- Email: [email protected]
Epigenetic modifications as new targets for liver disease therapies
Clinical Application of Basic Science
Epigenetic modifications as new targets for liver disease therapies Müjdat Zeybel, Derek A. Mann, Jelena Mann⇑ Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
Summary An important discovery from the human genome mapping project was that it is comprised of a surprisingly low number of genes, with recent estimates suggesting they are as few as 25,000 [1]. This supported an alternative hypothesis that our complexity in comparison with lower order species is likely to be determined by regulatory mechanisms operating at levels above the fundamental DNA sequences of the genome [2]. One set of mechanisms that dictate tissue and cellular complexity can be described by the overarching term ‘‘epigenetics’’. In the 1940s, Conrad Waddington described epigenetics as ‘‘the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being’’. Today we understand epigenetics as a gene regulatory system comprised of 3 major mechanisms including DNA modifications (e.g., methylation), use of histone variants and post-translational modifications of the amino acid tails of histones and non-coding RNAs of which microRNAs are the best characterized [3,4]. Together, these mechanisms orchestrate numerous sets of chemical reactions that switch parts of the genome on and off at specific times and locations. Epigenetic marks, or the epigenome, exhibit a high degree of cellular-specificity and developmental or environmentally driven dynamic plasticity. Due to being at the interface between genome and the environment, the epigenome evolves at a very high rate compared to genetic mutations. Indeed, the differences in the epigenome account for most of the phenotypic uniqueness between closely related species, especially primates. More interestingly, the epigenetic changes, or epimutations, within an individual are not only maintained over cellular generations, but may also be transmitted between generations, such that adaptive epimutations generated in response to a particular environmental cue can influence phenotypes in our children and grandchildren [5]. Chromatin structure and non-coding RNAs Genomic DNA in the nucleus is not present in a naked form, but rather it is associated with small proteins called histones to make a structure termed chromatin. Both DNA and histones are subject Keywords: Epigenetic modifications; Liver disease; Chromatin; microRNA. Received 8 April 2013; received in revised form 23 May 2013; accepted 28 May 2013 ⇑ Corresponding author. Address: Institute of Cellular Medicine, Faculty of Medical Sciences, 4th Floor, William Leech Building, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. Tel.: +44 191 222 5902; fax: +44 191 222 0723. E-mail address: [email protected] (J. Mann).
to epigenetic regulation; DNA by attachment of methyl group to cytosines (DNA methylation) within a cytosine-phospho-guanine dinucleotide (CpG) and histones by attachment of a number of chemical groups to specific amino acid residues (histone modifications) [4,6]. These two major epigenetic mechanisms will be considered in further detail in the latter sections. Non-coding RNAs play an increasingly appreciated and crucial role in epigenetic regulation. Broadly, non-coding RNAs can be divided into long and short species, both of which play prominent but very separate roles in gene regulation. It is now known that only 1% of the mammalian genome codes for proteins, however 70–90% of the entire genome is transcribed at some point during the lifetime to produce a large transcriptome of long non-coding RNAs (lncRNAs) [7,8]. They are a class of messenger RNA-like transcripts that lack any discernible open reading frame [7]. Long ncRNAs are often poorly conserved, characterized by low copy number, fast turnover rates as well as very long length, ranging from 200 nucleotides up to 100 kilobases, sometimes encompassing a whole chromosome [9]. Due to their length, these RNA species offer the possibility of allele-specific action and targeting of a single location within the genome. This is in contrast to transcription factors or small non-coding RNAs that bind a short DNA sequence which may be repeated numerous times throughout the genome [7]. Although function of many lncRNAs is yet to be illuminated, it is now clear that at least some lncRNAs play a role in processes that lead both to gene activation and repression. This is at least in part achieved by the ability of lncRNAs to guide the catalytic function of chromatin-modifying enzymes to specific sites in the genome, and these complexes then act on chromatin modifications and structure [7,10]. Currently, there are no lncRNAs with assigned role in liver disease; however, there have been excellent examples within other disease contexts, such as MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), which is a highly conserved lncRNA that regulates metastasis development in lung cancer by activating expression of metastasis-associated genes [11]. Latest studies have shown that antisense oligonucleotides can be used to silence MALAT1 and thus prevent metastatic disease [11,12]. It will be exciting to learn the potential roles of lncRNAs in liver disease and to find out whether similar antisense silencing regimes may have a therapeutic impact. MicroRNAs (miR) are a class of short (20–24 nucleotide) noncoding RNAs that regulate gene expression at a post-transcriptional level, through binding to the 30 -untranslated region of targeted messenger RNA and causing its degradation or destabilization; alternatively miRs can also effect translational
Journal of Hepatology 2013 vol. 59 j 1349–1353
Clinical Application of Basic Science repression [13]. The observed outcome is a modest change in gene expression, widely defined as a ‘‘fine-tuning effect’’ [14].
