings suggest that inhibition of hepatic p300 activity may be beneficial for treating metabolic diseases. Farnesoid X receptor (FXR) 2The abbreviations used are: FXR, farnesoid X receptor; SHP, small heterodimer partner; CYP7A1, cytochrome P450 7A1; CA, cholic acid; CDCA, chenodeoxy cholic acid; ChIP, chromatin immunoprecipitation; HDAC, histone deacetylase; HAT, histone acetylase; TSA, trichostatin A; Nam, nicotine amide; Ad, adenovirus; HDL, high density lipoprotein; VLDL, very low density lipoprotein; WT, wild type; MOI, multiplicity of infection; GST, glutathione S-transferase. is a member of the nuclear receptor superfamily and the primary biosensor for endogenous bile acids (1.Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2182) Google Scholar, 2.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar, 3.Wang H. Chen J. Hollister K. Sowers L. Forman B.M. Mol. Cell. 1999; 3: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar, 4.Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1857) Google Scholar). Upon activation by physiological concentrations of bile acids, FXR regulates expression of a number of bile acid-responsive genes in liver, kidney, and intestines (5.Kalaany N.Y. Mangelsdorf D.J. Annu. Rev. Physiol. 2006; 68: 159-191Crossref PubMed Scopus (491) Google Scholar). Previous studies utilizing synthetic FXR ligands and FXR-null mice have demonstrated that FXR plays a central role in maintaining whole body lipid and glucose homeostasis (6.Willson T.M. Jones S.A. Moore J.T. Kliewer S.A. Med. Res. Rev. 2001; 21: 513-522Crossref PubMed Scopus (101) Google Scholar, 7.Sinal C. Tohkin M. Miyata M. Ward J. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar). It has been established that bile acid-activated FXR regulates cholesterol and bile acid levels by induction of the orphan nuclear receptor and metabolic repressor, small heterodimeric partner (SHP) (8.Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar, 9.Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Wilson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar). SHP inhibits the transcription of cholesterol 7α-hydroxylase (CYP7A1), a key enzyme in the conversion of cholesterol to bile acids in the classical bile acid biosynthetic pathway (10.Chiang J.Y.L. Front. Biosci. 1998; 3: D176-D193Crossref PubMed Scopus (262) Google Scholar, 11.Russell D.W. Cell. 1999; 97: 539-542Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). In addition to its crucial role in cholesterol and bile acid homeostasis, functions of FXR have recently been extended to glucose and fatty acid metabolism, prevention of gallstone formation, liver regeneration, and inhibition of intestinal bacterial growth (12.Ma K. Saha P.K. Chan L. Moore D.D. J. Clin. Investig. 2006; 116: 1102-1109Crossref PubMed Scopus (660) Google Scholar, 13.Watanabe M. Houten S.M. Wang L. Moschetta A. Mangelsdorf D.J. Heyman R.A. Moore D.D. Auwerx J. J. Clin. Investig. 2004; 113: 1408-1418Crossref PubMed Scopus (997) Google Scholar, 14.Huang W. Ma K. Zhang J. Qatanani M. Cuvillier J. Liu J. Dong B. Huang X. Moore D.D. Science. 2006; 312: 233-236Crossref PubMed Scopus (537) Google Scholar, 15.Inagaki T. Moschetta A. Lee Y.K. Peng L. Zhao G. Downes M. Yu R.T. Shelton J.M. Richardson J.A. Repa J.J. Mangelsdorf D.J. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3920-3925Crossref PubMed Scopus (836) Google Scholar, 16.Zhang Y. Lee F.Y. Barrera G. Lee H. Vales C. Gonzalez F.J. Willson T.M. