Bile acid hepatotoxicity: Epigenetics comes to the rescue
Authors: Frederick J. Suchy, Meenakshisundaram Ananthanarayanan
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. See Article on Page 220 It is well known that type II nuclear receptors such as farnesoid X receptor (FXR), pregnane X receptor, constitutive androstane receptor, and small heterodimer partner (SHP) regulate the biosynthesis, detoxification, and transport of bile acids and other cholephiles.1 Critically important in regulating bile acid concentrations within hepatocytes, SHP inhibits the genes encoding the enzymes controlling bile acid synthesis, CYP7a1 and CYP8b1. Also, SHP suppresses expression of the sodium taurocholate cotransporting polypeptide, which mediates bile acid uptake. These processes are central to normal bile acid homeostasis and assume even greater importance in coping with intracellular bile acid levels in cholestatic liver disease. The small heterodimer partner does not directly catalyze enzymatic modifications of histones or bind to DNA but instead coordinately recruits transcriptional modifiers of chromatin including histone deacetylases (HDACs), the G9a lysine methyltransferase, and the Swi/Snf chromatin‐remodeling complex. The G9a lysine methyltransferase suppresses transcription through mono‐ and dimethylation of histone H3K9. In the current issue of Hepatology, Kim et al. add to the complexity of SHP‐mediated transcriptional repression involved in fine‐tuning of bile acid homeostasis by linking demethylation of a histone activation mark with FXR‐dependent gene repression through the induction of SHP.2 They showed convincingly that FXR induces, with the help of SHP, the expression and recruitment of the lysine‐specific histone demethylase LSD1 to the CYP7a1 and CYP8b1 loci, leading to epigenetic repression of bile synthesis. Lysine (K)–specific demethylase 1 (LSD1) is a histone demethylase that demethylates both lysine 4 (H3K4me) and lysine 9 (H3K9me) of histone H3 NH2‐terminal tails, potentially acting as a coactivator or a corepressor, depending on the cellular context.3 First, they discovered functional FXR binding motifs, inverted repeat element 1, in the intron regions of the Lsd1 gene.2 They also found that pharmacological or physiological activation of FXR signaling induced expression of Lsd1 in mouse liver. Farnesoid X receptor–induced Lsd1 directly interacted with SHP and enhanced gene repression by SHP in reporter assays. Importantly, on chromatin immunoprecipitation analysis occupancy of LSD1 and SHP at the CYP7A1 and CYP8B1 loci was increased by treatment of HepG2 cells with chenodeoxycholic acid and associated with depletion of the H3K4‐me3 mark. Previously defined SHP‐dependent H3K9/K14 deacetylation and H3K9 methylation, which are repressive modifications, could also be related to the expression of LSD1.4 The Lsd1‐mediated repression of bile acid synthetic and uptake genes could be confirmed in vivo. Inhibition of the gene encoding the sodium taurocholate cotransporting polypeptide (Ntcp) was completely reversed by depletion of Lsd1, while inhibition of CYP7a1 and CYP8b1 was only partially reversed, indicating that in vivo pathways independent of Lsd1 contribute to the suppression of CYP7a1 and CYP8b1 by bile acids. The authors went on to show that adenoviral short hairpin RNA–mediated down‐regulation of Lsd1 increased hepatic bile levels and toxicity in mice after bile acid feeding. On chromatin immunoprecipitation assays, cholic acid feeding increased recruitment of SHP and Lsd1 and decreased H3K4‐me3 at the CYP7a1, CYP8b1, and Ntcp promoters. Overall, these studies show that LSD‐mediated regulation of CYP7a1, CYP8b1, and Ntcp in conjunction with FXR/SHP signaling provides a mechanism for feedback inhibition of bile synthesis and uptake in the liver. A recent study showed that Prox1 interacts with LSD1 to recruit the repressive LSD1/NuRD complex to the CYP7A1 promoter.5 In the article by Kim et al. down‐regulation of Prox1 partially inhibited recruitment of LSD1 and HDAC1 to the CYP7A1 promoter, whereas down‐regulation of SHP completely inhibited the recruitment, suggesting that both SHP and Prox1 contribute to recruitment of LSD1 and HDAC1 but that SHP is likely the dominant factor in LSD1 recruitment.2 Why does the expression of Lsd1 involving modification of only a single histone lysine residue have such a profound effect on bile acid homeostasis? The answer is not entirely clear, but inferences may be drawn from the known critically important effects of the H3K4 site on transcription. Histone posttranslational modifications primarily function in gene expression by providing points of recognition for transcriptional