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Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation

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    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
    ,
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    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
    Sarah Snykers
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    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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    # These authors contributed equally to this work.
    Tom Henkens
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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  • Evelien De Rop
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
    Mathieu Vinken
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    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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  • Joanna Fraczek
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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  • Joery De Kock
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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  • Evi De Prins
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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    † Deceased.
    Albert Geerts
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    † Deceased.
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    Department of Cell Biology, Vrije Universiteit Brussels, Brussels, Belgium
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  • Vera Rogiers
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
    Tamara Vanhaecke
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    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
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    Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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    # These authors contributed equally to this work.
    ‡ These authors are post doctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen) Belgium.
    † Deceased.
Open AccessPublished:April 02, 2009DOI:https://doi.org/10.1016/j.jhep.2009.03.009
      Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cell’s differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions. In this review, we discuss the epigenetic control of (liver-specific) gene-transcription and the intricate interplay between chromatin modulation, including histone (de)acetylation and DNA (de)methylation, and liver-enriched transcription factors. Special attention is paid to their role in directing hepatic differentiation of primary hepatocytes and stem cells in vitro.

      Abbreviations:

      ADSC (adipose tissue-derived stem cells), ALB (albumin), AFP (alpha-fetoprotein), AhR (aryl hydrocarbon receptor), 5-AzaC (5-Azacytidine or azacytidine), 5-Aza-dC (5-Aza-2′-deoxycytidine or decitabine), C/EBP (CCAAT/Enhancer Binding Protein), CYP (cytochrome P450), CBP (CREB-binding protein), CpGs (cytosine-guanine dinucleotides), DHAC (5-6-Dihydro-5-azacytidine), 4-Me2N-BAVAH (5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide), DNMTs (DNA methyltransferases), DNMTi (DNMT inhibitors), ES (embryonic stem cells), EGF (epidermal growth factor), EGCG ((-)-epigallocatechin-3-gallate), HCC (hepatocellular carcinoma), HNF (hepatocyte nuclear factor), HATs (histone acetyl transferases), HDACs (histone deacetylases), HDACi (hydroxamate-based HDAC inhibitors), iPS (induced pluripotent cells), LETFs (liver-specific transcription factors), MSC (mesenchymal stem/progenitor cells), MBD (methylated DNA-binding protein), P/CAF (p300/CBP-associated factor), PGC-1α (PPAR-gamma coactivator 1alpha), zebularine (2-Pyrimidone-1-β-D-riboside), TSA (Trichostatin A), VPA (valproic acid)

      Keywords

      1. Introduction

      Hepatocytes contain a rich source of xenobiotic biotransformation enzymes and consequently, the liver represents a primary target for xenobiotic-induced acute and systemic toxicity. Hence, hepatocytes are the ultimate source for toxicological screening/profiling of potential drug candidates. The drawback, however, is that upon isolation and subsequent culture of hepatocytes, a rapid and substantial decline of hepatic functionality occurs. In particular, the xenobiotic biotransformation capacity undergoes phenotypic changes [
      • Papeleu P.
      • Vanhaecke T.
      • Rogiers V.
      Histone deacetylase inhibition: a differentiation therapy for cultured primary hepatocytes.
      ]. This progressive loss of a differentiated hepatic phenotype in vitro plausibly results from a proliferative response, elicited during hepatocyte isolation from the liver. In fact, the cell cycle entrance triggers the activation of several signal transduction pathways, ultimately leading to profound alterations in gene expression [
      • Papeleu P.
      • Loyer P.
      • Vanhaecke T.
      • Elaut G.
      • Geerts A.
      • Guguen-Guillouzo C.
      • et al.
      Trichostatin A induces cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.
      ]. The acquisition and stabilisation of a differentiated hepatic geno- and consequently phenotype, i.e. liver-specific gene/protein expression, very often rely on the concerted binding of liver-enriched transcription factors (LETFs) and other trans-acting DNA-binding proteins to well-defined regulatory and coding regions of target genes [
      • Kyrmizi I.
      • Hatzis P.
      • Katrakili N.
      • Tronche F.
      • Gonzalez F.J.
      • Talianidis I.
      Plasticity and expanding complexity of the hepatic transcription factor network during liver development.
      ,
      • Akiyama T.E.
      • Gonzalez F.J.
      Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors.
      ,
      • Costa R.H.
      • Kalinichenko V.V.
      • Holterman A.X.
      • Wang X.
      Transcription factors in liver development, differentiation, and regeneration.
      ]. As DNA is tightly condensed into chromatin fibers by histones and other proteins, modulation of chromatin compaction is a prerequisite to facilitate binding of transcription factors and consequential transcriptional activation [
      • Turner B.M.
      Reading signals on the nucleosome with a new nomenclature for modified histones.
      ,
      • Wolffe A.P.
      • Guschin D.
      Review: chromatin structural features and targets that regulate transcription.
      ]. Epigenetic events, including covalent histone modifications and DNA methylation, are therefore broadly acknowledged to play a fundamental role in the organisation of chromatin architecture and hence in the strict control of gene transcription [
      • Wang G.G.
      • Allis C.D.
      • Chi P.
      Chromatin remodeling and cancer, part I: covalent histone modifications.
      ,
      • Collas P.
      Epigenetic states in stem cells.
      ]. For example, in proliferating hepatocellular carcinoma (HCC) and HCC-derived hepatoma cell lines, inhibition of histone deacetylation and DNA methylation is found to drastically down- and up-regulate genes involved in cellular proliferation and xenobiotic metabolism, respectively [
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
      ,
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
      ]. Consequently, it was thought that epigenetic events may display a predominant role in the acquisition and maintenance of the hepatocyte’s differentiated phenotype of dedifferentiating primary hepatocytes in vitro. Alternatively, stem cells have been proposed to produce functional hepatocytes as well. New insights into the molecular mechanisms governing the balance between self-renewal/proliferation and lineage-directed differentiation of embryonic stem cells have unveiled the presence of epigenetic marks as being key regulatory players [
      • Collas P.
      Epigenetic states in stem cells.
      ,
      • Atkinson S.
      • Armstrong L.
      Epigenetics in embryonic stem cells: regulation of pluripotency and differentiation.
      ]. In fact, progression from unsoiled stem cells towards their differentiated progeny is characterized by alterations in the epigenetic landscapes of gene regulatory and coding regions. [
      • Collas P.
      Epigenetic states in stem cells.
      ,
      • Atkinson S.
      • Armstrong L.
      Epigenetics in embryonic stem cells: regulation of pluripotency and differentiation.
      ,
      • Reik W.
      Stability and flexibility of epigenetic gene regulation in mammalian development.
      ,
      • Yeo S.
      • Jeong S.
      • Kim J.
      • Han J.S.
      • Han Y.M.
      • Kang Y.K.
      Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.
      ,
      • Perry P.
      • Sauer S.
      • Billon N.
      • Richardson W.D.
      • Spivakov M.
      • Warnes G.
      • et al.
      A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction.
      ]. More specifically, locus-specific modifications on histones and DNA, progressively silence the transcription of pluripotent genes (euchromatic → heterochromatic state), whilst typical differentiated, lineage-specific genes become activated (heterochromatic → euchromatic state) [
      • Collas P.
      Epigenetic states in stem cells.
      ,
      • Atkinson S.
      • Armstrong L.
      Epigenetics in embryonic stem cells: regulation of pluripotency and differentiation.
      ,
      • Reik W.
      Stability and flexibility of epigenetic gene regulation in mammalian development.
      ,
      • Yeo S.
      • Jeong S.
      • Kim J.
      • Han J.S.
      • Han Y.M.
      • Kang Y.K.
      Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.
      ,
      • Perry P.
      • Sauer S.
      • Billon N.
      • Richardson W.D.
      • Spivakov M.
      • Warnes G.
      • et al.
      A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction.
      ]. Anticipation with nuclear chromatin might thus involve a key strategy for cell fate re-programmation.
      In this review, we will discuss the key regulatory role of epigenetic modification in gene transcription, with particular focus on the maintenance and the acquisition of a differentiated geno/phenotype of primary hepatocytes and stem cells, i.e. pluripotent embryonic stem cells (ESCs) and multipotent mesenchymal stem/progenitor cells (MSC), respectively.