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1006-1011Crossref PubMed Scopus (729) Google Scholar, 17.Moschetta A. Bookout A.L. Mangelsdorf D.J. Nat. Med. 2004; 10: 1352-1358Crossref PubMed Scopus (255) Google Scholar). Although such important biological roles of FXR have now been established, molecular mechanisms by which FXR activity is modulated are largely unknown. Nuclear receptors, including FXR, collaborate with a number of transcriptional cofactors to effectively modulate transcription of their target genes (18.Rosenfeld M. Glass C. J. Biol. Chem. 2001; 276: 36865-36868Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 19.Lazar M.A. Nucl. Recept. Signal. 2003; 1: 2001Crossref Google Scholar). A recent study demonstrated that FXR activity is modulated by a metabolic coactivator, PGC-1α (PPARγ coactivator α) in response to fasting (20.Zhang Y. Castellani L.W. Sinal C.J. Gonzalez F.J. Edwards P.A. Genes Dev. 2004; 18: 157-169Crossref PubMed Scopus (296) Google Scholar). PGC-1α enhances FXR gene transcription by coactivation of PPARγ and HNF-4 (hepatic nuclear factor 4) and also acts as a coactivator of FXR (20.Zhang Y. Castellani L.W. Sinal C.J. Gonzalez F.J. Edwards P.A. Genes Dev. 2004; 18: 157-169Crossref PubMed Scopus (296) Google Scholar). Furthermore, histone arginine methyltransferases, CARM1 (coactivator-associated arginine methyltransferase 1, Ref. 21.Ananthanarayanan M. Li S. Balasubramaniyan N. Suchy F.J. Walsh M.J. J. Biol. Chem. 2004; 279: 54348-54357Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and PRMT1 (protein arginine methyltransferase1, Ref. 22.Rizzo G. Renga B. Antonelli E. Passeri D. Pellicciari R. Fiorucci S. Mol. Pharmacol. 2005; 68: 551-558Crossref PubMed Scopus (69) Google Scholar), and a transcriptional mediator, DRIP205, (23.Pineda Torra I. Freedman L.P. Garabedian M.J. J. Biol. Chem. 2004; 279: 36184-36191Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), have been shown to interact with FXR in vitro and coactivate FXR in cell-based reporter assays. Whether these cofactors identified from in vitro and cultured cell studies could regulate FXR activity in metabolic pathways in vivo needs to be established. The transcription cofactor p300 functions in diverse biological pathways, including differentiation, development, and proliferation (24.Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar, 25.Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.E. Newsome D. Bronson R.T. Li E. Livingston D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar) and expression of p300 is altered in human gastric, colorectal, and prostate carcinomas (26.Muraoka M. Konishi M. Kikuchi-Yanoshita R. Tanaka K. Shitara N. Chong J.M. Iwama T. Miyaki M. Oncogene. 1996; 12: 1565-1569PubMed Google Scholar). Mice lacking the p300 gene die at early mid-gestation, suggesting that p300 is critical for embryonic development and organogenesis (27.Shikama N. Lutz W. Kretzschmar R. Sauter N. Roth J.F. Marino S. Wittwer J. Scheidweiler A. Eckner R. EMBO J. 2003; 22: 5175-5185Crossref PubMed Scopus (158) Google Scholar). p300 is a histone acetyl transferase (HAT) that catalyzes the acetylation of lysine residues not only in nucleosomal histones, but also in non-histone proteins, such as nuclear receptors, cofactors, and basal transcription factors, resulting in enhanced gene transcription (28.Fu M. Rao M. Wang C. Sakamaki T. Wang J. Di Vizio D. Zhang X. Albanese C. Balk S. Chang C. Fan S. Rosen E. Palvimo J.J. Janne O.A. Muratoglu S. Avantaggiati M.L. Pestell R.G. Mol. Cell. Biol. 2003; 23: 8563-8575Crossref PubMed Scopus (227) Google Scholar, 29.Kraus W.L. Wong J. Eur. J. Biochem. 