Nucleus
DNA methylation Me
microRNAs and liver disease: intriguing transformation from basic science into clinics In humans, over 1500 miRs have been identified which in turn target hundreds of genes within numerous biological pathways including liver development, regeneration, and fibrosis. miRNA profiling of distinctive liver diseases including chronic hepatitis C and B, NAFLD (nonalcoholic fatty liver disease), ALD (alcoholic liver disease), and drug-induced liver injury have revealed miRs that are now becoming novel therapeutic targets in various clinical conditions [15,16]. One of the best examples of such new developments is the discovery of miR-122 role in hepatitis C [17]. miR-122 was shown to facilitate HCV replication by binding to multiple sites in the 50 untranslated region of HCV RNA genome and enhances translation of the viral proteins and protect HCV RNA against nucleolytic degradation. In vivo targeting of miR122 was initially tested in chimpanzees where 12-week intravenous delivery of locked-nucleic-acid-[LNA]-modified complementary oligonucleotide led to depletion of miR-122 and durable reduction in HCV RNA levels as well as lower serum cholesterol levels [19]. Phase I and IIa human trials with Miravirsen (LNAmodified-antimiR-122) have also revealed good safety profiles and tolerability with prolonged reduction in HCV RNA following 18 weeks of treatment [20]. In addition, miR-122 is a mediator of insulin, lipid metabolism, and iron homeostasis. Antagonism of miR-122 reduces cholesterol and LDL levels through affecting hepatic cholesterol and fatty acid biosynthesis genes while inducing b oxidation of fatty acids as well as causing iron deficiency with lower levels of plasma and liver iron [18]. As such, antagonising miR-122 may have multiple beneficial outcomes. While antagonism of miR-122 shows very encouraging results, two remarkable papers, using germline and liver-specific knockout mice, highlighted some unexpected roles of miR-122 in the liver. Mice with miR-122 deletions had reduced serum cholesterol levels similar to the pharmacological inhibition of miR122, however, they developed microsteatosis with triglyceride accumulation, steatohepatitis with inflammatory cell recruitment, and elevated pro-inflammatory mediators (IL-6, TNF-a) [21]. Liver fibrosis, via the activation of KLF6 axis, and hepatocellular carcinoma-like tumours were also evident in this phenotype, indicating tumour suppressor properties of miR-122 [22]. With our current knowledge, the reason for the biological distinction between the temporary pharmacological inhibition and the global deletion of miR-122 is not yet known. However, it is likely that our current understanding of the mechanisms by which microRNAs operate in these systems is rather one-dimensional. With further research and improved insight into the ways in which various epigenetic regulatory aspects integrate with one another, we might discover means of manipulating homeostasis as well as disease states. Histone modifications and liver disease: still on the bench As already stated, DNA inside the nuclei is complexed with histones, forming a structure termed chromatin. The main histone proteins are called histone H2A, histone H2B, histone H3, and histone H4 [4,6]. The smallest unit of chromatin is a mononucleosome, which consists of 146 base pairs of genomic DNA 1350
Me
Me
Histone modifications Me
Me
Ac
Ac Me
Me
Cytoplasm
Non-coding RNAs
Fig. 1. Epigenetic mechanisms that regulate gene expression. The mammalian genome is packaged in the nucleus via wrapping of DNA around histones, forming a structure known as chromatin. The on and off state of gene expression is regulated by DNA methylation, post-translational modifications of various residues within histone tails, and non-coding RNAs.
sequence which is wrapped around an octamer of histones- two copies of each of the histones H2A, H2B, H3, and H4 (Fig. 1). Histones are small, globular proteins with long ’tails’ on one end of the amino acid structure, this being the location of post-translational modifications carried out by numerous enzymes. There are in excess of 60 sites within each octamer of histones that are subject to chemical modification. Modifications include methylation, citrullination, acetylation, phosphorylation, SUMOylation, ubiquitination, and ADP-ribosylation. Combinations of various chemical groups are attached to the histone tails which then form binding sites for protein complexes that are able to affect transcriptional activity of a particular gene. Histone modifications are also able to induce conformational changes in chromatin such to promote transcription or silencing of gene expression in varying degrees. Both of these processes depend on the site, type, and number of chemical groups attached to the histone tails [4,6,23]. As there are strings of histone octamers at every 146 base pairs of DNA sequence along the gene, and each one can have a particular set of modifications at one of over 60 sites, the possible combinations that can be achieved are huge [4]. Each combination of histone marks potentially provides a specific recognition site for a protein complex that can alter chromatin structure. In that sense, the histone modifications provide a complex blueprint that regulates transcriptional activity, which is also referred to as the histone code [4]. In terms of a functional outcome, histone acetylation almost invariably underpins induction of gene transcription; however, methylation of lysine residues within the histone tail can either stimulate (histone 3 lysine 4) or inhibit gene expression (histone
Journal of Hepatology 2013 vol. 59 j 1349–1353
JOURNAL OF HEPATOLOGY HDAC inhibitors
HAT inhibitors
● Vorinostat ● Romidepsin ● Valproic acid ● Trichostatin A
● Anacardic acid ● Curcumin ● CPHT2
Ac Ac Ac
Ac
Ac
Ac
HAT HDAC
ical trials links under Refs. 26,27]. In vitro and in vivo studies suggest that HDACs are considerably upregulated in chronic liver disease; this led to research using HDAC inhibitors (HDACi) to block myofibroblast generation and fibrogenesis. Laboratory evidence suggests that various HDACi attenuate the activation of hepatic stellate cells and fibrosis in animal models. However, the mechanism of action of the HDACi was not clearly understood and, of note, it is now known that HDACs and their inhibitors have effects on non-histone proteins such as cytoskeleton proteins. For HDACs to be fully exploited for therapeutic purposes, it will be necessary to have a much deeper understanding of their activities and substrates. In addition, we will need histone modifiers with greater specificity; two examples of this approach were recently reported using highly selective inhibitors of EZH2 histone methyltransferase and jumonji H3K27 histone demethylase, which demonstrated antitumorigenic activity in lymphoma cells and modulated macrophage response, respectively [28,29].
KDM KMT Me Me Me
DNA methylation, methyl-binding proteins and liver disease: astonishing roles – prepare for lift off
Me
KMT inhibitors-targets ● DZNep-H3K27me ● BIX-01294-H3K9me ● PDB4e47-H3K4me ● Chaetocin-SUV39H1
Me
Me
KDM inhibitors ● Phenelzine ● Pargyline hydrochloride ● GSK_J1 ● Tranylcypromine
Fig. 2. Enzymes that control histone lysine methylation and acetylation are targets for numerous drugs that are being developed or already used clinically. HDAC, histone deacetylase; HAT, histone acetyltransferase; KMT, histone lysine methyltransferas; KDM, histone lysine demethylase.