      2. Epigenetic control of gene transcription

      2.1 Structural chromatin modifications by histone acetylation/deacetylation

      The chromatin higher order structure can be subjected to a number of reversible posttranslational modifications [
      • Moggs J.G.
      • Goodman J.L.
      • Trosko J.E.
      • Roberts R.A.
      Epigenetics and cancer: implications for drug discovery and safety assessment.
      ]. Although the functional relevance of the individual reactions is often unclear, it is generally believed that the global repertoire of histone tail modifications constitutes a (epigenetic) code, which affects chromatin structure and/or gene expression [
      • Szyf M.
      DNA methylation and demethylation as targets for anticancer therapy.
      ]. To date, histone acetylation, methylation, phosphorylation/ubiquination/sumoylation, ADP-ribosylation and glycosylation of histones were reported [
      • Turner B.M.
      Reading signals on the nucleosome with a new nomenclature for modified histones.
      ,
      • Moggs J.G.
      • Goodman J.L.
      • Trosko J.E.
      • Roberts R.A.
      Epigenetics and cancer: implications for drug discovery and safety assessment.
      ]. Histone acetylation, the best-understood posttranslational histone modification, is discussed in the following paragraphs.

      2.1.1 Role of histone deacetylases in the regulation of gene expression

      Two opposing enzyme activities, i.e. histone acetyl transferases (HATs) (recently also referred to as lysine (K)-acetyltransfereases or briefly KATs [
      • Allis C.D.
      • Berger S.L.
      • Cote J.
      • Dent S.
      • Jenuwien T.
      • Kouzarides T.
      • et al.
      New nomenclature for chromatin-modifying enzymes.
      ]) and histone deacetylases (HDACs), determine the acetylation status of the lysine residues at the N-terminal histone tails extending out of the nucleosome [
      • Turner B.M.
      Reading signals on the nucleosome with a new nomenclature for modified histones.
      ]. Upon acetylation (Fig. 1), the positive charges on the side chains of these lysine residues are partially neutralised, thereby weakening the interaction with the negatively charged phosphate groups in the DNA backbone and affecting the nucleosome stability. The degree of acetylation of core histones can thus modulate DNA accessibility and chromatin activity in transcription, replication, recombination and repair [
      • Weidle U.H.
      • Grossmann A.
      Inhibition of histone deacetylases: a new strategy to target epigenetic modifications for anticancer treatment.
      ]. Whereas actively transcribed genes are characterized by highly acetylated core histones, hypoacetylated histones are preferentially found in transcriptionally silenced chromatin regions [
      • Grunstein M.
      Histone acetylation in chromatin structure and transcription.
      ]. Consequently, the long-standing paradigm existed that HDAC inhibition, leading to histone hyperacetylation, was exclusively associated with transcriptional activation. Yet, evidence is accumulating of HDACs functioning as both transcriptional activators or repressors. Indeed, by removal of acetyl groups from histone tails, HDACs do not only modulate the physical interaction between histones and DNA in the nucleosomal units, but also the message encrypted in the histones’ posttranscriptional modifications, and thus the epigenetic/histone code [
      • Yang X.J.
      • Seto E.
      HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention.
      ]. Consequently, specific effector proteins, e.g. transcription factors, are recruited resulting in further transcriptional stimulation or silencing, depending on the message comprised. Additionally, HDACs have targets other than histones, including the transcription factors p53, c-myc, NF-κB, YY-1, E2F and GATA family [
      • Xu W.S.
      • Parmigiani R.B.
      • Marks P.A.
      Histone deacetylase inhibitors: molecular mechanisms of action.
      ]. HDAC-mediated deacetylation of these non-histone proteins may affect their stability, localization, DNA-binding activity or ability to interact with other proteins [
      • Minucci S.
      • Pellici P.G.
      Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer.
      ]. As a result, the activity of the target protein might be augmented/reduced (Fig. 1).
      Figure thumbnail gr1
      Fig. 1HAT/HDAC-mediated transcriptional control. (A), Histone-related pathway: HATs/HDACs acetylate/deacetylate histones resulting in reduced/augmented chromatin compaction and alternations of the histone code, respectively. (B) Non-histone related pathway: HDACs directly interfere with non-histone protein targets, including transcription factors, nuclear hormone receptors, nuclear import factors, structural proteins and adhesion proteins. Deacetylation of latter non-histone proteins might affect diverse aspects of their protein physiology, resulting in either decreased or increased activity of the target protein. Both pathways interconnect with each other. The transcriptional outcome thus relies on the sum of all – transcription-stimulating/inhibiting – actions.
      In light of these data, it is clear that HDACs operate at more than one level in the transcriptional regulation scheme. We refer to Smith for a scrupulous review regarding the transcriptional machinery underlying HDACs-mediated transcriptional (in)activation [
      • Smith C.L.
      A shifting paradigm: histone deacetylases and transcriptional activation.
      ].