2002; 269: 2275-2283Crossref PubMed Scopus (64) Google Scholar). Despite its functions in diverse biological processes, a role for p300 in metabolic regulation has not been reported. Small heterodimer partner (SHP) is a well known FXR target and metabolic regulator (8.Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar, 9.Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Wilson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar). SHP is an unusual orphan nuclear receptor, which lacks a DNA binding domain but contains a putative ligand binding domain (30.Seol W. Choi H. Moore D.D. Science. 1996; 272: 1336-1339Crossref PubMed Scopus (446) Google Scholar). SHP interacts with and inhibits the activity of numerous nuclear receptors that are involved in regulation of diverse metabolic pathways (31.Lee Y. Moore D.D. J. Biol. Chem. 2002; 277: 2463-2467Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 32.Kemper J. Kim H. Miao J. Bhalla S. Bae Y. Mol. Cell. Biol. 2004; 24: 7707-7719Crossref PubMed Scopus (91) Google Scholar, 33.Bavner A. Sanyal S. Gustafsson J.A. Treuter E. Trends Endocrinol. Metab. 2005; 16: 478-488Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). We recently reported that bile acid-induced SHP inhibits transcription of its target genes, including CYP7A1, by coordinately recruiting chromatin-modifying cofactors, such as mSin3A/HDACs corepressors, G9a histone lysine methyltransferase, and Swi/Snf-Brm remodeling complex to the promoter, resulting in chromatin remodeling and histone modification (32.Kemper J. Kim H. Miao J. Bhalla S. Bae Y. Mol. Cell. Biol. 2004; 24: 7707-7719Crossref PubMed Scopus (91) Google Scholar, 34.Fang S. Miao J. Xiang L. Ponugoti B. Treuter E. Kemper J.K. Mol. Cell. Biol. 2007; 27: 1407-1424Crossref PubMed Scopus (83) Google Scholar). Marked alterations in cholesterol and bile acid levels in SHP-null mice have established a role for SHP in lipid homeostasis (35.Kerr T.A. Saeki S. Schneider M. Schaefer K. Berdy S. Redder T. Shan B. Russell D.W. Schwarz M. Dev. Cell. 2002; 2: 713-720Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 36.Wang L. Lee Y. Bundman D. Han Y. Thevananther S. Kim C. Chua S. Wei P. Heyman R. Karin M. Moore D. Dev. Cell. 2002; 2: 721-731Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Interestingly, chronically elevated expression of SHP has been shown to associate with development of fatty liver and related metabolic disorders (37.Boulias K. Katrakili N. Bamberg K. Underhill P. Greenfield A. Talianidis I. EMBO J. 2005; 24: 2624-2633Crossref PubMed Scopus (120) Google Scholar, 38.Huang J. Iqbal J. Saha P.K. Liu J. Chan L. Hussain M.M. Moore D.D. Wang L. Hepatology. 2007; 46: 147-157Crossref PubMed Scopus (121) Google Scholar, 39.Wang L. Huang J. Saha P. Kulkarni R.N. Hu M. Kim Y.D. Park K.G. Chan L. Rajan A.S. Lee I. Moore D.D. Mol. Endocrinol. 2006; 11: 2671-2681Crossref Scopus (38) Google Scholar). Despite the established function of SHP in maintaining cholesterol and bile acid levels in health and disease states, how SHP is induced by bile acid-activated FXR remains relatively unknown. From molecular, cellular, and mouse in vivo studies, we have obtained evidence indicating that p300 is critically involved in ligand-activated FXR signaling, particularly in SHP gene induction, by acetylating histones at the SHP promoter and FXR itself. Down-regulation of p300 substantially reduced SHP expression and further, altered expression of other hepatic FXR target genes, such that beneficial lipid and glucose profiles would be expected. We propose that inhibition of hepatic p300 activity may be beneficial for treating fatty liver disease and related metabolic