3 lysine 9 or 27 methylation) (please see Fig. 2 for a list of compounds that target histone methylation and acetylation). The acetylation of histones is carried out by two groups of enzymes, histone acetyl transferases (HAT) and histone deacetylases (HDAC) (Fig. 2). Among histone modifying enzymes, HDACs have received most attention. The role of HDAC3 in circadian regulation, hepatic energy metabolism, and hepatosteatosis has now been clearly demonstrated [24]. HDAC3 negatively regulates lipogenic genes and directs metabolic sources towards gluconeogenesis to maintain normal blood glucose during daytime. In contrast, HDAC3 is significantly downregulated at night, which stimulates lipid synthesis. Interestingly, liver-specific knockdown of HDAC3 leads to the development of marked steatosis, with triglyceride accumulation and ‘‘insulin hypersensitivity’’. Similarly, alcohol-induced microsteatosis is associated with reduced HDAC3 expression. Furthermore, alcohol and its metabolite acetate induce acetylation of histones globally in macrophages and hepatocytes, particularly in promoters of pro-inflammatory cytokines [25]. Pharmacological targeting of histone modifying enzymes, particularly inhibitors of HDACs, constitutes a rapidly growing component of the epigenetic drug market. Two HDAC inhibitors are currently licensed for use in haematological malignancies and similar drugs are in clinical trials for various hematogenous malignancies and solid organ tumors (Fig. 2) [please refer to clin-
Methylation of DNA occurs at the 5th carbon in the pyrimidine ring of cytosine nucleotides and most commonly in cytosine phosphoguanine dinucleotides, often referred to as CpGs. Large (longer than 200 bp) and dense (>55%) clusters of CpG sequences are termed CpG islands. Many human gene promoters contain clusters of CpG islands in the unmethylated state; in contrast, most non-promoter CpGs are methylated [30]. The functional role of DNA methylation is relatively complicated, but generally speaking, methylation within the promoter regions ordinarily leads to transcriptional repression, whereas methylation within gene body can exert transcriptional activation. Two main mechanisms have been described for DNA methylation-mediated longterm gene silencing. Methylated DNA can either repress gene expression by recruiting methyl-binding proteins (MeCP2, MBD1–4) that in turn interact with chromatin silencing protein complexes, or it may directly interfere with the binding of transcription factors to their DNA sequence recognition motifs [30]. Methylation of DNA is executed by enzymes known as DNA methyltransferases (Dnmt) using S-adenosyl methionine as the methyl donor [30]. It is not clear how DNA methylation is targeted to a particular region, but it is likely to involve interaction of DNMTs with other chromatin associated factors [30]. The transdifferentiation of hepatic stellate cells (HSC) into a myofibroblast-like phenotype is a pivotal event in hepatic wound-healing and fibrosis. The dramatic change in phenotype associated with HSC transdifferentiation is underpinned by a global change in gene expression. DNA methylation exists in a number of genes that are expressed at a high level in quiescent HSCs, which are then silenced as the cells activate. At present it is unclear as to whether the DNA methylation pattern of the HSC genome alters with transactivation, however, there is evidence that the way in which the cell ‘‘reads’’ the so-called epigenome does change [31]. As HSCs transdifferentiate, they begin to express MeCP2, a DNA methyl-binding protein that is recruited to methyl-CpG marks present on the DNA and upon binding can subsequently recruit chromatin remodeling complexes that silence gene transcription. This type of gene silencing mechanism occurs at the IjBa and PPAR-c genes in activated HSCs, which is required for these cells to acquire their inflammatory and
Journal of Hepatology 2013 vol. 59 j 1349–1353
1351
Clinical Application of Basic Science fibrogenic phenotype [31,32]. In addition to its repressive action, MeCP2 can also act as a transcriptional activator, although the mechanisms by which it achieves this apparently paradoxical effect are not yet clear. A recent report suggested that modification of methyl-CpG to hydroxymethyl-CpG enables MeCP2 to function as a stimulator of transcription [33]. We have previously reported that MeCP2 enhances the expression of other histone modifying enzymes such as the histone methyltransferases EZH2 and ASH1 [31,34]. These enzymes in turn regulate the attachment of methyl groups to histone H3 lysine 27 (EZH2) and histone H3 lysine 4 (ASH1). Because H3K27 methylation is a transcriptionally repressive mark, EZH2 therefore regulates silencing of target genes, such as PPAR-c [31]. In contrast, methylation of lysine 4 of histone H3 is a signature of actively transcribed genes [6]. During HSC transdifferentiation, ASH1 is recruited to the regulatory regions of the collagen 1A1, TIMP1 and TGFb1 where it facilitates their transcription [34]. Taken together, MeCP2 acts as a pivotal regulator of HSC activation, and it is worth noting that MeCP2 gene deletion or drug mediated EZH2-inhibition attenuates hepatic fibrosis. Rosemarinic acid and baicalin (active ingredient of Yang-Gan-Wan, Chinese herbal preparation) also repress MeCP2-EZH2 relay, thus allowing re-expression of PPAR-c, which in turn prevents hepatic fibrosis [35]. DNA methylation, MeCP2, EZH2, and ASH1 may be bona fide therapeutic targets for prevention and/or treatment of liver fibrosis. It is very well worth noting the manner in which DNA methylation, methyl binding proteins and enzymes that control histone modifications are involved in crosstalk, and it will be crucial to understand these interactions in order to develop drugs that will target these pathways.
to injurious stimuli to allow rapid adaptation to limit the fibrogenic response and preserve normal liver architecture. It also raises the exciting possibility of epigenetic screening of liver patients for prognosis of disease progression.
Conclusions Complex genetic and environmental interactions give rise to the phenotype of an individual. This phenotype is then subject to lifelong change through remodeling of the individual’s epigenome. The underlying epigenetic mechanisms can also impact on the course of diseases or chronic conditions that the individual may suffer, such that they can restrict or promote their progression. The challenge now lies in fully understanding the epigenetic mechanisms and their interaction, which is a tall order due to the complexity of the system. With much improved knowledge, we might be able to selectively modulate epigenome through the use of drugs, alteration in diet, or applying other types of interventions in order to alter the course of disease and ageing.