      2.1.2 Classification of histone deacetylases and their role in differentiation programs

      Today, 18 HDACs have been characterized. Based on sequence similarity, they can be classified into four distinct classes. Class I (HDAC-1,-2,-3,-8) and class II HDACs (HDAC-4,-5,-6,-7,-9,-10) closely resemble the yeast RPD3 and HDA1 proteins, respectively. Class III HDACs or ‘sirtuins’ are structurally unrelated to the other HDACs and apparently insensitive to hydroxamate-based HDAC inhibitors (HDACi) such as Trichostatin A (TSA) [
      • Xu W.S.
      • Parmigiani R.B.
      • Marks P.A.
      Histone deacetylase inhibitors: molecular mechanisms of action.
      ]. HDAC-11 is the single representative of mammalian class IV HDACs. It displays conserved residues in the catalytic core that share some, yet insufficient, homology to both class I and II enzymes [
      • Gray S.G.
      • Ekström T.J.
      The human histone deacetylase family.
      ,
      • Bolden J.E.
      • Peart M.J.
      • Johnstone R.W.
      Anticancer activities of histone deacetylase inhibitors.
      ,
      • Mariadason J.M.
      HDACs and HDAC inhibitors in colon cancer.
      ]. Most HDACs lack intrinsic DNA-binding activities and are therefore capable of homo- and hetero-dimerisation. The HDAC catalytic domain is formed by a stretch of ca. 390 amino acids consisting of a set of conserved amino acids, which differ between class I and class II HDACs [
      • Turner B.M.
      Reading signals on the nucleosome with a new nomenclature for modified histones.
      ,
      • Grozinger C.M.
      • Hassig C.A.
      • Schreiber S.L.
      Three proteins define a class of human histone deacetylases related to yeast Hda1p.
      ]. The active site consists of a gently curved tubular pocket, a zinc-binding site and two Asp-His charge relay systems [
      • Finnin M.S.
      • Donigian J.R.
      • Cohen A.
      • Richon V.M.
      • Rifkind R.A.
      • Marks P.A.
      • et al.
      Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors.
      ].
      Class I HDACs (HDACs-1,-2,-3,-8) are generally small nuclear enzymes [
      • Mariadason J.M.
      HDACs and HDAC inhibitors in colon cancer.
      ] that participate in cell cycle progression [
      • Gray S.G.
      • Ekström T.J.
      The human histone deacetylase family.
      ] and the regulation of housekeeping genes [
      • Grozinger C.M.
      • Hassig C.A.
      • Schreiber S.L.
      Three proteins define a class of human histone deacetylases related to yeast Hda1p.
      ]. Class II HDACs (HDACs-4,-5,-6,-7,-9,-10) share some domain similarity with yeast HDA1 [
      • Gray S.G.
      • Ekström T.J.
      The human histone deacetylase family.
      ,
      • Mariadason J.M.
      HDACs and HDAC inhibitors in colon cancer.
      ]. They can be subdivided into class IIa HDACs (HDACs-4,-5,-7,-9) and class IIb HDACs (HDAC-6 and -10) [
      • Yang X.J.
      • Grégoire S.
      Class II histone deacetylases: from sequence to function, regulation, and clinical implication.
      ]. With the exception of HDAC-10, class II HDACs show a restricted tissue-specific expression pattern with the highest expression found in heart, brain and skeletal muscle [
      • Grozinger C.M.
      • Hassig C.A.
      • Schreiber S.L.
      Three proteins define a class of human histone deacetylases related to yeast Hda1p.
      ]. They are larger than class I HDACs and shuttle between cytoplasm and nucleus. In the nucleus, they mediate cellular proliferation and transcriptional repression of differentiation-related genes, leading to loss of the differentiated phenotype [
      • Gray S.G.
      • Ekström T.J.
      The human histone deacetylase family.
      ]. This was particularly shown for class IIa HDACs-4,-5,-7,-9 isoforms in muscle cell differentiation [
      • Zhang C.L.
      • McKinsey T.A.
      • Olsen E.N.
      Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation.
      ]. Class IIa HDACs, and principally HDAC-4, are also implemented in stress signaling processes, such as cardiac/chondrocyte hypertrophy and neuronal cell death. [
      • Martin M.
      • Kettmann R.
      • Dequiedt F.
      Class IIa histone deacetylases: regulating the regulators.
      ,
      • Vega R.B.
      • Matsuda K.
      • Oh J.
      • Barbosa A.C.
      • Yang X.
      • Meadows E.
      • et al.
      Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis.
      ,
      • Bolger T.A.
      • Yao T.P.
      Intracellular trafficking of histone deacetylase 4 regulates neuronal cell death.
      ]. Most of these functions are mediated by interactions with the MEF2 transcription factor family [
      • Martin M.
      • Kettmann R.
      • Dequiedt F.
      Class IIa histone deacetylases: regulating the regulators.
      ]. In contrast to other HDACs, Class IIb HDAC-6 can accomplish cytoplasm-located functions as well. More specifically, HDAC-6 functions at the crossroads between two cellular signaling systems, i.e. protein lysine acetylation and ubiquitination [
      • Boyault C.
      • Sadoel K.
      • Pabion M.
      • Khochbin S.
      HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination.
      ]. This unique feature expounds its protective role against the accumulation of cytotoxic misfolded protein aggregates within cells [
      • Boyault C.
      • Sadoel K.
      • Pabion M.
      • Khochbin S.
      HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination.
      ]. In addition, HDAC-6 and class I HDAC-3 regulate osteoblast differentiation and bone formation via interaction with transcriptional regulators such as Runx2 [
      • Boyault C.
      • Sadoel K.
      • Pabion M.
      • Khochbin S.
      HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination.
      ,
      • Schroeder T.M.
      • Kahler R.A.
      • Li X.
      • Westendorf J.J.
      Histone deacetylase 3 interacts with runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation.
      ]. Likewise, class I HDAC-1, in complex with myoD, mediates repression of muscle-specific gene expression in undifferentiated myoblasts [
      • Puri P.L.
      • Iezzi S.
      • Stiegler P.
      • Chen T.T.
      • Schiltz R.L.
      • Muscat G.E.
      • et al.
      Class I histone deacetylases sequentially interact with MyoD and pRb during skeletal myogenesis.
      ]. In general, class I HDACs overexpression coincides with increased cell proliferation and a concomitant shift towards dedifferentiation, while levels drop during differentiation processes. For example, in normal small intestine, the HDAC-3 expression is maximal in proliferating cells at the crypt base and is markedly decreased at the villus tip, harbouring more differentiated cells [
      • Wilson A.J.
      • Byun D.S.
      • Popova N.
      • Murray L.B.
      • L’Italien K.
      • Sowa Y.
      • et al.
      Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer.
      ]. In comparison, 90% of cells residing in adenomas of small intestine are HDAC-3+. Basically, overexpression of distinct HDACs appears in various tumor specimens, e.g. HDAC-1 in prostate, gastric and colon tumors, HDAC-2 in colorectal, cervical and gastric cancer [
      • Bolden J.E.
      • Peart M.J.
      • Johnstone R.W.
      Anticancer activities of histone deacetylase inhibitors.
      ], and HDAC-3 in colon cancer [
      • Wilson A.J.
      • Byun D.S.
      • Popova N.
      • Murray L.B.
      • L’Italien K.
      • Sowa Y.
      • et al.
      Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer.
      ]. Specific inhibition of HDAC-3 by RNA interference could inhibit proliferation of colon cancer cell lines and increase both expression and activity of the differentiation marker alkaline phosphatase [
      • Wilson A.J.
      • Byun D.S.
      • Popova N.
      • Murray L.B.
      • L’Italien K.
      • Sowa Y.
      • et al.
      Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer.
      ]. As for liver-specific functions of HDACs, recently a crucial role was credited to HDAC-3 in liver homeostasis and development. In this respect, HDAC-3 absence in zebrafish leads to abnormalities in liver development, [
      • Farooq M.
      • Sulochana K.N.
      • Pan X.
      • To J.
      • Sheng D.
      • Gong Z.
      • et al.
      Histone deacetylase 3 (hdac3) is specifically required for liver development in zebrafish.
      ] whilst conditioned deletion of HDAC-3 in mice induces severe disruption of carbohydrate and lipid metabolism, resulting in organ hypertrophy and hepatocellular damage [
      • Knutson S.K.
      • Chyla B.J.
      • Amann J.M.
      • Bhaskara S.
      • Huppert S.S.
      • Hiebert S.W.
      Liver-specific deletion of histone deacetylase 3 disrupts metabolic transcriptional networks.
      ]. HDAC-1 overexpression in transgenic mice, on the other hand, results in a high incidence of hepatic steatosis and nuclear pleomorphism concomitant with altered expression of genes involved in cell cycle, apoptosis, and lipid metabolism such as p53, PPARγ, Bak and p21 [
      • Wang A.G.
      • Seo S.B.
      • Moon H.B.
      • Shin H.J.
      • Kim D.H.
      • Kim J.M.
      • et al.
      Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1.
      ,
      • Wang A.G.
      • Kim S.U.
      • Lee S.H.
      • Kim S.K.
      • Seo S.B.
      • Yu D.Y.
      • et al.
      Histone deacetylase 1 contributes to cell cycle and apoptosis.
      ]. Also, a number of studies provide evidence for the involvement of HDACs and HATs in the transcriptional regulation of liver-specific genes by LETFs. This issue will be thoroughly discussed in a later section.
      Briefly, these data indicate that modulating the expression of specific HDACs might involve a strategy to (re)activate differentiation programs.