disorders. Cell Culture—Human hepatoma HepG2 cells (ATCC HB8065) were grown in phenol red-free Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1). COS-1 cells were grown in DMEM media. Media were supplemented with 100 units/ml penicillin G-streptomycin sulfate and 10% heat-inactivated fetal bovine serum. For down-regulation of p300, HepG2 cells were infected with 5-25 MOI of Ad-empty or Ad-sip300, and 2 days later, cells were transfected with reporter and expression plasmids as indicated in the figure legends, and reporter assays were done. Mouse in Vivo Experiments—Eight-week-old male mice were maintained on a 12-h light and 12-h dark cycle. Cholic acid (CA) feeding was started at 5:00 PM to reduce variability between experiments, and food intake was monitored. For GW4064 experiments, mice were treated with GW4064 (2 mg/20 g mouse in 1% Tween 80 and 1% methylcellulose) or vehicle using oral gavage and 3 h later, livers were collected for further studies. For adenoviral experiments, mice were injected with about 0.5 × 109 active viral particles (Ad-empty, Ad-3Flag-FXR) in 200 μl of phosphate-buffered saline via the tail vein, and 4-7 days after infection, the mice were fed normal chow or chow supplemented with 0.5% CA for 3-24 h. All the animal use and adenoviral protocols were approved by the Institutional Animal Care and Use and Institutional Biosafety Committees at University of Illinois at Urbana-Champaign and were in accordance with National Institutes of Health guidelines. Plasmids and Adenoviral Vectors—Expression vectors for Gal4-FXR and 3Flag-FXR (40.Nakahara M. Furuya N. Takagaki K. Sugaya T. Hirota K. Fukamizu A. Kanda T. Fujii H. Sato R. J. Biol. Chem. 2005; 280: 42283-42289Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) were kindly provided by R. Sato, for p300 wild type and mutant (41.Lee Y. Koh S. Zhang X. Cheng X. Stallcup M. Mol. Cell. Biol. 2002; 22: 3621-3632Crossref PubMed Scopus (153) Google Scholar) by M. Stallcup, and for GCN5 wild type and mutant by P. Puigserver. Reporter plasmids, SHP promoter-luc (42.Lee Y. Dell H. Dowhan D.H. Hadzopoulou-Cladaras M. Moore D.D. Mol. Cell. Biol. 2000; 20: 187-195Crossref PubMed Scopus (265) Google Scholar) and FXRE-tk-luc (21.Ananthanarayanan M. Li S. Balasubramaniyan N. Suchy F.J. Walsh M.J. J. Biol. Chem. 2004; 279: 54348-54357Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), were provided by Y. Lee and M. Ananthanarayanan, respectively. An adenoviral vector expressing p300 siRNA (Ad-sip300) was kindly provided by Dr. P. Rotwein (44.Kuninger D. Stauffer D. Eftekhari S. Wilson E. Thayer M. Rotwein P. Hum. Gene Ther. 2004; 15: 1287-1292Crossref PubMed Scopus (23) Google Scholar). Adenoviral vectors were amplified, purified, and titered as described (34.Fang S. Miao J. Xiang L. Ponugoti B. Treuter E. Kemper J.K. Mol. Cell. Biol. 2007; 27: 1407-1424Crossref PubMed Scopus (83) Google Scholar, 45.Ponugoti B. Fang S. Kemper J.K. Mol. Endocrinol. 2007; 11: 2698-2712Crossref Scopus (42) Google Scholar). Real-time RTPCR—Total RNA was isolated using TRIzol reagent, and cDNA was synthesized and RTPCR was performed with an iCycler iQ (Bio-Rad). The amount of PCR product for each mRNA was normalized by dividing by the amount of 36B4 PCR product. Sequences of the primers are available upon request. In Vitro and in Cell Acetylation Assays—For in vitro acetylation assays, p300, CBP, pCAF, and GCN5, were purified from Sf9 insect cells infected with baculovirus encoding each of these proteins, as described (43.Thomas M.C. Chiang C.M. Mol. Cell. 2005; 17: 251-264Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). 