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Hepatocyte Nuclear Factor 4 Alpha and Farnesoid X Receptor Co-regulates Gene Transcription in Mouse Livers on a Genome-Wide Scale

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Abstract

Purpose

Farnesoid X receptor (Fxr) is a ligand-activated nuclear receptor critical for liver function. Reports indicate that the functions of Fxr in the liver may overlap with those of hepatocyte nuclear factor 4α (Hnf4α), but studies of their precise genome-wide interaction to regulate gene transcription in the liver are lacking. Thus, we compared the genome-wide binding of Fxr and Hnf4α in the liver of mice and characterized their cooperative activity on binding to and activating target gene transcription.

Methods

Genome-wide ChIP-Seq data of Fxr and Hnf4α in mouse liver were analyzed by MACS, BEDTools, and DAVID. Co-immunoprecipitation, ChIP-qPCR, and luciferase assays were done to test for protein-protein interaction and cooperative binding.

Results

ChIP-seq analysis showed nearly 50% binding sites of Fxr and Hnf4α in mouse liver overlap and Hnf4α bound to shared target sites upstream and in close proximity to Fxr. Moreover, genes co-bound by Fxr and Hnf4α are enriched in complement and coagulation cascades and drug metabolism. A direct Fxr-Hnf4α protein interaction dependent on Fxr activity was detected and transcriptional assays suggest that Hnf4α can increase Fxr transcriptional activity. Conversely, binding assays showed Hnf4α can be either Fxr-dependent or -independent at different shared binding sites.

Conclusion

Our results showed that Fxr cooperates with Hnf4α in the liver to modulate gene transcription. This study provides the first evidence on a genome-wide scale of both cooperative and independent interactions between Fxr and Hnf4α in regulating gene transcription in the liver.

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Abbreviations

ApoC-III:

apolipoprotein C-III

CA:

cholic acid

ChIP-qPCR:

chromatin immunoprecipitation followed by quantitative polymerase chain reaction

ChIP-Seq:

chromatin immunoprecipitation followed by massively parallel sequencing

Co-IP:

co-immunoprecipitation

Cyp7a1:

cholesterol 7 alpha-hydroxylase

DR-1:

direct hexanucleotide repeat separated by 1 nucleotide

FXR/Fxr:

farnesoid X receptor

HNF4α/Hnf4α:

hepatocyte nuclear factor 4 alpha

IR-1:

inverted hexanucleotide repeat separated by 1 nucleotide

KO:

knockout

RXRα:

retinoid x receptor alpha

Shp:

small heterodimer partner

Sr-b1:

scavenger receptor class B type 1

TSS:

transcriptional start site

REFERENCES

  1. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, et al. Bile acids: natural ligands for an orphan nuclear receptor. Science. 1999;284:1365–8.

    Article  PubMed  CAS  Google Scholar 

  2. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, et al. Identification of a nuclear receptor for bile acids. Science. 1999;284:1362–5.

    Article  PubMed  CAS  Google Scholar 

  3. Wang H, Chen J, Hollister K, Sowers LC, Forman BM. Endogenous bile acids are ligands for the nuclear receptor Fxr/Bar. Mol Cell. 1999;3:543–53.

    Article  PubMed  CAS  Google Scholar 

  4. Seol W, Choi HS, Moore DD. Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors. Mol Endocrinol. 1995;9:72–85.

    Article  PubMed  CAS  Google Scholar 

  5. Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, et al. Targeted disruption of the nuclear receptor Fxr/Bar impairs bile acid and lipid homeostasis. Cell. 2000;102:731–44.

    Article  PubMed  CAS  Google Scholar 

  6. Kok T, Hulzebos CV, Wolters H, Havinga R, Agellon LB, et al. Enterohepatic circulation of bile salts in farnesoid X receptor-deficient mice. J Biol Chem. 2003;278:41930–7.

    Article  PubMed  CAS  Google Scholar 

  7. Chen WS, Manova K, Weinstein DC, Duncan SA, Plump AS, et al. Disruption of the Hnf-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos. Genes Dev. 1994;8:2466–77.

    Article  PubMed  CAS  Google Scholar 

  8. Inoue Y, Yu AM, Yim SH, Ma X, Krausz KW, et al. Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4alpha. J Lipid Res. 2006;47:215–27.

    Article  PubMed  CAS  Google Scholar 

  9. Inoue Y, Yu AM, Inoue J, Gonzalez FJ. Hepatocyte nuclear factor 4alpha is a central regulator of bile acid conjugation. J Biol Chem. 2004;279:2480–9.

    Article  PubMed  CAS  Google Scholar 

  10. Claudel T, Inoue Y, Barbier O, Duran-Sandoval D, Kosykh V, et al. Farnesoid X receptor agonists suppress hepatic apolipoprotein Ciii expression. Gastroenterology. 2003;125:544–55.

    Article  PubMed  CAS  Google Scholar 

  11. Shih DQ, Dansky HM, Fleisher M, Assmann G, Fajans SS, et al. Genotype/phenotype relationships in Hnf-4alpha/Mody1: haploinsufficiency is associated with reduced apolipoprotein (Aii), apolipoprotein (Ciii), lipoprotein (a), and triglyceride levels. Diabetes. 2000;49:832–7.

