Background

Retinoid x receptor α (RXRα) is abundantly expressed in the liver and is essential for the function of other nuclear receptors. Using chromatin immunoprecipitation sequencing and mRNA profiling data generated from wild type and RXRα-null mouse livers, the current study identifies the bona-fide hepatic RXRα targets and biological pathways. In addition, based on binding and motif analysis, the molecular mechanism by which RXRα regulates hepatic genes is elucidated in a high-throughput manner.

Principal findings

Close to 80% of hepatic expressed genes were bound by RXRα, while 16% were expressed in an RXRα-dependent manner. Motif analysis predicted direct repeat with a spacer of one nucleotide as the most prevalent RXRα binding site. Many of the 500 strongest binding motifs overlapped with the binding motif of specific protein 1. Biological functional analysis of RXRα-dependent genes revealed that hepatic RXRα deficiency mainly resulted in up-regulation of steroid and cholesterol biosynthesis-related genes and down-regulation of translation- as well as anti-apoptosis-related genes. Furthermore, RXRα bound to many genes that encode nuclear receptors and their cofactors suggesting the central role of RXRα in regulating nuclear receptor-mediated pathways.

Conclusions

This study establishes the relationship between RXRα DNA binding and hepatic gene expression. RXRα binds extensively to the mouse genome. However, DNA binding does not necessarily affect the basal mRNA level. In addition to metabolism, RXRα dictates the expression of genes that regulate RNA processing, translation, and protein folding illustrating the novel roles of hepatic RXRα in post-transcriptional regulation.

The current study tests the hypothesis that peroxisome proliferator-activated receptor β (PPARβ) has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. The role of PPARβ in liver regeneration was studied using two-third partial hepatectomy (PH) in Wild-type (WT) and PPARβ-null (KO) mice. In KO mice, liver regeneration was delayed and the number of Ki-67 positive cells reached the peak at 60 hr rather than at 36-48 hr after PH shown in WT mice. RNA-sequencing uncovered 1344 transcriptomes that were differentially expressed in regenerating WT and KO livers. About 70% of those differentially expressed genes involved in glycolysis and fatty acid synthesis pathways failed to induce during liver regeneration due to PPARβ deficiency. The delayed liver regeneration in KO mice was accompanied by lack of activation of phosphoinositide-dependent kinase 1 (PDK1)/Akt. In addition, cell proliferation-associated increase of genes encoding E2f transcription factor (E2f) 1-2 and E2f7-8 as well as their downstream target genes were not noted in KO livers 36-48 hr after PH. E2fs have dual roles in regulating metabolism and proliferation. Moreover, transient steatosis was only found in WT, but not in KO mice 36 hr after PH. These data suggested that PPARβ-regulated PDK1/Akt and E2f signaling that controls metabolism and proliferation is involved in the normal progression of liver regeneration.

Abstract Background The eyes and skin are obvious retinoid target organs. Vitamin A deficiency causes night blindness and retinoids are widely used to treat acne and psoriasis. However, more than 90% of total body retinol is stored in liver stellate cells. In addition, hepatocytes produce the largest amount of retinol binding protein and cellular retinoic acid binding protein to mobilize retinol from the hepatic storage pool and deliver retinol to its receptors, respectively. Furthermore, hepatocytes express the highest amount of retinoid x receptor alpha (RXRα) among all the cell types. Surprisingly, the function of endogenous retinoids in the liver has received very little attention. Results Based on the data generated from chromatin immunoprecipitation followed by sequencing, the global DNA binding of transcription factors including retinoid x receptor α (RXRα) along with its partners i.e. retinoic acid receptor α (RARα), pregnane x receptor (PXR), liver x receptor (LXR), farnesoid x receptor (FXR), and peroxisome proliferator-activated receptor α (PPARα) has been established. Based on the binding, functional annotation illustrated the role of those receptors in regulating hepatic lipid homeostasis. To correlate the DNA binding data with gene expression data, the expression patterns of 576 genes that regulate lipid homeostasis were studied in wild type and liver RXRα-null mice treated with and without RA. The data showed that RA treatment and RXRα-deficiency had opposite effects in regulating lipid homeostasis. A subset of genes (114), which could clearly differentiate the effect of ligand treatment and receptor deficiency, were selected for further functional analysis. The expression data suggested that RA treatment could produce unsaturated fatty acids and induce triglyceride breakdown, bile acid secretion, lipolysis, and retinoids elimination. In contrast, RXRα deficiency might induce the synthesis of saturated fatty acids, triglyceride, cholesterol, bile acids, and retinoids. In addition, DNA binding data indicated extensive cross-talk among RARα, PXR, LXR, FXR, and PPARα in regulating those RA/RXRα-dependent gene expression levels. Moreover, RA reduced serum cholesterol, triglyceride, and bile acid levels in mice. Conclusions We have characterized the role of hepatic RA for the first time. Hepatic RA mediated through RXRα and its partners regulates lipid homeostasis.

Background & aims

Farnesoid X receptor (FXR, NR1H4) is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq), with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.

Methods

In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs) treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq) and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.

Results

ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.

Conclusions

In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.