1 μg of purified GST, GST-FXR, or core histones were incubated with each of the purified HATs in the presence of [3H]acetyl-CoA (0.25 μCi) in acetylation buffer (50 mm Hepes, pH 7.9, 10% glycerol, 1 μm GW4064). After incubation at 30 °C for 1 h, the proteins were separated by SDS-PAGE, proteins were detected by Coomassie Blue staining, and radioactivity was detected by fluorography. To detect acetylated FXR in cells, HepG2 or COS-1 cells were transfected with expression plasmids for p300 (or infected with Ad-p300 wild type), along with Ad-Flag-FXR (5 MOI). Cells were treated with histone deacetylase inhibitors such as 0.5 μm trichostatin A (TSA) and 5 mm nicotinamide (Nam), in the presence of 200 nm GW4064 for 5 h and collected for co-IP assays as described (46.Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar, 47.Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (570) Google Scholar, 48.Lerin C. Rodgers J.T. Kalume D.E. Kim S.H. Pandey A. Puigserver P. Cell Metab. 2006; 3: 429-438Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 49.Fu M. Liu M. Sauve A.A. Jiao X. Zhang X. Wu X. Powell M.J. Yang T. Gu W. Avantaggiati M.L. Pattabiraman N. Pestell T.G. Wang F. Quong A.A. Wang C. Pestell R.G. Mol. Cell. Biol. 2006; 26: 8122-8135Crossref PubMed Scopus (203) Google Scholar, 50.Lagouge M. Argmann C. Gerhart-Hines Z. Meziane H. Lerin C. Daussin F. Messadeq N. Milne J. Lambert P. Elliott P. Geny B. Laakso M. Puigserver P. Auwerx J. Cell. 2006; 127: 1109-1122Abstract Full Text Full Text PDF PubMed Scopus (3370) Google Scholar, 51.Baur J.A. Pearson K.J. Price N.L. Jamieson H.A. Lerin C. Kalra A. Prabhu V.V. Allard J.S. Lopez-Lluch G. Lewis K. Pistell P.J. Poosala S. Becker K.G. Boss O. Gwinn D. Wang M. Ramaswamy S. Fishbein K.W. Spencer R.G. Lakatta E.G. Le Couteur D. Shaw R.J. Navas P. Puigserver P. Ingram D.K. de Cabo R. Sinclair D.A. Nature. 2006; 444: 337-342Crossref PubMed Scopus (3704) Google Scholar). Briefly, 3× Flag-FXR was immunoprecipitated in post-translational modification (PTM) buffer (50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 10% glycerol, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, protease inhibitors, 1 μm TSA, 10 mm sodium butyrate, 10 mm Nam, 1 mm dithiothreitol, and phosphatase inhibitors) with 1 μg of either M2 antibody (Sigma, Inc) or goat FXR antibody (Santa Cruz Biotechnology, sc-1204) and immunoprecipitates were stringently washed with PTM buffer. Acetylated Flag-FXR in the immunoprecipitates was detected by Western blotting using acetyl lysine antibody (Cell Signaling, Inc). Membranes were stripped and Flag-FXR was detected by Western blotting using M2 or rabbit FXR (sc-13063) antibody. Coimmunoprecipitation (Co-IP) Assays—To examine protein-protein interactions in mouse liver, co-IP assays were performed essentially as described (32.Kemper J. Kim H. Miao J. Bhalla S. Bae Y. Mol. Cell. Biol. 2004; 24: 7707-7719Crossref PubMed Scopus (91) Google Scholar, 34.Fang S. Miao J. Xiang L. Ponugoti B. Treuter E. Kemper J.K. Mol. Cell. Biol. 2007; 27: 1407-1424Crossref PubMed Scopus (83) Google Scholar, 45.Ponugoti B. Fang S. Kemper J.K. Mol. Endocrinol. 2007; 11: 2698-2712Crossref Scopus (42) Google Scholar). Briefly, 0.25-0.5 mg of mouse liver nuclear extracts was incubated with 1 μg of p300 antibody (sc-584) at 4 °C overnight. The immune complex was isolated by incubation with protein G-agarose, and proteins in the immunoprecipitates were detected by Western blotting. Chromatin Immunoprecipitation (ChIP) Assays—ChIP assays in HepG2, normal mice, and FXR-null mice were carried out essentially as described (32.Kemper J. Kim H. Miao J. Bhalla S. Bae Y. Mol. Cell. Biol. 2004; 24: 7707-7719Crossref PubMed Scopus (91) Google Scholar, 34.Fang S. Miao J. Xiang L. Ponugoti B. Treuter E. Kemper J.K. Mol. Cell. Biol. 2007; 27: 1407-1424Crossref PubMed Scopus (83) Google Scholar, 45.Ponugoti B. Fang S. Kemper J.K. Mol. Endocrinol. 