      2.1.3 HDAC inhibitors: types and effects

      Currently, several structurally diverse compounds both natural and synthetic, are known as HDACi. These include short-chain fatty acids, (non)-cyclic hydroxamates, (non)-epoxyketone-containing cyclic tetrapeptides, benzamides and miscellaneous structures [
      • Jung M.
      Inhibitors of histone deacetylase as new anticancer agents.
      ]. Hydroxamate-based inhibitors of classes I and II are promising since they were repeatedly shown to selectively inhibit tumor growth in animals at low (micromolar) and apparently non-toxic doses [
      • Martin M.
      • Kettmann R.
      • Dequiedt F.
      Class IIa histone deacetylases: regulating the regulators.
      ]. Basically, in recent years, HDACi have emerged as promising therapeutics for the treatment of several malignancies, including leukaemia, solid tumors and non-solid cancers such as multiple myeloma [
      • Mariadason J.M.
      HDACs and HDAC inhibitors in colon cancer.
      ]. In that respect, Vorinostat® has recently been approved by the FDA for the treatment of advanced primary cutaneous T-cell lymphoma, whilst several other hydroxamate-containing HDACi are being tested in phases I and II clinical trials for their therapeutic potential [
      • Johnstone R.A.
      The evolution of inaccurate mimics.
      ,
      • Duvic M.
      • Vu J.
      Vorinostat: a new oral histone deactylase inhibitor approved for cutaneous T-cell ymphoma.
      ]. Having seen the growth-inhibiting and differentiation-promoting features of hydroxamate-based HDACi in tumor cells, including hepatoma cells [
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
      ,
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
      ,
      • Lu Y.S.
      • Kashida Y.
      • Kulp S.K.
      • Wang Y.C.
      • Wang D.
      • Hung J.H.
      • et al.
      Efficacy of a novel histone deacetylase inhibitor in murine models of hepatocellular carcinoma.
      ,
      • Herold C.
      • Ganslmayer M.
      • Ocker M.
      • Hermann M.
      • Geerts A.
      • Hahn E.G.
      • et al.
      The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells.
      ,
      • Wakabayashi K.
      • Saito H.
      • Kaneko F.
      • Nakamoto N.
      • Tada S.
      • Hibi T.
      Gene expression associated with the decrease in malignant phenotype of human liver cancer cells following stimulation with a histone deacetylase inhibitor.
      ], our group successfully applied these compounds to stabilize the differentiated phenotype of normal primary hepatocytes in vitro[
      • Papeleu P.
      • Vanhaecke T.
      • Rogiers V.
      Histone deacetylase inhibition: a differentiation therapy for cultured primary hepatocytes.
      ,
      • Papeleu P.
      • Loyer P.
      • Vanhaecke T.
      • Elaut G.
      • Geerts A.
      • Guguen-Guillouzo C.
      • et al.
      Trichostatin A induces cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.
      ,
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      ,
      • Papeleu P.
      • Wullaert A.
      • Elaut G.
      • Henkens T.
      • Vinken M.
      • Laus G.
      • et al.
      Inhibition of NF-kappaB activation by the histone deacetylase inhibitor 4-Me2N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytes.
      ,
      • Vinken M.
      • Henkens T.
      • Snykers S.
      • Lukaszuk A.
      • Tourwé D.
      • Rogiers V.
      • et al.
      The novel histone deacetylase inhibitor 4-Me2N-BAVAH differentially affects cell junctions between primary hepatocytes.
      ,
      • Vinken M.
      • Henkens T.
      • Vanhaecke T.
      • Papeleu P.
      • Geerts A.
      • Van Rossen E.
      • et al.
      Trichostatin A enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes.
      ]. This will be discussed later in this review.

      2.2 DNA methylation

      2.2.1 Role of DNA methyltransferases in the regulation of gene expression

      Reversible DNA methylation occurs at the cytosine–guanine dinucleotides (CpGs) in the DNA and includes addition of a methyl group to the carbon-5 position of cytosine [
      • Baylin S.B.
      DNA methylation and gene silencing in cancer.
      ]. DNA methylation patterns are established by DNA methyltransferases (DNMTs), catalyzing the addition of a methyl group derived from the methyl donor S-adenosyl methionine [
      • Szyf M.
      DNA methylation and demethylation as targets for anticancer therapy.
      ,
      • Rice K.L.
      • Hormaeche I.
      • Licht J.D.
      Epigenetic regulation of normal and malignant hematopoiesis.
      ]. The catalytic activity of these enzymes is accomplished by a highly conserved C-terminal domain, present in all DNMTs.
      In the mammalian genome, CpGs are not uniformly distributed. CpG islands, comprising >1 CpG per 80 base pairs [
      • Costello J.F.
      • Plass C.
      Methylation matters.
      ], are particulary present in/near gene promotor regions. They are usually unmethylated, thereby allowing gene expression [
      • Baylin S.B.
      DNA methylation and gene silencing in cancer.
      ,
      • Rice K.L.
      • Hormaeche I.
      • Licht J.D.
      Epigenetic regulation of normal and malignant hematopoiesis.
      ,
      • Costello J.F.
      • Plass C.
      Methylation matters.
      ]. The distribution of (un)methylated CpGs differs, however, within distinct cell types due to the interplay between DNA methylation/demethylation, giving a cell-type specific DNA methylation pattern [
      • Szyf M.
      DNA methylation and demethylation as targets for anticancer therapy.
      ]. Passive DNA demethylation occurs during DNA replication by chemically blocking DNMTs [
      • Szyf M.
      DNA methylation and demethylation as targets for anticancer therapy.
      ]. The exact mechanism of active DNA replication-independent demethylation still remains elusive. Recent data suggest the involvement of identical enzymes in both the establishment of DNA methylation and demethylation patterns [
      • Métivier R.
      • Gallais R.
      • Tiffoche C.
      • Le Peron C.
      • Jurkowska R.Z.
      • Carmouche R.P.
      • et al.
      Cyclical DNA methylation of a transcriptionally active promoter.
      ]. For example, the methylated DNA-binding protein MBD2 also displays demethylase activity [
      • Massillon D.
      • Arinze I.J.
      • Xu C.
      • Bone F.
      Regulation of glucose-6-phosphatase gene expression in cultured hepatocytes and H4IIE cells by short-chain fatty acids: role of hepatic nuclear factor-4alpha.
      ]. Additional information is needed to unravel this tangled web.

      2.2.2 Classification of DNA methyltransferases and their role in differentiation programs

      Based on structural differences in their regulatory N-terminal domain, three distinct families of DNMTs, i.e. DNMT1, DNMT2 and DNMT3, have currently been identified. All are expressed in human liver tissue [
      • Park H.J.
      • Yu E.
      • Shim Y.H.
      DNA methyltransferase expression and DNA hypermethylation in human hepatocellular carcinoma.
      ].
      DNMT1 is the most abundant DNMT in mammals and mainly methylates hemimethylated GpGs. This ‘maintenance’ DNMT is particulary involved in maintaining DNA methylation patterns during DNA replication [
      • Rice K.L.
      • Hormaeche I.
      • Licht J.D.
      Epigenetic regulation of normal and malignant hematopoiesis.
      ,
      • Park H.J.
      • Yu E.
      • Shim Y.H.
      DNA methyltransferase expression and DNA hypermethylation in human hepatocellular carcinoma.
      ,
      • Pradhan S.
      • Esteve P.O.
      Mammalian DNA (cytosine-5) methyltransferases and their expression.
      ]. It also shows activity towards unmethylated DNA and plays a role in de novo DNA methylation [
      • Bestor T.H.
      The DNA methyltransferases of mammals.
      ]. DNMT2 is the least distinguished DNMT and lacks the regulatory N-terminal domain present in other DNMT enzyme families [
      • Pradhan S.
      • Esteve P.O.
      Mammalian DNA (cytosine-5) methyltransferases and their expression.
      ]. Its associated intrinsic DNMT activity and potential RNA methyltransferase activity suggest a possible role in epigenetic regulation [
      • Kuhlmann M.
      • Borisova B.E.
      • Kaller M.
      • Larsson P.
      • Stach D.
      • Na J.
      • et al.
      Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi.
      ,
      • Jurkowski T.P.
      • Meusburger M.
      • Phalke S.
      • Helm M.
      • Nellen W.
      • Reuter G.
      • et al.
      Human DNMT2 methylates tRNAAsp molecules using a DNA methyltransferase-like catalytic mechanism.
      ]. The DNMT3 family contains three different DNMTs, i.e. DNMT3a, DNMT3b and DNMT3L. DNMT3a and DNMT3b, characterized as de novo DNMTs, mainly methylate unmethylated CpGs and establish new DNA methylation patterns during early embryonal development [
      • Rice K.L.
      • Hormaeche I.
      • Licht J.D.
      Epigenetic regulation of normal and malignant hematopoiesis.
      ]. They plausibly cooperate with DNMT1 to maintain the DNA methylation pattern [
      • Liang G.
      • Chan M.F.
      • Tomigahara Y.
      • Tsai Y.C.
      • Gonzales F.A.
      • Li E.
      • et al.
      Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements.
      ]. DNMT3L is a methyltransferase-like protein without intrinsic DNMT activity [
      • Pradhan S.
      • Esteve P.O.
      Mammalian DNA (cytosine-5) methyltransferases and their expression.
      ,
      • Klose R.J.
      • Bird A.P.
      Genomic DNA methylation: the mark and its mediators.
      ]. It interacts with DNMT3a/DNMT3b and directly modulates their catalytic activity [
      • Suetake I.
      • Shinozaki F.
      • Miyagawa J.
      • Takeshima H.
      • Tajima S.
      DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction.
      ].
      DNMTs play a crucial role in the onset of chromatin remodelling and gene expression regulation. They are responsible for maintaining telomere integrity [
      • Gonzalo S.
      • Jaco I.
      • Fraga M.F.
      • Chen T.
      • Li E.
      • Esteller M.
      • et al.
      DNA methyltransferases control telomere length and telomere recombination in mammalian cells.
      ] and methylation pattern acquisition during gametogenesis, embryogenesis and somatic tissue development [
      • Turek-plewa J.
      • Jagodzinski P.P.
      The role of mammalian DNA methyltransferases in the regulation of gene expression.
      ]. Several studies performed in tumor cell lines, including HepG2 cells [
      • Qiu W.
      • Zhou B.
      • Zou H.
      • Liu X.
      • Chu P.G.
      • Lopez R.
      • et al.
      Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promotor in human heptocellualr carcinoma.
      ,
      • Zhu W.G.
      • Otterson G.A.
      The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells.
      ], also indicate the occurrence of cell cycle arrest, apoptosis and cellular differentiation upon DNMT inhibition. Accordingly, it has been suggested that modulation of the expression of specific DNMTs might involve a strategy to target the differentiation status in developing and proliferating and consequently dedifferentiating cells.