    Article  PubMed  CAS  Google Scholar 

  12. Stroup D, Chiang JY. Hnf4 and Coup-Tfii interact to modulate transcription of the cholesterol 7alpha-hydroxylase gene (Cyp7a1). J Lipid Res. 2000;41:1–11.

    PubMed  CAS  Google Scholar 

  13. Tirona RG, Lee W, Leake BF, Lan LB, Cline CB, et al. The orphan nuclear receptor Hnf4alpha determines Pxr- and car-mediated xenobiotic induction of Cyp3a4. Nat Med. 2003;9:220–4.

    Article  PubMed  CAS  Google Scholar 

  14. Zhang Y, Lee FY, Barrera G, Lee H, Vales C, et al. Activation of the nuclear receptor Fxr improves hyperglycemia and hyperlipidemia in diabetic mice. Proc Natl Acad Sci U S A. 2006;103:1006–11.

    Article  PubMed  CAS  Google Scholar 

  15. Watanabe M, Houten SM, Wang L, Moschetta A, Mangelsdorf DJ, et al. Bile acids lower triglyceride levels via a pathway involving Fxr, Shp, and Srebp-1c. J Clin Invest. 2004;113:1408–18.

    PubMed  CAS  Google Scholar 

  16. Staels B, Kuipers F. Bile acid sequestrants and the treatment of type 2 diabetes mellitus. Drugs. 2007;67:1383–92.

    Article  PubMed  CAS  Google Scholar 

  17. Cariou B, van Harmelen K, Duran-Sandoval D, van Dijk TH, Grefhorst A, et al. The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice. J Biol Chem. 2006;281:11039–49.

    Article  PubMed  CAS  Google Scholar 

  18. Ma K, Saha PK, Chan L, Moore DD. Farnesoid X receptor is essential for normal glucose homeostasis. J Clin Invest. 2006;116:1102–9.

    Article  PubMed  CAS  Google Scholar 

  19. Maran RR, Thomas A, Roth M, Sheng Z, Esterly N, et al. Fxr deficiency in mice leads to increased intestinal epithelial cell proliferation and tumor development. J Pharmacol Exp Ther. 2009;328:469-77.

    Google Scholar 

  20. Modica S, Murzilli S, Salvatore L, Schmidt DR, Moschetta A. Nuclear bile acid receptor Fxr protects against intestinal tumorigenesis. Cancer Res. 2008;68:9589–94.

    Article  PubMed  CAS  Google Scholar 

  21. Kim I, Morimura K, Shah Y, Yang Q, Ward JM, et al. Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice. Carcinogenesis. 2007;28:940–6.

    Article  PubMed  CAS  Google Scholar 

  22. Yang F, Huang X, Yi T, Yen Y, Moore DD, et al. Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor. Cancer Res. 2007;67:863–7.

    Article  PubMed  CAS  Google Scholar 

  23. Thomas AM, Hart SN, Kong B, Fang J, Zhong XB, et al. Genome-wide tissue-specific farnesoid X receptor binding in mouse liver and intestine. Hepatology. 2010;51:1410–9.

    Article  PubMed  CAS  Google Scholar 

  24. Chong HK, Infante AM, Seo Y-K, Jeon T-I, Zhang Y, et al. Genome-wide interrogation of hepatic Fxr reveals an asymmetric Ir-1 motif and synergy with Lrh-1. Nucleic Acids Res. 2010;38:6007–17.

    Article  PubMed  CAS  Google Scholar 

  25. Gonzalez FJ. Regulation of hepatocyte nuclear factor 4 alpha-mediated transcription. Drug Metab Pharmacokinet. 2008;23:2–7.

    Article  PubMed  CAS  Google Scholar 

  26. Maloney PR, Parks DJ, Haffner CD, Fivush AM, Chandra G, et al. Identification of a chemical tool for the orphan nuclear receptor Fxr. J Med Chem. 2000;43:2971–4.

    Article  PubMed  CAS  Google Scholar 

  27. Hayhurst GP, Lee YH, Lambert G, Ward JM, Gonzalez FJ. Hepatocyte nuclear factor 4alpha (nuclear receptor 2a1) is essential for maintenance of hepatic gene expression and lipid homeostasis. Mol Cell Biol. 2001;21:1393–403.

    Article  PubMed  CAS  Google Scholar 

  28. Schmidt D, Wilson MD, Ballester B, Schwalie PC, Brown GD, et al. Five-vertebrate Chip-Seq reveals the evolutionary dynamics of transcription factor binding. Science. 2010;328:1036–40.

    Article  PubMed  CAS  Google Scholar 

  29. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, et al. Model-based analysis of Chip-Seq (Macs). Genome Biol. 2008;9:R137.

