The objective of this research is to define how modifications to chromatin and recruitment of coregulators associated with ligand-induced activation of FXR, a prototypical nuclear receptor for bile acids, regulate the expression of liver transport proteins in the normal liver and during cholestasis. We propose that epigenetic modification of chromatin into repressive or permissive configurations provides a precise mechanism for coordinating liver transporters that participate in bile formation.
In Aim 1 we will focus on how CARM 1 (coactivator-associated arginine methyl transferase) and SRC-2 (steroid receptor co-activator 2) and other coactivators are recruited to the nucleosome in response to bile acid-dependent activation of FXR, regulating the genes encoding BSEP (the Bile Salt Export Pump), the sodium taurocholate cotransporting polypeptide (Ntcp) and the organic solute transporter (OST1-OST2). Additional coactivators and histone modifications will be identified in HepG2 cells and in mouse liver using the complementary techniques of chromatin immunoprecipitation (ChIP) assays, in vivo ChIP assays and mass spectrometry. The integration, synergy and validity of identified components will be tested in liver cell transfection assays with promoters of FXR-regulated genes and by siRNA knockdown of individual coactivators and histone modifying enzymes. The effect of SRC- 2 loss on ligand-dependent activation and assembly of other coactivators at the promoters of FXR target genes will be studied in SRC-2 knockout mice.
Aim 2 will demonstrate the functional significance of FXR acetylation and deacetylation in the regulation of the genes encoding liver transporters. Our studies will test whether mutation of acetylation sites alters the subcellular distribution of GFP-labeled FXR, whether the protein abundance of FXR is decreased, whether its biochemical association with RXR1 and coactivators is altered.
Aim 3 will define if aberrant histone methylation and histone deacetylation in experimental cholestasis (induced in the mouse by bile duct ligation or injection of endotoxin) change chromatin structure, impair recruitment of coactivators, and contribute to dysregulation of transcription and progression of cholestasis. Alternatively or in addition, we predict that there will be active recruitment of potent repressor complexes composed of histone deacetylases and corepressor molecules to the promoters of most FXR-target genes. We will also determine how epigenetic modifications contribute to the marked induction of OST1-OST2, as a potential efflux mechanism to reduce bile acid injury in obstructive cholestasis. A customized PCR microarray will be used to obtain an unbiased view of changes in expression of histone modifying enzymes and coactivators that may enhance the cholestatic process or contribute to adaptive changes that may protect the hepatocyte. These studies will provide insight into an unexplored area of hepatic biology and pathobiology, and suggest potential new targets for drug development.

Public Health Relevance

The objective of this research is to define how modifications to chromatin and recruitment of coregulators associated with ligand-induced activation of FXR, a prototypical nuclear receptor for bile acids, regulate the expression of liver transport proteins in the normal liver and during cholestasis. Chromatin is a complex of proteins (histones) and DNA that is tightly bundled to fit into the nucleus and may be chemically modified to influence gene expression. This proposal will define how these modifications regulate the genes encoding liver transport proteins that contribute to bile secretion, excrete many xenobiotics, and are altered during cholestasis;these studies will provide insight into a poorly understood area of hepatic biology and pathobiology, and suggest potential new targets for drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK084434-28
Application #
8282880
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Sherker, Averell H
Project Start
1985-09-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
28
Fiscal Year
2012
Total Cost
$386,039
Indirect Cost
$129,302
Name
University of Colorado Denver
Department
Pediatrics
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Xu, Shuhua; Sun, An-Qiang; Suchy, Frederick J (2014) A novel RAR*/CAR-mediated mechanism for regulation of human organic solute transporter-* gene expression. Am J Physiol Gastrointest Liver Physiol 306:G154-62
Balasubramaniyan, Natarajan; Luo, Yuhuan; Sun, An-Qiang et al. (2013) SUMOylation of the farnesoid X receptor (FXR) regulates the expression of FXR target genes. J Biol Chem 288:13850-62
Sun, An-Qiang; Luo, Yuhuan; Backos, Donald S et al. (2013) Identification of functionally relevant lysine residues that modulate human farnesoid X receptor activation. Mol Pharmacol 83:1078-86
Balasubramaniyan, Natarajan; Ananthanarayanan, Meena; Suchy, Frederick J (2012) Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes. Am J Physiol Gastrointest Liver Physiol 302:G937-47
Ananthanarayanan, M; Li, Yanfeng; Surapureddi, S et al. (2011) Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis. Am J Physiol Gastrointest Liver Physiol 300:G771-81