2007; 11: 2698-2712Crossref Scopus (42) Google Scholar, 52.Miao J. Fang S. Bae Y. Kemper J.K. J. Biol. Chem. 2006; 281: 14537-14546Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In re-ChIP assays, chromatin was first immunoprecipitated with antisera to FXR and then eluted with 100 μl of elution buffer with 10 mm dithiothreitol at 37 °C for 30 min and then, diluted (25-fold) with dilution buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 2 mm EDTA, 1% Triton X-100), and re-immunoprecipitated with IgG or p300 antibody. ChIP experiments were repeated at least three times with reproducible results. Sequences of the ChIP primers for mouse and human SHP promoters are available upon request. Cholic Acid Feeding Increases the Interaction of p300 with FXR in Mouse Liver—To determine if the p300 acetylase is involved in bile acid signaling in vivo, we first examined the effects of cholic acid (CA), a primary bile acid and natural FXR agonist, on p300 interaction with FXR in mouse liver. Mice were infected with Ad-Flag-FXR or control Ad-empty and then, fed normal chow or 0.5% CA-supplemented chow for 6 h. In Western analysis of liver extracts, Flag-FXR was not detected in the Ad-empty-infected mice and was present at similar levels in the two Ad-Flag-FXR groups (supplemental Fig. S1). p300 was immunoprecipitated from liver nuclear extracts, and Flag-FXR in the immunoprecipitates was detected by Western blotting. The levels of Flag-FXR were similar in input samples, but were substantially increased in the anti-p300 immunoprecipitates after CA feeding and not detected in the IgG control precipitates (Fig. 1A). We confirmed these results with endogenous FXR without overexpression of Flag-FXR. First, to confirm that CA feeding is operating in vivo, we measured the mRNA levels of Shp, a well known FXR target and metabolic corepressor, and Cyp7a1, a key bile acid biosynthetic enzyme and inhibited by Shp. The mRNA levels of Shp were significantly increased, whereas those of Cyp7a1 were decreased, after 6 h of CA feeding (Fig. 1B). Endogenous FXR was immunoprecipitated with FXR antisera or IgG and p300 was detected. While p300 was not detected in the IgG precipitates, the amount of p300 in the anti-FXR immunoprecipitates was increased by CA feeding (Fig. 1C). Endogenous p300 levels were not changed after 6 h of CA feeding (supplemental Fig. S2). These results indicate that CA feeding increases the interaction of p300 with FXR in mouse liver, suggesting that p300 may be involved in bile acid-activated FXR signaling in vivo. p300 Enhances FXR Transactivation—To determine whether the p300/FXR interaction is functionally relevant, we tested the effects of down-regulation of p300 on FXR activity. Infection with Ad-sip300 decreased the endogenous p300 mRNA levels by 80% in HepG2 cells (supplemental Fig. S3). It was shown that infection with this Ad-sip300 did not reduce mRNA levels of the acetylase CBP, which is highly conserved and functionally related with p300 (44.Kuninger D. Stauffer D. Eftekhari S. Wilson E. Thayer M. Rotwein P. Hum. Gene Ther. 2004; 15: 1287-1292Crossref PubMed Scopus (23) Google Scholar). Expression of FXR and RXRα activated the FXRE reporter in the presence of chenodeoxy cholic acid (CDCA), a primary