Key Points Clinical application of epigenetics in Hepatology •
May be used as biomarkers of liver injury or fibrosis from blood/plasma-based on necrotic or apoptotic cells released from injured liver, free microRNA released from cells or exosome associated microRNA
•
May be used as prognostic indicators of disease activity from liver/blood - tight regulation compared to gene expression changes, which may solve sample variability
•
May predict treatment response
•
Can provide new avenues for therapeutic applicationmicroRNA inhibition by oligonucleotide inhibitors or microRNA delivery/potentiating the effect by miRNA mimics, inhibitors of histone modifying enzymes, DNMT inhibitors
Epigenetic marks are heritable by future generations Genetic studies exploring the association of single nucleotide polymorphisms and chronic liver diseases created fundamental data regarding the variability of disease development (e.g., NAFLD and PNPLA3) and treatment responses (e.g., hepatitis C and IL28B). However, there remains a significant gap in our understanding of liver patho-physiology. Liver fibrosis is a final common pathway of liver injury irrespective of aetiology, however, it is currently not clear if predisposition to development of fibrosis exists or indeed if existence of disease in previous generations alters such predisposition. We recently described the potential for transgenerational epigenetic transmission of susceptibility traits for liver fibrosis in male rats [5]. Offspring from a lineage of two consecutive generations of ancestors that suffered liver fibrosis, upon injury with the hepatotoxin carbon tetrachloride, developed less fibrosis compared with rats that had no ancestral history of liver disease. The adapted response was characterized by reduced numbers of myofibroblastic HSC, reduced expression of profibrogenic TGFb1, and elevated expression of antifibrogenic PPAR-c and PPAR-a. Interrogation of DNA methylation at the PPAR-c and PPAR-a promoters revealed that ancestral liver disease leads to hypomethylation of these regulatory regions in the livers of offspring. Intriguingly, in a NAFLD cohort, patients with severe fibrosis or cirrhosis had higher levels of PPAR-c promoter DNA methylation as compared to patients with mild fibrosis [5]. This study therefore provides evidence of heritable epigenetic signatures that can be modified in response
1352
Conflict of interest DAM consults for and is in receipt of grant funding from GlaxoSmithKline. References [1] Available at: http://www.ornl.gov/sci/techresources/Human_Genome/project/about.shtml. [2] Turner BM. Defining an epigenetic code. Nat Cell Biol 2007;9:2–6. [3] Costa FF. Non-coding RNAs, epigenetics and complexity. Gene 2008;410: 9–17. [4] Kouzarides T. Chromatin modifications and their function. Cell 2007;128:693–705. [5] Zeybel M, Hardy T, Wong YK, Mathers JC, Fox CR, Gackowska A, et al. Multigenerational epigenetic adaptation of the hepatic wound-healing response. Nat Med 2012:1369–1377, [Erratum in: Nat Med 2012;18(10):159]. [6] Richards EJ, Elgin SC. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 2002;108:489–500.
Journal of Hepatology 2013 vol. 59 j 1349–1353
JOURNAL OF HEPATOLOGY [7] Mercer TR, Mattick JS. Structure and function of long noncoding RNAs in epigenetic regulation. Nat Struct Mol Biol 2013;20:300–307. [8] Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, et al. The transcriptional landscape of the mammalian genome. Science 2005;309: 1559–1563, [Erratum in: Science 2006;311:1713]. [9] Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell 2009;136:629–641. [10] Lee JT. Epigenetic regulation by long noncoding RNAs. Science 2012;338: 1435–1439. [11] Gutschner T, Hämmerle M, Eissmann M, Hsu J, Kim Y, Hung G, et al. The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res 2013;73:1180–1189. [12] Gutschner T, Diederichs S. The hallmarks of cancer: a long non-coding RNA point of view. RNA Biol 2012;9:703–719. [13] He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 2004;5:522–531. [14] Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature 2008;455:64–71. [15] Rottiers V, Näär AM. MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol 2012;13:239–250, [Review]. [16] Szabo G, Sarnow P, Bala S. MicroRNA silencing and the development of novel therapies for liver disease. J Hepatol 2012;57:462–466. [17] Machlin ES, Sarnow P, Sagan SM. Combating hepatitis C virus by targeting microRNA-122 using locked nucleic acids. Curr Gene Ther 2012;12:301–306. [18] Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 2010;327:198–201. [19] Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, Patel K, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med 2013, [Epub ahead of print] PubMed PMID: 23534542. [20] Castoldi M, Vujic Spasic M, Altamura S, Elmén J, Lindow M, Kiss J, et al. The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice. J Clin Invest 2011;121:1386–1396. [21] Hsu SH, Wang B, Kota J, Yu J, Costinean S, Kutay H, et al. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest 2012;122:2871–2883. [22] Tsai WC, Hsu SD, Hsu CS, Lai TC, Chen SJ, Shen R, et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J Clin Invest 2012;122:2884–2897.
[23] Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:1074–1080. [24] Feng D, Liu T, Sun Z, Bugge A, Mullican SE, Alenghat T, et al. A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism. Science 2011;331:1315–1319. [25] Sun Z, Miller RA, Patel RT, Chen J, Dhir R, Wang H, et al. Hepatic Hdac3 promotes gluconeogenesis by repressing lipid synthesis and sequestration. Nat Med 2012;18:934–942. [26] Clinical trials for HDAC inhibitors: Available at: http://www.clinicaltrials.gov/ct2/show/NCT01486277, http://www.clinicaltrials.gov/ct2/show/ NCT01344707, http://www.clinicaltrials.gov/ct2/show/NCT00686218. [27] Burridge S. Target watch: drugging the epigenome. Nat Rev Drug Discov 2013;12:92–93. [28] Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus, et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol 2012;8:890–896. [29] Kruidenier L, Chung CW, Cheng Z, Liddle J, Che K, Joberty G, et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 2012;488:404–408. [30] Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16:6–21. [31] Mann J, Chu DC, Maxwell A, Oakley F, Zhu NL, Tsukamoto H, et al. MeCP2 controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. Gastroenterology 2010;138:705–714. [32] Mann J, Oakley F, Akiboye F, Elsharkawy A, Thorne AW, Mann DA. Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis. Cell Death Differ 2007;14:275–285. [33] Mellén M, Ayata P, Dewell S, Kriaucionis S, Heintz N. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell 2012;151:1417–1430. [34] Perugorria MJ, Wilson CL, Zeybel M, Walsh M, Amin S, Robinson S, et al. Histone methyltransferase ASH1 orchestrates fibrogenic gene transcription during myofibroblast transdifferentiation. Hepatology 2012;56: 1129–1139. [35] Yang MD, Chiang YM, Higashiyama R, Asahina K, Mann DA, Mann J, et al. Rosmarinic acid and baicalin epigenetically derepress peroxisomal proliferator-activated receptor c in hepatic stellate cells for their antifibrotic effect. Hepatology 2012;55:1271–1281.