      2.2.3 DNMT inhibitors: types and effects

      Today, a number of synthetic and natural DNMT inhibitors (DNMTi) exist. (i) The group of nucleoside analogue DNMTi contains several structural analogues of deoxycytidine, including 5-azacytidine (azacytidine, 5-AzaC), 5-Aza-2′-deoxycytidine (decitabine, 5-Aza-dC), arabinosyl-5-azacytidine (fazarabine), 5-6-dihydro-5-azacytidine (DHAC) and 2-pyrimidone-1-β-d-riboside (zebularine) [
      • Laird P.W.
      Cancer epigenetics.
      ] (Fig. 2). These analogues, with the exception of zebularine, are modified at the carbon-5 position of the pyrimidine base cytosine [
      • Yoo C.B.
      • Cheng J.C.
      • Jones P.A.
      Zebularine: a new drug for epigenetic therapy.
      ]. After phosphorylation and incorporation in DNA/RNA, they form covalent bounds with DNA methyltransferases, resulting in passive demethylation upon replication [
      • Stresemann C.
      • Lyko F.
      Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine.
      ]. The use of nucleoside analogue DNMTi in tumor cells results, like HDACi, in cell cycle arrest, induction of apoptosis and differentiation [
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
      ,
      • Zhu W.G.
      • Otterson G.A.
      The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells.
      ]. In addition, latter DNMTi were applied by our group to maintain differentiation in normal primary hepatocytes [

      Rogiers V, Vanhaecke T, De Rop E, Fraczek J. Title of invention: stabilisation of the phenotypic properties of isolated primary cells. International patent number: PCT/EP2008/056706.

      ]. This issue will be discussed in the next section.
      (ii) The non-nucleoside analogue DNMTi represent a heterogeneous group of DNMTi enclosing molecules such as derivatives of 4-aminobenzoic acid (procaine and procainamide), the main compound in green tea, (-)-epigallocatechin-3-gallate (EGCG) [
      • Lin X.
      • Asgari K.
      • Putzi M.J.
      • Gage W.R.
      • Yu X.
      • Cornblatt B.S.
      • et al.
      Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide.
      ,
      • Villar-Garea A.
      • Fraga M.F.
      • Espada J.
      • Esteller M.
      Procaine is a DNA-demethylating agent with growth-inhibitory effects in human cancer cells.
      ] and psammaplins from the sponge Pseudoceratina purpurea[
      • Piña I.C.
      • Gautschi J.T.
      • Wang G.Y.
      • Sanders M.L.
      • Schmitz F.J.
      • France D.
      • et al.
      Psammaplins from the sponge Pseudoceratina purpurea: inhibition of both histone deacetylase and DNA methyltransferase.
      ]. Procain and procainamide directly bind to CpG-rich DNA, interrupting the interaction between DNMTs and their target DNA sequences. EGCG and psammaplins are both involved in several cellular processes and also affect enzymes other than DNMT [
      • Villar-Garea A.
      • Fraga M.F.
      • Espada J.
      • Esteller M.
      Procaine is a DNA-demethylating agent with growth-inhibitory effects in human cancer cells.
      ]. Until now, the DNMTs inhibition mechanisms of EGCG and psammaplins remain unclear.

      2.3 Interplay between DNA methylation and histone acetylation

      A tight correlation exists between [gene expression and DNA methylation] and [chromatin structure and DNA methylation]. Suppression of gene expression is frequently associated with methylated DNA and a dense chromatin structure, whereas active transcription is associated with unmethylated DNA and hyperacetylated open chromatin [
      • Szyf M.
      DNA methylation and demethylation as targets for anticancer therapy.
      ] (Fig. 3). Initially, DNA methylation was thought to unidirectionally affect chromatin structure. However, recent data in cancer cells now suggest a mutual interplay between both epigenetic modifications [
      • Szyf M.
      DNA methylation and demethylation as targets for anticancer therapy.
      ]. For example, in several cancer cell lines, combinations of DNMTi and HDACi have synergistic effects on the cellular homeostasis [
      • Zhu W.G.
      • Otterson G.A.
      The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells.
      ,
      • Belinsky S.A.
      • Klinge D.M.
      • Stidley C.A.
      • Issa J.P.
      • Herman J.G.
      • March T.H.
      • et al.
      Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer.
      ,
      • Klisovic M.I.
      • Maghraby E.A.
      • Parthun M.R.
      • Guimond M.
      • Sklenar A.R.
      • Whitman S.P.
      • et al.
      Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells.
      ]. Accordingly, so far, several ‘cocktails’ are in clinical trials as chemotherapeutics [
      • Brueckner B.
      • Lyko F.
      DNA methyltransferase inhibitors: old and new drugs for an epigenetic cancer therapy.
      ]. Also, in vitro, our group discovered a synergistic effect of DNMTi and HDACi with respect to the differentiated phenotype of normal primary cells [

      Rogiers V, Vanhaecke T, De Rop E, Fraczek J. Title of invention: stabilisation of the phenotypic properties of isolated primary cells. International patent number: PCT/EP2008/056706.

      ]. This will be further explained in the next section.
      Figure thumbnail gr3
      Fig. 3Epigenetic control of gene transcription. Inhibition of gene transcription typically corresponds to hypermethylated CpG islands in gene promoter regions and deacetylated histone tails at local chromatin domains. The indirect mechanism of gene silencing may involve binding of methyl-binding proteins (MeCp) to methylated cytosine and subsequent recruitment of HDAC-corepressor (CoR) complexes, resulting in a non-permissive heterochromatin status that blocks binding of transcription factors (TF) and polymerase II RNA complexes (PolII) to target promoter sequences. The direct mechanism may involve the direct interference of TF with HDAC or methylated CpG sites within the promoter. HDAC inhibitors (HDACi) and DNMT inhibitors (DNMTi) modulate the chromatin structure. They create an open, transcriptionally active euchromatin configuration at gene coding and regulatory regions, accessible for TF, thereby facilitating gene transcription. Abbreviations: 5-AzaC, decitabine; M, 5-methyl cytosine at CpGs; SB, sodium butyrate; TSA, trichostatin A; VPA, valproic acid.
      In summary, our findings show that transcription of genes, governing maintenance of a cell’s differentiated status in adult life and development, can be accomplished via targeting the expression of DNMTs and/or HDACs with increased chromatin accessibility of transcription factors to their target DNA as a result.