    Article  PubMed  Google Scholar 

  30. Quinlan AR, Hall IM. Bedtools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26:841–2.

    Article  PubMed  CAS  Google Scholar 

  31. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, et al. The human genome browser at UCSC. Genome Res. 2002;12:996–1006.

    PubMed  CAS  Google Scholar 

  32. Dennis Jr G, Sherman BT, Hosack DA, Yang J, Gao W, et al. David: database for annotation, visualization, and integrated discovery. Genome Biol. 2003;4:P3.

    Article  PubMed  Google Scholar 

  33. Li G, Thomas AM, Hart SN, Zhong X, Wu D, et al. Farnesoid X receptor activation mediates head-to-tail chromatin looping in the Nr0b2 gene encoding small heterodimer partner. Mol Endocrinol. 2010;24:1404–12.

    Article  PubMed  CAS  Google Scholar 

  34. Li G, Thomas AM, Williams JA, Kong B, Liu J, et al. Farnesoid X receptor induces murine scavenger receptor class B type I via intron binding. PLoS One. 2012;7:e35895.

    Article  PubMed  CAS  Google Scholar 

  35. Williams JA, Thomas AM, Li G, Kong B, Zhan L, et al. Tissue specific induction of P62/Sqstm1 by farnesoid X receptor. PLoS One. 2012;7:e43961.

    Article  PubMed  CAS  Google Scholar 

  36. Huber RM, Murphy K, Miao B, Link JR, Cunningham MR, et al. Generation of multiple farnesoid-X-receptor isoforms through the use of alternative promoters. Gene. 2002;290:35–43.

    Article  PubMed  CAS  Google Scholar 

  37. Li T, Chiang JY. Rifampicin induction of Cyp3a4 requires pregnane X receptor cross talk with hepatocyte nuclear factor 4alpha and coactivators, and suppression of small heterodimer partner gene expression. Drug Metab Dispos. 2006;34:756–64.

    Article  PubMed  CAS  Google Scholar 

  38. Podvinec M, Kaufmann MR, Handschin C, Meyer UA. Nubiscan, an in silico approach for prediction of nuclear receptor response elements. Mol Endocrinol. 2002;16:1269–79.

    Article  PubMed  CAS  Google Scholar 

  39. Sladek FM, Zhong WM, Lai E, Darnell Jr JE. Liver-enriched transcription factor Hnf-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev. 1990;4:2353–65.

    Article  PubMed  CAS  Google Scholar 

  40. Bolotin E, Schnabl JM, Sladek FM, in Yusuf D et al. Hnf4a: the transcription factor encyclopedia. Genome Biol. 2012;13:R24.

    Google Scholar 

  41. Modica S, Gadaleta RM, Moschetta A. Deciphering the nuclear bile acid receptor Fxr paradigm. Nucl Recept Signal. 2010;8:e005.

    PubMed  CAS  Google Scholar 

  42. Hurtado A, Holmes KA, Ross-Innes CS, Schmidt D, Carroll JS. Foxa1 is a key determinant of estrogen receptor function and endocrine response. Nat Genet. 2011;43:27–33.

    Article  PubMed  CAS  Google Scholar 

  43. Lupien M, Eeckhoute J, Meyer CA, Wang Q, Zhang Y, et al. Foxa1 translates epigenetic signatures into enhancer-driven lineage-specific transcription. Cell. 2008;132:958–70.

    Article  PubMed  CAS  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

This work was supported by CPRIT Training Grants RP101502 to The University of Texas MD Anderson Cancer Center (AMT), DK031343 (GLG), DK090036 (GLG), and a Madison and Lila Self Graduate Fellowship from the University of Kansas (SNH).

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Authors and Affiliations

  1. Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, 66160, USA

    Ann M. Thomas, Steve N. Hart, Guodong Li, Hong Lu, Xiao-bo Zhong & Grace L. Guo

  2. Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, 77054, USA

    Ann M. Thomas

  3. Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota, 55905, USA

    Steve N. Hart

  4. Department of Abdominal Surgery, Cancer Treatment Center, Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150000, China

    Guodong Li

  5. Department of Pharmacology, Upstate Medical School, Syracuse, New York, 13210, USA

    Hong Lu

  6. Applied Bioinformatics Laboratory, Structural Biology Center, University of Kansas, Lawrence, Kansas, 66047, USA

    Yaping Fang & Jianwen Fang

  7. Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut, 06269, USA

    Xiao-bo Zhong

  8. Department of Pharmacology and Toxicology, School of Pharmacy, Rutgers University, 170 Frelinghuysen Rd./EOHSI Bldg. - Rm322, Piscataway, New Jersey, 08854, USA

    Grace L. Guo

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  1. Ann M. Thomas
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Correspondence to Grace L. Guo.

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Thomas, A.M., Hart, S.N., Li, G. et al. Hepatocyte Nuclear Factor 4 Alpha and Farnesoid X Receptor Co-regulates Gene Transcription in Mouse Livers on a Genome-Wide Scale. Pharm Res 30, 2188–2198 (2013). https://doi.org/10.1007/s11095-013-1006-7

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  • DOI: https://doi.org/10.1007/s11095-013-1006-7

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