Journal of Hepatology 2013 vol. 59 j 1349–1353
1353
Epigenetic modifications as new targets for liver disease therapies Müjdat Zeybel, Derek A. Mann, Jelena Mann⇑ Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
Summary An important discovery from the human genome mapping project was that it is comprised of a surprisingly low number of genes, with recent estimates suggesting they are as few as 25,000 [1]. This supported an alternative hypothesis that our complexity in comparison with lower order species is likely to be determined by regulatory mechanisms operating at levels above the fundamental DNA sequences of the genome [2]. One set of mechanisms that dictate tissue and cellular complexity can be described by the overarching term ‘‘epigenetics’’. In the 1940s, Conrad Waddington described epigenetics as ‘‘the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being’’. Today we understand epigenetics as a gene regulatory system comprised of 3 major mechanisms including DNA modifications (e.g., methylation), use of histone variants and post-translational modifications of the amino acid tails of histones and non-coding RNAs of which microRNAs are the best characterized [3,4]. Together, these mechanisms orchestrate numerous sets of chemical reactions that switch parts of the genome on and off at specific times and locations. Epigenetic marks, or the epigenome, exhibit a high degree of cellular-specificity and developmental or environmentally driven dynamic plasticity. Due to being at the interface between genome and the environment, the epigenome evolves at a very high rate compared to genetic mutations. Indeed, the differences in the epigenome account for most of the phenotypic uniqueness between closely related species, especially primates. More interestingly, the epigenetic changes, or epimutations, within an individual are not only maintained over cellular generations, but may also be transmitted between generations, such that adaptive epimutations generated in response to a particular environmental cue can influence phenotypes in our children and grandchildren [5]. Chromatin structure and non-coding RNAs Genomic DNA in the nucleus is not present in a naked form, but rather it is associated with small proteins called histones to make a structure termed chromatin. Both DNA and histones are subject Keywords: Epigenetic modifications; Liver disease; Chromatin; microRNA. Received 8 April 2013; received in revised form 23 May 2013; accepted 28 May 2013 ⇑ Corresponding author. Address: Institute of Cellular Medicine, Faculty of Medical Sciences, 4th Floor, William Leech Building, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. Tel.: +44 191 222 5902; fax: +44 191 222 0723. E-mail address: [email protected] (J. Mann).
to epigenetic regulation; DNA by attachment of methyl group to cytosines (DNA methylation) within a cytosine-phospho-guanine dinucleotide (CpG) and histones by attachment of a number of chemical groups to specific amino acid residues (histone modifications) [4,6]. These two major epigenetic mechanisms will be considered in further detail in the latter sections. Non-coding RNAs play an increasingly appreciated and crucial role in epigenetic regulation. Broadly, non-coding RNAs can be divided into long and short species, both of which play prominent but very separate roles in gene regulation. It is now known that only 1% of the mammalian genome codes for proteins, however 70–90% of the entire genome is transcribed at some point during the lifetime to produce a large transcriptome of long non-coding RNAs (lncRNAs) [7,8]. They are a class of messenger RNA-like transcripts that lack any discernible open reading frame [7]. Long ncRNAs are often poorly conserved, characterized by low copy number, fast turnover rates as well as very long length, ranging from 200 nucleotides up to 100 kilobases, sometimes encompassing a whole chromosome [9]. Due to their length, these RNA species offer the possibility of allele-specific action and targeting of a single location within the genome. This is in contrast to transcription factors or small non-coding RNAs that bind a short DNA sequence which may be repeated numerous times throughout the genome [7]. Although function of many lncRNAs is yet to be illuminated, it is now clear that at least some lncRNAs play a role in processes that lead both to gene activation and repression. This is at least in part achieved by the ability of lncRNAs to guide the catalytic function of chromatin-modifying enzymes to specific sites in the genome, and these complexes then act on chromatin modifications and structure [7,10]. Currently, there are no lncRNAs with assigned role in liver disease; however, there have been excellent examples within other disease contexts, such as MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), which is a highly conserved lncRNA that regulates metastasis development in lung cancer by activating expression of metastasis-associated genes [11]. Latest studies have shown that antisense oligonucleotides can be used to silence MALAT1 and thus prevent metastatic disease [11,12]. It will be exciting to learn the potential roles of lncRNAs in liver disease and to find out whether similar antisense silencing regimes may have a therapeutic impact. MicroRNAs (miR) are a class of short (20–24 nucleotide) noncoding RNAs that regulate gene expression at a post-transcriptional level, through binding to the 30 -untranslated region of targeted messenger RNA and causing its degradation or destabilization; alternatively miRs can also effect translational
Journal of Hepatology 2013 vol. 59 j 1349–1353
Clinical Application of Basic Science repression [13]. The observed outcome is a modest change in gene expression, widely defined as a ‘‘fine-tuning effect’’ [14].