regulators that facilitate transcriptional activation or repression. Methylation of H3 lysine 4 by multiple methyltransferases of the mixed lineage leukemia family and the SET‐domain proteins correlates with active gene transcription.6 It is not understood why there are so many methyltransferases that target H3K4, but their role is likely context‐ and gene‐specific. Some, such as Set7/9, not only target H3K4 but also methylate nonhistone proteins such as FXR itself in an essential, ligand‐dependent regulatory step.7 Also, H3K4me3 can interact with RNA polymerase–associated proteins, with adenosine triphosphate–dependent chromatin remodelers, and recruit other proteins with histone‐modifying activity, particularly histone acetyltransferases and the histone demethylase JMJD2A.4 It could be easily accepted that the demethylation of H3K4 by LSD1 would abrogate transcription of select FXR target genes by solely inferring with the many interactions cited above. However, the scenario is more complex as the demethylated H3K4 has an additional role in suppressing transcription. Unmethylated H3K4 and DNA methylation are closely linked to form silent heterochromatin.9 Two related lysine‐specific demethylases, LSD1 and LSD2, whose substrates include H3K4me1/2, are absolutely essential in maintaining DNA methylation, which is commonly associated with transcriptional repression. The DNA methyltransferases DNMT3A and DNMT3B bind to H3K4 only in the unmethylated state through their ADD domains.10 Also, LSD1 is closely associated with recruitment of the NuRD complex, which includes histone deacetylase (HDAC1/HDAC2) and chromatin remodeling adenosine triphosphatase activities. The relative importance and possible redundancy of the various inhibitory pathways associated with the induction of SHP remain enigmatic and may be context‐specific. Although there may be sequential and parallel recruitment of chromatin modifiers, the central role of H3K4me3 in gene activation would indicate that Lsd1 and its associated activities are of great importance. It should be mentioned that there are other signaling pathways involved in SHP‐dependent inhibition of bile acid synthesis. Expression of CYP7A1 is negatively regulated by both the hepatic FXR/SHP and the intestinal FXR‐FGF15/19 pathways. The small heterodimer partner interacts with several transcription factors, including LRH‐1, to inhibit transcription of CYPA1 and CYP8B1.4 Fibroblast growth factors 15 and 19 bind to the fibroblast growth factor receptor 4/β‐Klotho complex in hepatocytes, activating both the c‐Jun N‐terminal kinase 1/2 and the extracellular signal–regulated kinase 1/2 signaling cascades. However, it remains unknown how epigenetic mechanisms, including the role of LSD1, are integrated in regulating bile acid homeostasis in health and disease. The authors mention the possibility of developing small molecules that specifically modulate LSD1 function for the treatment of hepatobiliary diseases. However, the challenges of such an approach will be considerable unless an agent could target only the FXR‐induced LSD1 recruited along with SHP to the promoters of bile acid synthetic and uptake genes. Nonspecific interference with the ubiquitous and important H3K4me3 site could be associated with undesirable side effects affecting many, if not most, genes. For example, up‐regulation of LSD1 would be expected to adversely affect the function of critical FXR‐dependent transporters including the bile salt export pump and the organic solute transporter OSTα‐OSTβ, leading to an exacerbation of cholestasis. The challenge will be to understand the complexities of epigenetic mechanisms and their interactions which are exemplified in this report.Figure 1: Lsd1 protects the liver from bile acid–induced injury. Bile acid feeding to mice led to activation of FXR/RXR that bound to inverted repeat elements (FXRE) within the introns of the Lsd1 gene, resulting in increased Lsd1 transcription. Lsd1 was recruited to the proximal promoters of Cyp7a1, Cyp8b1, and Ntcp mediated by Shp, leading to demethylation of H3K4me3, followed by deacetylation of H3K9/14 and methylation of H3K9, resulting in suppression of their expression. Thus, activation of Lsd1 protected the liver from bile acid–induced injury by down‐regulating the genes involved in bile acid synthesis and uptake. Silencing of Lsd1 expression using an adenoviral short hairpin RNA reversed these effects, resulting in a cholestatic phenotype (not shown). BA, bile acid; Cyp7a1, cholesterol 7α‐hydroxylase; Cyp8b1, sterol 12α‐hydroxylase; IR1, inverted repeat element 1; Lsd1, lysine‐specific demethylase 1; Ntcp, sodium taurocholate cotransporting polypeptide.