      3. Epigenetic modifiers as potent differentiation-promoting compounds in vitro

      3.1 Acquisition and stabilisation of a differentiated hepatic phenotype in vitro: an interplay between chromatin remodelling and liver-enriched transcription factors

      Of major interest, at least from a pharmaco-toxicological point of view, is the ability of hepatocytes to protect the organism from toxic chemical insults. Hepatocytes dispose of an ingenious multi-step enzymatic clearance system, i.e. xenobiotic biotransformation, and therefore constitute the main cell type of interest for in vitro hepatotoxicity and drug metabolism studies to date [

      Vanhaecke T, Rogiers V. Hepatocyte cultures in drug metabolism and toxicological research and testing. In: Phillips IR, Shephard EA, editors. Cytochrome P450 Protocols. Methods in molecular biology. 2nd ed. Totowa, NJ: Humana Press Inc.; 2006. p. 209–27.

      ].
      Ex vivo, though, cell–cell and cell–extracellular matrix disruptions, resulting from collagenase perfusion and subsequent oxidative stress response, trigger the activation of several ‘proliferative’ signaling cascades [
      • Elaut G.
      • Henkens T.
      • Papeleu P.
      • Snykers S.
      • Vinken M.
      • Vanhaecke T.
      • et al.
      Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures.
      ]. Unlike hepatocytes in vivo, primary hepatocytes in culture are unable to completely redifferentiate upon proliferation, resulting in a loss of the differentiated phenotype and concomitant deterioration of cytochrome P450 (CYP)-mediated xenobiotic biotransformation capacity [
      • Papeleu P.
      • Loyer P.
      • Vanhaecke T.
      • Elaut G.
      • Geerts A.
      • Guguen-Guillouzo C.
      • et al.
      Trichostatin A induces cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.
      ,
      • Elaut G.
      • Henkens T.
      • Papeleu P.
      • Snykers S.
      • Vinken M.
      • Vanhaecke T.
      • et al.
      Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures.
      ]. Another essential factor is the substantial decline in LETFs, controlling the transcription of numerous liver-specific genes [
      • Kyrmizi I.
      • Hatzis P.
      • Katrakili N.
      • Tronche F.
      • Gonzalez F.J.
      • Talianidis I.
      Plasticity and expanding complexity of the hepatic transcription factor network during liver development.
      ,
      • Akiyama T.E.
      • Gonzalez F.J.
      Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors.
      ,
      • Costa R.H.
      • Kalinichenko V.V.
      • Holterman A.X.
      • Wang X.
      Transcription factors in liver development, differentiation, and regeneration.
      ,
      • Baylin S.B.
      DNA methylation and gene silencing in cancer.
      ,
      • Elaut G.
      • Henkens T.
      • Papeleu P.
      • Snykers S.
      • Vinken M.
      • Vanhaecke T.
      • et al.
      Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures.
      ,
      • Schrem H.
      • Klempnauer J.
      • Borlak J.
      Liver-enriched transcription factors in liver function and development. Part I: the hepatocyte nuclear factor network and liver-specific gene expression.
      ,
      • Padgham C.R.
      • Boyle C.C.
      • Wang X.J.
      • Raleigh S.M.
      • Wright M.C.
      • Paine A.J.
      Alteration of transcription factor mRNAs during the isolation and culture of rat hepatocytes suggests the activation of a proliferative mode underlies their de-differentiation.
      ,
      • Rodriguez-Antona C.
      • Donato M.T.
      • Boobis A.
      • Edwards R.J.
      • Watts P.S.
      • Castell J.V.
      • et al.
      Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells.
      ]. Indeed, hepatocyte proliferation and differentiation are predominantly regulated at the transcriptional level [
      • Zaret K.S.
      • Watts J.
      • Xu J.
      • Wandzioch E.
      • Smale S.T.
      • Sekiya T.
      Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm.
      ]. Basically, eukaryotic gene transcription relies on the combinatorial binding of multiple specific trans-acting DNA-binding proteins, i.e. transcription factors, to particular DNA-sequence motifs in regulatory elements of a specific gene. Efficient gene expression is further often determined by interplays between different transcription factors, either adjacently or distantly located on the promoter, and by protein–protein interactions between transcription factors and coactivators/corepressors [
      • Wolffe A.P.
      • Guschin D.
      Review: chromatin structural features and targets that regulate transcription.
      ,
      • Reik W.
      Stability and flexibility of epigenetic gene regulation in mammalian development.
      ,
      • Schrem H.
      • Klempnauer J.
      • Borlak J.
      Liver-enriched transcription factors in liver function and development. Part I: the hepatocyte nuclear factor network and liver-specific gene expression.
      ]. Additionally, efficient binding of transcription factors and associated proteins to their cognate DNA-sequences requires a permissive chromatin configuration in order to drive gene expression The dynamic modulation of the chromatin architecture by e.g. DNA methylation and/or covalent histone modifications represents thus a basic machinery for transcriptional activation, repression and derepression [
      • Wolffe A.P.
      • Guschin D.
      Review: chromatin structural features and targets that regulate transcription.
      ,
      • Wang G.G.
      • Allis C.D.
      • Chi P.
      Chromatin remodeling and cancer, part I: covalent histone modifications.
      ,
      • Collas P.
      Epigenetic states in stem cells.
      ,
      • Reik W.
      Stability and flexibility of epigenetic gene regulation in mammalian development.
      ,
      • Yeo S.
      • Jeong S.
      • Kim J.
      • Han J.S.
      • Han Y.M.
      • Kang Y.K.
      Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells.
      ,
      • Perry P.
      • Sauer S.
      • Billon N.
      • Richardson W.D.
      • Spivakov M.
      • Warnes G.
      • et al.
      A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction.
      ] (Fig. 3).
      In hepatocytes, the liver-enriched transcription factors play an elemental role in hepatocyte-specific gene expression [
      • Kyrmizi I.
      • Hatzis P.
      • Katrakili N.
      • Tronche F.
      • Gonzalez F.J.
      • Talianidis I.
      Plasticity and expanding complexity of the hepatic transcription factor network during liver development.
      ,
      • Akiyama T.E.
      • Gonzalez F.J.
      Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors.
      ,
      • Costa R.H.
      • Kalinichenko V.V.
      • Holterman A.X.
      • Wang X.
      Transcription factors in liver development, differentiation, and regeneration.
      ,
      • Schrem H.
      • Klempnauer J.
      • Borlak J.
      Liver-enriched transcription factors in liver function and development. Part I: the hepatocyte nuclear factor network and liver-specific gene expression.
      ,
      • Zaret K.S.
      • Watts J.
      • Xu J.
      • Wandzioch E.
      • Smale S.T.
      • Sekiya T.
      Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm.
      ], and are as such key regulators of liver development, architecture and physiology. These trans-acting DNA-binding proteins are predominantly, but not exclusively, expressed in liver. It is hypothesized that the coordinated and timely expression of LETFs, in concert with ubiquitously expressed transcription factors such as NF1, Oct-1, Hex and other LETFs, is prerequisite for hepatocyte differentiation and constitutive liver-specific gene expression, including CYP-mediated xenobiotic biotransformation [