Nucleus
DNA methylation Me
microRNAs and liver disease: intriguing transformation from basic science into clinics In humans, over 1500 miRs have been identified which in turn target hundreds of genes within numerous biological pathways including liver development, regeneration, and fibrosis. miRNA profiling of distinctive liver diseases including chronic hepatitis C and B, NAFLD (nonalcoholic fatty liver disease), ALD (alcoholic liver disease), and drug-induced liver injury have revealed miRs that are now becoming novel therapeutic targets in various clinical conditions [15,16]. One of the best examples of such new developments is the discovery of miR-122 role in hepatitis C [17]. miR-122 was shown to facilitate HCV replication by binding to multiple sites in the 50 untranslated region of HCV RNA genome and enhances translation of the viral proteins and protect HCV RNA against nucleolytic degradation. In vivo targeting of miR122 was initially tested in chimpanzees where 12-week intravenous delivery of locked-nucleic-acid-[LNA]-modified complementary oligonucleotide led to depletion of miR-122 and durable reduction in HCV RNA levels as well as lower serum cholesterol levels [19]. Phase I and IIa human trials with Miravirsen (LNAmodified-antimiR-122) have also revealed good safety profiles and tolerability with prolonged reduction in HCV RNA following 18 weeks of treatment [20]. In addition, miR-122 is a mediator of insulin, lipid metabolism, and iron homeostasis. Antagonism of miR-122 reduces cholesterol and LDL levels through affecting hepatic cholesterol and fatty acid biosynthesis genes while inducing b oxidation of fatty acids as well as causing iron deficiency with lower levels of plasma and liver iron [18]. As such, antagonising miR-122 may have multiple beneficial outcomes. While antagonism of miR-122 shows very encouraging results, two remarkable papers, using germline and liver-specific knockout mice, highlighted some unexpected roles of miR-122 in the liver. Mice with miR-122 deletions had reduced serum cholesterol levels similar to the pharmacological inhibition of miR122, however, they developed microsteatosis with triglyceride accumulation, steatohepatitis with inflammatory cell recruitment, and elevated pro-inflammatory mediators (IL-6, TNF-a) [21]. Liver fibrosis, via the activation of KLF6 axis, and hepatocellular carcinoma-like tumours were also evident in this phenotype, indicating tumour suppressor properties of miR-122 [22]. With our current knowledge, the reason for the biological distinction between the temporary pharmacological inhibition and the global deletion of miR-122 is not yet known. However, it is likely that our current understanding of the mechanisms by which microRNAs operate in these systems is rather one-dimensional. With further research and improved insight into the ways in which various epigenetic regulatory aspects integrate with one another, we might discover means of manipulating homeostasis as well as disease states. Histone modifications and liver disease: still on the bench As already stated, DNA inside the nuclei is complexed with histones, forming a structure termed chromatin. The main histone proteins are called histone H2A, histone H2B, histone H3, and histone H4 [4,6]. The smallest unit of chromatin is a mononucleosome, which consists of 146 base pairs of genomic DNA 1350
Me
Me
Histone modifications Me
Me
Ac
Ac Me
Me
Cytoplasm
Non-coding RNAs
Fig. 1. Epigenetic mechanisms that regulate gene expression. The mammalian genome is packaged in the nucleus via wrapping of DNA around histones, forming a structure known as chromatin. The on and off state of gene expression is regulated by DNA methylation, post-translational modifications of various residues within histone tails, and non-coding RNAs.
sequence which is wrapped around an octamer of histones- two copies of each of the histones H2A, H2B, H3, and H4 (Fig. 1). Histones are small, globular proteins with long ’tails’ on one end of the amino acid structure, this being the location of post-translational modifications carried out by numerous enzymes. There are in excess of 60 sites within each octamer of histones that are subject to chemical modification. Modifications include methylation, citrullination, acetylation, phosphorylation, SUMOylation, ubiquitination, and ADP-ribosylation. Combinations of various chemical groups are attached to the histone tails which then form binding sites for protein complexes that are able to affect transcriptional activity of a particular gene. Histone modifications are also able to induce conformational changes in chromatin such to promote transcription or silencing of gene expression in varying degrees. Both of these processes depend on the site, type, and number of chemical groups attached to the histone tails [4,6,23]. As there are strings of histone octamers at every 146 base pairs of DNA sequence along the gene, and each one can have a particular set of modifications at one of over 60 sites, the possible combinations that can be achieved are huge [4]. Each combination of histone marks potentially provides a specific recognition site for a protein complex that can alter chromatin structure. In that sense, the histone modifications provide a complex blueprint that regulates transcriptional activity, which is also referred to as the histone code [4]. In terms of a functional outcome, histone acetylation almost invariably underpins induction of gene transcription; however, methylation of lysine residues within the histone tail can either stimulate (histone 3 lysine 4) or inhibit gene expression (histone
Journal of Hepatology 2013 vol. 59 j 1349–1353
JOURNAL OF HEPATOLOGY HDAC inhibitors
HAT inhibitors
● Vorinostat ● Romidepsin ● Valproic acid ● Trichostatin A
● Anacardic acid ● Curcumin ● CPHT2
Ac Ac Ac
Ac
Ac
Ac
HAT HDAC
ical trials links under Refs. 26,27]. In vitro and in vivo studies suggest that HDACs are considerably upregulated in chronic liver disease; this led to research using HDAC inhibitors (HDACi) to block myofibroblast generation and fibrogenesis. Laboratory evidence suggests that various HDACi attenuate the activation of hepatic stellate cells and fibrosis in animal models. However, the mechanism of action of the HDACi was not clearly understood and, of note, it is now known that HDACs and their inhibitors have effects on non-histone proteins such as cytoskeleton proteins. For HDACs to be fully exploited for therapeutic purposes, it will be necessary to have a much deeper understanding of their activities and substrates. In addition, we will need histone modifiers with greater specificity; two examples of this approach were recently reported using highly selective inhibitors of EZH2 histone methyltransferase and jumonji H3K27 histone demethylase, which demonstrated antitumorigenic activity in lymphoma cells and modulated macrophage response, respectively [28,29].
KDM KMT Me Me Me
DNA methylation, methyl-binding proteins and liver disease: astonishing roles – prepare for lift off
Me
KMT inhibitors-targets ● DZNep-H3K27me ● BIX-01294-H3K9me ● PDB4e47-H3K4me ● Chaetocin-SUV39H1
Me
Me
KDM inhibitors ● Phenelzine ● Pargyline hydrochloride ● GSK_J1 ● Tranylcypromine
Fig. 2. Enzymes that control histone lysine methylation and acetylation are targets for numerous drugs that are being developed or already used clinically. HDAC, histone deacetylase; HAT, histone acetyltransferase; KMT, histone lysine methyltransferas; KDM, histone lysine demethylase.
