Gene selective regulation by hepatic Farnesoid x receptor (fxr) in health and disease (2011)

Metabolic syndrome is a clustered condition including obesity, insulin resistance, hypertension, dyslipidemia, and liver steatosis. The incidence of metabolic syndrome and the accompanying risk of developing cardiovascular disease and type II diabetes is dramatically increasing. Disrupted homeostasis of metabolites contributes to metabolic disease. Farnesoid X Receptor (FXR) which is known as a primary bile acid nuclear receptor plays important roles for maintaining levels of metabolites like triglycerides, fatty acids and glucose by regulating its targets genes in liver. Over the past decades, compelling studies establishing the function of FXR were performed using a synthetic FXR ligand, GW4064 and FXR null mice. These studies revealed that FXR inhibits triglyceride biosynthesis and glucose production to regulate glucose and lipid homeostasis. However, it is not known how FXR regulates its targets in metabolic diseases, like fatty livers associated with elevated triglyceride and glucose levels. As a ligand-dependent transcriptional regulator, FXR regulates numerous target genes by binding to FXR-responsive DNA elements (FXRE) with a heterodimer partner, RXRα, in response to bile acids or other physiological stimuli. Interestingly, our group reported that FXR is aberrantly acetylated in fatty liver induced by a high fat diet, and acetylation of FXR impaired its ability to form a heterodimer with RXRα or to bind to DNA. Taken together, these observations led us to ask how FXR maintains metabolic homeostasis by regulating its target genes in normal and disease state. Despite the known FXR functions in liver metabolism, whether FXR mediated transcriptional responses are altered in metabolic disease state is not known. To begin to address this question, I analyzed FXR regulation of miRNAs by gene array analysis and binding of FXR genome-wide by ChIP-seq studies.\n\nFXR regulates many target genes in the liver in different metabolic pathways. It is not clear how this is accomplished, but one possibility is that FXR regulates expression of miRNAs which are ideal candidates to regulate expression of multiple groups of genes. miRNA-mediated regulation by hepatic FXR were examined by miRNA-microarrays using livers of FXR null mice. miRNAs are small non-coding regulatory RNAs which act by binding to 3’-untranslated regions (UTRs) of target mRNAs to regulate expression of the gene products. In this study, the most significantly downregulated miRNA in FXR null mice was miR-34a, which was also shown to be abnormally expressed miRNA in human liver steatosis. FXR activation by bile acid treatment resulted in decreased miR-34a levels through induction of small heterodimer partner (SHP). miR-34a inhibits expression of SIRT1 which is a key metabolic regulator as well as the best known longevity molecule at the post-transcriptional level. Theses events link SIRT1 regulation by FXR to miR-34a inhibition in healthy liver. Interestingly, this molecular link was impaired in the metabolic diseased liver which has markedly elevated miR-34a levels and reduced SIRT1 levels. This study provides evidence for the regulation of the levels of SIRT1, a metabolic switch, by FXR through repression of miRNA expression in healthy liver. Furthermore, this finding supports the idea that activation of FXR by ligand treatment in diseased mice positively regulates glucose and lipid homeostasis by increasing hepatic SIRT1 levels via miR-34a suppression.\n\nIn liver disease states such as fatty liver, FXR is hyperacetylated and regulation of FXR target genes is altered. It is possible that modification of FXR may affect its binding to its target genes. To determine whether hepatic FXR differentially interacts with target binding sites in the metabolic disease state compared to healthy states, genomic FXR binding sites in normal and fatty livers were examined by ChIP-sequencing. The total number of FXR binding sites detected was 5,272 in fatty livers induced by high fat diet (HFD), compared to 15,263 in normal diet (ND) livers. The differences in number of binding sites may be partially explained by reduced FXR levels, reduced DNA binding of FXR by acetylation in the metabolic disease liver or by experimental variations including false positive signals. Most of the FXR binding sites were located in intergenic- or intronic region with an IR-1 motif (AGGTCA) in both diet groups. Interestingly, 7,440 or 2,344 unique FXR binding sites were identified in ND or HFD, respectively. Gene ontology (GO) annotated these unique FXR binding sites to groups of genes including Ca2+/K+ channels, S/T kinases, and lipid oxidation genes in the ND group and iron-sulfur binding proteins, Toll-like receptors, and oncogenic proteins in the HFD group. Randomly selected genes occupied by FXR uniquely in either ND or HFD were analyzed by standard ChIP and 90% of FXR binding sites detected in the ChIP-seq studies were validated in for ChIP analysis. In addition, altered gene expression was associated with FXR binding suggesting that FXR binding is functionally significant and that FXR regulates different sets of genes in ND or HFD mice. \n\nThe miRNA studies advance our understanding of how FXR regulates its target genes using miRNAs to maintain metabolic homeostasis for healthy liver and how the regulation is abnormal in metabolic disease states. In addition, genomic FXR binding analysis broadened our insight into the biological functions of FXR suggesting that FXR is highly involved not only in the liver metabolism but also in the inflammation, apoptosis, and liver cancer. Importantly, these findings may provide useful information for the development of therapeutic reagent treating liver diseases, metabolic syndrome.