      • Akiyama T.E.
      • Gonzalez F.J.
      Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors.
      ,
      • Schrem H.
      • Klempnauer J.
      • Borlak J.
      Liver-enriched transcription factors in liver function and development. Part I: the hepatocyte nuclear factor network and liver-specific gene expression.
      ,
      • Rodriguez-Antona C.
      • Donato M.T.
      • Boobis A.
      • Edwards R.J.
      • Watts P.S.
      • Castell J.V.
      • et al.
      Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells.
      ,
      • Zaret K.S.
      • Watts J.
      • Xu J.
      • Wandzioch E.
      • Smale S.T.
      • Sekiya T.
      Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm.
      ]. Evidence is accumulating that recruitment of coactivators/corepressors, able to modulate the local chromatin configuration through post-translational histone modifications, mainly determine their transactivation potential. In this context, the transcriptional activation of LETFs critically depends on the recruitment of co-activator proteins with intrinsic HAT activity, such as CREB-binding protein (CBP), p300/CBP-associated factor (P/CAF) and SRC1, whereas co-repressor complexes containing HDAC negatively regulate liver-specific gene expression [
      • Kistanova E.
      • Dell H.
      • Tsantili P.
      • Falvey E.
      • Cladaras C.
      • Hadzopoulou-Cladaras M.
      The activation function-1 of hepatocyte nuclear factor-4 is an acidic activator that mediates interactions through bulky hydrophobic residues.
      ,
      • Green V.J.
      • Kokkotou E.
      • Ladias J.A.
      Critical structural elements and multitarget protein interactions of the transcriptional activator AF-1 of hepatocyte nuclear factor 4.
      ,
      • Wang J.C.
      • Stafford J.M.
      • Granner D.K.
      SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4.
      ,
      • Sladek F.M.
      • Ruse Jr., M.D.
      • Nepomuceno L.
      • Huang S.M.
      • Stallcup M.R.
      Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1.
      ,
      • Yoshida E.
      • Aratani S.
      • Itou H.
      • Miyagishi M.
      • Takiguchi M.
      • Osumu T.
      • et al.
      Functional association between CBP and HNF4 in trans-activation.
      ,
      • Dell H.
      • Hadzopoulou-Cladaras M.
      CREB-binding protein is a transcriptional coactivator for hepatocyte nuclear factor-4 and enhances apolipoprotein gene expression.
      ,
      • Torres-Padilla M.E.
      • Sladek F.M.
      • Weiss M.C.
      Developmentally regulated N-terminal variants of the nuclear receptor hepatocyte nuclear factor 4alpha mediate multiple interactions through coactivator and corepressor-histone deacetylase complexes.
      ,
      • Torres-Padilla M.E.
      • Weiss M.C.
      Effects of interactions of hepatocyte nuclear factor 4alpha isoforms with coactivators and corepressors are promoter-specific.
      ,
      • Martinez-Jimenez C.P.
      • Castell J.V.
      • Gomez-Lechon M.J.
      • Jover R.
      Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4alpha requires coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha and steroid receptor coactivator 1.
      ,
      • Puigserver P.
      • Adelmant G.
      • Wu Z.
      • Fan M.
      • Xu J.
      • O’Malley B.
      • et al.
      Activation of PPARgamma coactivator-1 through transcription factor docking.
      ,
      • Batsche E.
      • Desroches J.
      • Bilodeau S.
      • Gauthier Y.
      • Drouin J.
      Rb enhances p160/SRC coactivator-dependent activity of nuclear receptors and hormone responsiveness.
      ,
      • Ruse Jr., M.D.
      • Privalsky M.L.
      • Sladek F.M.
      Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain.
      ,
      • Maeda Y.
      • Seidel S.D.
      • Wei G.
      • Liu X.
      • Sladek F.M.
      Repression of hepatocyte nuclear factor 4alpha tumor suppressor p53: involvement of the ligand-binding domain and histone deacetylase activity.
      ,
      • Ban N.
      • Yamada Y.
      • Someya Y.
      • Miyawaki K.
      • Ihara Y.
      • Hosokawa M.
      • et al.
      Hepatocyte nuclear factor-1alpha recruits the transcriptional co-activator p300 on the GLUT2 gene promoter.
      ,
      • Soutoglou E.
      • Papafotiou G.
      • Katrakili N.
      • Talianidis I.
      Transcriptional activation by hepatocyte nuclear factor-1 requires synergism between multiple coactivator proteins.
      ,
      • Dohda T.
      • Kaneoka H.
      • Inayoshi Y.
      • Kamihira M.
      • Miyake K.
      • Iijima S.
      Transcriptional coactivators CBP and p300 cooperatively enhance HNF-1alpha-mediated expression of the albumin gene in hepatocytes.
      ,
      • Soutoglou E.
      • Viollet B.
      • Vaxillaire M.
      • Yaniv M.
      • Pontoglio M.
      • Talianidis I.
      Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity.
      ,
      • Rausa F.M.
      • Tan Y.
      • Costa R.H.
      Association between hepatocyte nuclear factor 6 (HNF-6) and FoxA2 DNA binding domains stimulates FoxA2 transcriptional activity but inhibits HNF-6 DNA binding.
      ,
      • Yoshida Y.
      • Hughes D.E.
      • Rausa 3rd, F.M.
      • Kim I.M.
      • Tan Y.
      • Darlington G.J.
      • et al.
      C/EBPalpha and HNF6 protein complex formation stimulates HNF6-dependent transcription by CBP coactivator recruitment in HepG2 cells.
      ,
      • Erickson R.L.
      • Hemati N.
      • Ross S.E.
      • MacDougald O.A.
      p300 coactivates the adipogenic transcription factor CCAAT/enhancer-binding protein alpha.
      ,
      • Chen P.L.
      • Riley D.J.
      • Chen Y.
      • Lee W.H.
      Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs.
      ,
      • Charles A.
      • Tang X.
      • Crouch E.
      • Brody J.S.
      • Xiao Z.X.
      Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP-mediated transcription.
      ]. In detail, HNF-4α, directly interacts with SRC1, CBP and p300, resulting in its increased transcriptional activity [
      • Kistanova E.
      • Dell H.
      • Tsantili P.
      • Falvey E.
      • Cladaras C.
      • Hadzopoulou-Cladaras M.
      The activation function-1 of hepatocyte nuclear factor-4 is an acidic activator that mediates interactions through bulky hydrophobic residues.
      ,
      • Green V.J.
      • Kokkotou E.
      • Ladias J.A.
      Critical structural elements and multitarget protein interactions of the transcriptional activator AF-1 of hepatocyte nuclear factor 4.
      ,
      • Wang J.C.
      • Stafford J.M.
      • Granner D.K.
      SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4.
      ,
      • Sladek F.M.
      • Ruse Jr., M.D.
      • Nepomuceno L.
      • Huang S.M.
      • Stallcup M.R.
      Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1.
      ,
      • Yoshida E.
      • Aratani S.
      • Itou H.
      • Miyagishi M.
      • Takiguchi M.
      • Osumu T.
      • et al.
      Functional association between CBP and HNF4 in trans-activation.
      ,
      • Dell H.
      • Hadzopoulou-Cladaras M.
      CREB-binding protein is a transcriptional coactivator for hepatocyte nuclear factor-4 and enhances apolipoprotein gene expression.
      ,
      • Torres-Padilla M.E.
      • Sladek F.M.
      • Weiss M.C.
      Developmentally regulated N-terminal variants of the nuclear receptor hepatocyte nuclear factor 4alpha mediate multiple interactions through coactivator and corepressor-histone deacetylase complexes.
      ]. The level of upregulation is isoform-dependent [
      • Torres-Padilla M.E.
      • Sladek F.M.
      • Weiss M.C.