3 lysine 9 or 27 methylation) (please see Fig. 2 for a list of compounds that target histone methylation and acetylation). The acetylation of histones is carried out by two groups of enzymes, histone acetyl transferases (HAT) and histone deacetylases (HDAC) (Fig. 2). Among histone modifying enzymes, HDACs have received most attention. The role of HDAC3 in circadian regulation, hepatic energy metabolism, and hepatosteatosis has now been clearly demonstrated [24]. HDAC3 negatively regulates lipogenic genes and directs metabolic sources towards gluconeogenesis to maintain normal blood glucose during daytime. In contrast, HDAC3 is significantly downregulated at night, which stimulates lipid synthesis. Interestingly, liver-specific knockdown of HDAC3 leads to the development of marked steatosis, with triglyceride accumulation and ‘‘insulin hypersensitivity’’. Similarly, alcohol-induced microsteatosis is associated with reduced HDAC3 expression. Furthermore, alcohol and its metabolite acetate induce acetylation of histones globally in macrophages and hepatocytes, particularly in promoters of pro-inflammatory cytokines [25]. Pharmacological targeting of histone modifying enzymes, particularly inhibitors of HDACs, constitutes a rapidly growing component of the epigenetic drug market. Two HDAC inhibitors are currently licensed for use in haematological malignancies and similar drugs are in clinical trials for various hematogenous malignancies and solid organ tumors (Fig. 2) [please refer to clin-
Methylation of DNA occurs at the 5th carbon in the pyrimidine ring of cytosine nucleotides and most commonly in cytosine phosphoguanine dinucleotides, often referred to as CpGs. Large (longer than 200 bp) and dense (>55%) clusters of CpG sequences are termed CpG islands. Many human gene promoters contain clusters of CpG islands in the unmethylated state; in contrast, most non-promoter CpGs are methylated [30]. The functional role of DNA methylation is relatively complicated, but generally speaking, methylation within the promoter regions ordinarily leads to transcriptional repression, whereas methylation within gene body can exert transcriptional activation. Two main mechanisms have been described for DNA methylation-mediated longterm gene silencing. Methylated DNA can either repress gene expression by recruiting methyl-binding proteins (MeCP2, MBD1–4) that in turn interact with chromatin silencing protein complexes, or it may directly interfere with the binding of transcription factors to their DNA sequence recognition motifs [30]. Methylation of DNA is executed by enzymes known as DNA methyltransferases (Dnmt) using S-adenosyl methionine as the methyl donor [30]. It is not clear how DNA methylation is targeted to a particular region, but it is likely to involve interaction of DNMTs with other chromatin associated factors [30]. The transdifferentiation of hepatic stellate cells (HSC) into a myofibroblast-like phenotype is a pivotal event in hepatic wound-healing and fibrosis. The dramatic change in phenotype associated with HSC transdifferentiation is underpinned by a global change in gene expression. DNA methylation exists in a number of genes that are expressed at a high level in quiescent HSCs, which are then silenced as the cells activate. At present it is unclear as to whether the DNA methylation pattern of the HSC genome alters with transactivation, however, there is evidence that the way in which the cell ‘‘reads’’ the so-called epigenome does change [31]. As HSCs transdifferentiate, they begin to express MeCP2, a DNA methyl-binding protein that is recruited to methyl-CpG marks present on the DNA and upon binding can subsequently recruit chromatin remodeling complexes that silence gene transcription. This type of gene silencing mechanism occurs at the IjBa and PPAR-c genes in activated HSCs, which is required for these cells to acquire their inflammatory and
Journal of Hepatology 2013 vol. 59 j 1349–1353
1351
Clinical Application of Basic Science fibrogenic phenotype [31,32]. In addition to its repressive action, MeCP2 can also act as a transcriptional activator, although the mechanisms by which it achieves this apparently paradoxical effect are not yet clear. A recent report suggested that modification of methyl-CpG to hydroxymethyl-CpG enables MeCP2 to function as a stimulator of transcription [33]. We have previously reported that MeCP2 enhances the expression of other histone modifying enzymes such as the histone methyltransferases EZH2 and ASH1 [31,34]. These enzymes in turn regulate the attachment of methyl groups to histone H3 lysine 27 (EZH2) and histone H3 lysine 4 (ASH1). Because H3K27 methylation is a transcriptionally repressive mark, EZH2 therefore regulates silencing of target genes, such as PPAR-c [31]. In contrast, methylation of lysine 4 of histone H3 is a signature of actively transcribed genes [6]. During HSC transdifferentiation, ASH1 is recruited to the regulatory regions of the collagen 1A1, TIMP1 and TGFb1 where it facilitates their transcription [34]. Taken together, MeCP2 acts as a pivotal regulator of HSC activation, and it is worth noting that MeCP2 gene deletion or drug mediated EZH2-inhibition attenuates hepatic fibrosis. Rosemarinic acid and baicalin (active ingredient of Yang-Gan-Wan, Chinese herbal preparation) also repress MeCP2-EZH2 relay, thus allowing re-expression of PPAR-c, which in turn prevents hepatic fibrosis [35]. DNA methylation, MeCP2, EZH2, and ASH1 may be bona fide therapeutic targets for prevention and/or treatment of liver fibrosis. It is very well worth noting the manner in which DNA methylation, methyl binding proteins and enzymes that control histone modifications are involved in crosstalk, and it will be crucial to understand these interactions in order to develop drugs that will target these pathways.
to injurious stimuli to allow rapid adaptation to limit the fibrogenic response and preserve normal liver architecture. It also raises the exciting possibility of epigenetic screening of liver patients for prognosis of disease progression.
Conclusions Complex genetic and environmental interactions give rise to the phenotype of an individual. This phenotype is then subject to lifelong change through remodeling of the individual’s epigenome. The underlying epigenetic mechanisms can also impact on the course of diseases or chronic conditions that the individual may suffer, such that they can restrict or promote their progression. The challenge now lies in fully understanding the epigenetic mechanisms and their interaction, which is a tall order due to the complexity of the system. With much improved knowledge, we might be able to selectively modulate epigenome through the use of drugs, alteration in diet, or applying other types of interventions in order to alter the course of disease and ageing.