bile acid, or GW4046, a synthetic FXR agonist (Fig. 2A, lanes 2, 9, 16). Infection with increasing amounts of Ad-sip300 progressively and significantly inhibited FXR transactivation (Fig. 2A, lanes 9-22). These results indicate that p300, at endogenous levels, functions as a coactivator of ligand-activated FXR. Because SHP is a well known direct FXR target, we also tested if p300 coactivates FXR transactivation of the natural SHP promoter. Treatment with GW4064 or CDCA substantially increased FXR activity on the SHP promoter (Fig. 2B, lanes 2, 9, 16). Ad-sip300 significantly reduced the FXR activity in a dosedependent manner, whereas Ad-empty had little effect (lanes 9-22). These results indicate that p300 functions as a coactivator for FXR transactivation of the SHP promoter. CA Feeding Induces Recruitment of p300 to the Shp Promoter in Mouse Liver—To further determine whether p300 is involved in ligand-activated FXR signaling, we examined whether p300 can be recruited to the SHP promoter after CA feeding. In ChIP assays, FXR was associated with the Shp promoter in livers of control mice, and CA feeding significantly increased FXR occupancy at the promoter (Fig. 3, A and B). CA feeding also substantially increased recruitment of p300 to the promoter (Fig. 3A). Consistent with p300 recruitment, acetylation of histone H3 at K9/K14, a gene activation histone mark, was increased. These effects were not observed for the control Gapdh sequence (Fig. 3A). To monitor temporal association of FXR and p300 with the Shp promoter, time course ChIP assays were done. To confirm that CA feeding effectively altered expression of the Shp gene, mRNA levels were monitored by q-RTPCR. The Shp mRNA levels were increased by about 2-fold as early as 6 h of CA feeding (supplemental Fig. S4). Association of FXR with the promoter was detected in livers of untreated mice and was increased by 6 h after CA feeding (Fig. 3C). p300 was recruited to the Shp promoter at 3 h of CA feeding and reached a maximum at 6 h. Consistent with p300 recruitment, acetylated histone H3 levels were increased after CA feeding (Fig. 3C). To determine whether the p300 recruitment was dependent on FXR, we performed ChIP assays in FXR-null mice and normal mice in parallel. It has been demonstrated that Shp expression was substantially reduced in FXR-null mice fed either control chow or CA-supplemented chow (7.Sinal C. Tohkin M. Miyata M. Ward J. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar). In FXR-null mice, association of FXR was not observed, as expected, and the association of p300, acetylated histones, and RNA polymerase II was also largely eliminated (Fig. 3D). These results demonstrate that recruitment of p300 to the Shp promoter is FXR-dependent. Interestingly, association of RXR was observed in wild type and FXR-null mice independent of CA feeding, suggesting that another nuclear receptor that can form a heterodimer with RXR was present at the promoter. Ligand-regulated FXR Induction of SHP in HepG2 Cells—The mechanism of p300-mediated coactivation of FXR signaling was studied in more detail in HepG2 cells. The effects of FXR ligands on the levels of acetylated histones at the SHP promoter were first studied to determine if FXR activation resulted in the same histone modification in HepG2 cells as in vivo. Acetylated histone H3 K9/K14 was detected in untreated cells, and the levels were increased after treatment with CDCA, and even more dramatically by treatment with GW4046 (Fig. 4A). Interestingly, co-treatment with the reported FXR antagonist, gugglesterone (53.Urizar N.L. Liverm