Key Points Clinical application of epigenetics in Hepatology •
May be used as biomarkers of liver injury or fibrosis from blood/plasma-based on necrotic or apoptotic cells released from injured liver, free microRNA released from cells or exosome associated microRNA
•
May be used as prognostic indicators of disease activity from liver/blood - tight regulation compared to gene expression changes, which may solve sample variability
•
May predict treatment response
•
Can provide new avenues for therapeutic applicationmicroRNA inhibition by oligonucleotide inhibitors or microRNA delivery/potentiating the effect by miRNA mimics, inhibitors of histone modifying enzymes, DNMT inhibitors
Epigenetic marks are heritable by future generations Genetic studies exploring the association of single nucleotide polymorphisms and chronic liver diseases created fundamental data regarding the variability of disease development (e.g., NAFLD and PNPLA3) and treatment responses (e.g., hepatitis C and IL28B). However, there remains a significant gap in our understanding of liver patho-physiology. Liver fibrosis is a final common pathway of liver injury irrespective of aetiology, however, it is currently not clear if predisposition to development of fibrosis exists or indeed if existence of disease in previous generations alters such predisposition. We recently described the potential for transgenerational epigenetic transmission of susceptibility traits for liver fibrosis in male rats [5]. Offspring from a lineage of two consecutive generations of ancestors that suffered liver fibrosis, upon injury with the hepatotoxin carbon tetrachloride, developed less fibrosis compared with rats that had no ancestral history of liver disease. The adapted response was characterized by reduced numbers of myofibroblastic HSC, reduced expression of profibrogenic TGFb1, and elevated expression of antifibrogenic PPAR-c and PPAR-a. Interrogation of DNA methylation at the PPAR-c and PPAR-a promoters revealed that ancestral liver disease leads to hypomethylation of these regulatory regions in the livers of offspring. Intriguingly, in a NAFLD cohort, patients with severe fibrosis or cirrhosis had higher levels of PPAR-c promoter DNA methylation as compared to patients with mild fibrosis [5]. This study therefore provides evidence of heritable epigenetic signatures that can be modified in response
1352
Conflict of interest DAM consults for and is in receipt of grant funding from GlaxoSmithKline. References [1] Available at: http://www.ornl.gov/sci/techresources/Human_Genome/project/about.shtml. [2] Turner BM. Defining an epigenetic code. Nat Cell Biol 2007;9:2–6. [3] Costa FF. Non-coding RNAs, epigenetics and complexity. Gene 2008;410: 9–17. [4] Kouzarides T. Chromatin modifications and their function. Cell 2007;128:693–705. [5] Zeybel M, Hardy T, Wong YK, Mathers JC, Fox CR, Gackowska A, et al. Multigenerational epigenetic adaptation of the hepatic wound-healing response. Nat Med 2012:1369–1377, [Erratum in: Nat Med 2012;18(10):159]. [6] Richards EJ, Elgin SC. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 2002;108:489–500.
Journal of Hepatology 2013 vol. 59 j 1349–1353
JOURNAL OF HEPATOLOGY [7] Mercer TR, Mattick JS. Structure and function of long noncoding RNAs in epigenetic regulation. Nat Struct Mol Biol 2013;20:300–307. [8] Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, et al. The transcriptional landscape of the mammalian genome. Science 2005;309: 1559–1563, [Erratum in: Science 2006;311:1713]. [9] Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell 2009;136:629–641. [10] Lee JT. Epigenetic regulation by long noncoding RNAs. Science 2012;338: 1435–1439. [11] Gutschner T, Hämmerle M, Eissmann M, Hsu J, Kim Y, Hung G, et al. The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res 2013;73:1180–1189. [12] Gutschner T, Diederichs S. The hallmarks of cancer: a long non-coding RNA point of view. RNA Biol 2012;9:703–719. [13] He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 2004;5:522–531. [14] Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature 2008;455:64–71. [15] Rottiers V, Näär AM. MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol 2012;13:239–250, [Review]. [16] Szabo G, Sarnow P, Bala S. MicroRNA silencing and the development of novel therapies for liver disease. J Hepatol 2012;57:462–466. [17] Machlin ES, Sarnow P, Sagan SM. Combating hepatitis C virus by targeting microRNA-122 using locked nucleic acids. Curr Gene Ther 2012;12:301–306. [18] Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 2010;327:198–201. [19] Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, Patel K, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med 2013, [Epub ahead of print] PubMed PMID: 23534542. [20] Castoldi M, Vujic Spasic M, Altamura S, Elmén J, Lindow M, Kiss J, et al. The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice. J Clin Invest 2011;121:1386–1396. [21] Hsu SH, Wang B, Kota J, Yu J, Costinean S, Kutay H, et al. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest 2012;122:2871–2883. [22] Tsai WC, Hsu SD, Hsu CS, Lai TC, Chen SJ, Shen R, et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J Clin Invest 2012;122:2884–2897.
[23] Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:1074–1080. [24] Feng D, Liu T, Sun Z, Bugge A, Mullican SE, Alenghat T, et al. A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism. Science 2011;331:1315–1319. [25] Sun Z, Miller RA, Patel RT, Chen J, Dhir R, Wang H, et al. Hepatic Hdac3 promotes gluconeogenesis by repressing lipid synthesis and sequestration. Nat Med 2012;18:934–942. [26] Clinical trials for HDAC inhibitors: Available at: http://www.clinicaltrials.gov/ct2/show/NCT01486277, http://www.clinicaltrials.gov/ct2/show/ NCT01344707, http://www.clinicaltrials.gov/ct2/show/NCT00686218. [27] Burridge S. Target watch: drugging the epigenome. Nat Rev Drug Discov 2013;12:92–93. [28] Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus, et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol 2012;8:890–896. [29] Kruidenier L, Chung CW, Cheng Z, Liddle J, Che K, Joberty G, et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 2012;488:404–408. [30] Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16:6–21. [31] Mann J, Chu DC, Maxwell A, Oakley F, Zhu NL, Tsukamoto H, et al. MeCP2 controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. Gastroenterology 2010;138:705–714. [32] Mann J, Oakley F, Akiboye F, Elsharkawy A, Thorne AW, Mann DA. Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis. Cell Death Differ 2007;14:275–285. [33] Mellén M, Ayata P, Dewell S, Kriaucionis S, Heintz N. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell 2012;151:1417–1430. [34] Perugorria MJ, Wilson CL, Zeybel M, Walsh M, Amin S, Robinson S, et al. Histone methyltransferase ASH1 orchestrates fibrogenic gene transcription during myofibroblast transdifferentiation. Hepatology 2012;56: 1129–1139. [35] Yang MD, Chiang YM, Higashiyama R, Asahina K, Mann DA, Mann J, et al. Rosmarinic acid and baicalin epigenetically derepress peroxisomal proliferator-activated receptor c in hepatic stellate cells for their antifibrotic effect. Hepatology 2012;55:1271–1281.
Journal of Hepatology 2013 vol. 59 j 1349–1353
1353