      Developmentally regulated N-terminal variants of the nuclear receptor hepatocyte nuclear factor 4alpha mediate multiple interactions through coactivator and corepressor-histone deacetylase complexes.
      ,
      • Torres-Padilla M.E.
      • Weiss M.C.
      Effects of interactions of hepatocyte nuclear factor 4alpha isoforms with coactivators and corepressors are promoter-specific.
      ]. In human hepatoma cells, transactivation of CYP1A1, CYP1A2 and CYP2C9 by HNF-4α relies on the presence of the PPAR-gamma coactivator 1alpha (PGC-1α) [
      • Martinez-Jimenez C.P.
      • Castell J.V.
      • Gomez-Lechon M.J.
      • Jover R.
      Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4alpha requires coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha and steroid receptor coactivator 1.
      ]. PGC-1α, a key regulator of hepatic gluconeogenesis, lacks HAT-activity, but enables transcription through the assembly of a complex, containing SRC1 and CBP/p300 [
      • Puigserver P.
      • Adelmant G.
      • Wu Z.
      • Fan M.
      • Xu J.
      • O’Malley B.
      • et al.
      Activation of PPARgamma coactivator-1 through transcription factor docking.
      ]. Likewise, in differentiating Caco2-cells, Rb strengthens HNF-4-dependent activation of the α-antitrypsin gene through reinforcement of the SRC-coactivator function [
      • Batsche E.
      • Desroches J.
      • Bilodeau S.
      • Gauthier Y.
      • Drouin J.
      Rb enhances p160/SRC coactivator-dependent activity of nuclear receptors and hormone responsiveness.
      ]. Conversely, interaction with SMRT or p53, recruiting HDACs to transcription factors, represses HNF-4α activity [
      • Torres-Padilla M.E.
      • Sladek F.M.
      • Weiss M.C.
      Developmentally regulated N-terminal variants of the nuclear receptor hepatocyte nuclear factor 4alpha mediate multiple interactions through coactivator and corepressor-histone deacetylase complexes.
      ,
      • Ruse Jr., M.D.
      • Privalsky M.L.
      • Sladek F.M.
      Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain.
      ,
      • Maeda Y.
      • Seidel S.D.
      • Wei G.
      • Liu X.
      • Sladek F.M.
      Repression of hepatocyte nuclear factor 4alpha tumor suppressor p53: involvement of the ligand-binding domain and histone deacetylase activity.
      ]. HNF-1α, on the other hand, physically interacts with the HATs CBP/p300, P/CAF, SRC-1, and RAC3 [
      • Ban N.
      • Yamada Y.
      • Someya Y.
      • Miyawaki K.
      • Ihara Y.
      • Hosokawa M.
      • et al.
      Hepatocyte nuclear factor-1alpha recruits the transcriptional co-activator p300 on the GLUT2 gene promoter.
      ,
      • Soutoglou E.
      • Papafotiou G.
      • Katrakili N.
      • Talianidis I.
      Transcriptional activation by hepatocyte nuclear factor-1 requires synergism between multiple coactivator proteins.
      ]. CBP and PCAF, on one hand, and CBP and p300, on the other hand, synergistically upregulate HNF-1-mediated transactivation [
      • Soutoglou E.
      • Papafotiou G.
      • Katrakili N.
      • Talianidis I.
      Transcriptional activation by hepatocyte nuclear factor-1 requires synergism between multiple coactivator proteins.
      ,
      • Dohda T.
      • Kaneoka H.
      • Inayoshi Y.
      • Kamihira M.
      • Miyake K.
      • Iijima S.
      Transcriptional coactivators CBP and p300 cooperatively enhance HNF-1alpha-mediated expression of the albumin gene in hepatocytes.
      ], whilst association of HNF-1α with HDAC1 – through NCoR– impairs its transcriptional activity. Treatment with the HDACi TSA disrupts latter corepressor complex, enhancing in turn HNF-1α-mediated transcription [
      • Soutoglou E.
      • Viollet B.
      • Vaxillaire M.
      • Yaniv M.
      • Pontoglio M.
      • Talianidis I.
      Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity.
      ]. A good example of LETFs acting in a cooperative, synergistic regulatory network is the interaction between HNF-6 and HNF-3. In this respect, HNF-6 potentiates HNF-3β transcriptional activity by recruiting p300/CBP HAT proteins, [
      • Rausa F.M.
      • Tan Y.
      • Costa R.H.
      Association between hepatocyte nuclear factor 6 (HNF-6) and FoxA2 DNA binding domains stimulates FoxA2 transcriptional activity but inhibits HNF-6 DNA binding.
      ] whilst HNF-6-dependent transcription is stimulated by complex formation between HNF-6 and C/EBPα, also recruiting coactivator CBP [
      • Yoshida Y.
      • Hughes D.E.
      • Rausa 3rd, F.M.
      • Kim I.M.
      • Tan Y.
      • Darlington G.J.
      • et al.
      C/EBPalpha and HNF6 protein complex formation stimulates HNF6-dependent transcription by CBP coactivator recruitment in HepG2 cells.
      ]. The transactivation potential of C/EBPα, in turn, is promoted by direct interaction with either CBP/p300 or Rb [
      • Erickson R.L.
      • Hemati N.
      • Ross S.E.
      • MacDougald O.A.
      p300 coactivates the adipogenic transcription factor CCAAT/enhancer-binding protein alpha.
      ,
      • Chen P.L.
      • Riley D.J.
      • Chen Y.
      • Lee W.H.
      Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs.
      ,
      • Charles A.
      • Tang X.
      • Crouch E.
      • Brody J.S.
      • Xiao Z.X.
      Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP-mediated transcription.
      ]. Apparently, this binding to C/EBPα robustly stimulates nucleosomal HAT activity of CBP [
      • Chen C.J.
      • Deng Z.
      • Kim A.Y.
      • Blobel G.A.
      • Lieberman P.M.
      Stimulation of CREB binding protein nucleosomal histone acetyltransferase activity by a class of transcriptional activators.
      ]. C/EBPβ-dependent transactivation is further mediated by direct acetylation through association with the HATs p300 and PCAF [
      • Mink S.
      • Haenig B.
      • Klempnauer K.H.
      Interaction and functional collaboration of p300 and C/EBPbeta.
      ,
      • Cui T.X.
      • Piwien-Pilipuk G.
      • Huo J.S.
      • Kaplani J.
      • Kwok R.
      • Schwartz J.
      Endogenous CCAAT/enhancer binding protein beta and p300 are both regulated by growth hormone to mediate transcriptional activation.
      ,
      • Cesena T.I.
      • Cardinaux J.R.
      • Kwok R.
      • Schwartz J.
      CCAAT/enhancer-binding protein (C/EBP) beta is acetylated at multiple lysines: acetylation of C/EBPbeta at lysine 39 modulates its ability to activate transcription.
      ,
      • Wiper-Bergeron N.
      • Salem H.A.
      • Tomlinson J.J.
      • Wu D.
      • Hache R.J.
      Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPbeta by GCN5.
      ]. Conversely, interaction between SMRT or subcomponents of the Sin3 complex, e.g. Sin3a, and HDAC1 represses its transcriptional activity [
      • Wiper-Bergeron N.
      • Salem H.A.
      • Tomlinson J.J.
      • Wu D.
      • Hache R.J.
      Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPbeta by GCN5.
      ]. Of particular interest is HNF3 as, in contrast to other LETFs, it directly affects chromatin conformations of numerous hepatic genes such as albumin (ALB) and α-foetoprotein (AFP), likely without interference with intermediary coactivators bearing HAT-activity or ATP-dependent enzymes. More specifically, the C-terminal domain of the protein binds to histones H3/H4 within highly compacted chromatin, creating a local, open nucleosomal domain, which facilitates further interactions between transcription factors, such as GATA4 and other LETFs, and DNA [
      • Shim E.Y.
      • Woodcock C.
      • Zaret K.S.
      Nucleosome positioning by the winged helix transcription factor HNF3.
      ,
      • Cirillo L.A.
      • Lin F.R.
      • Cuesta I.
      • Friedman D.
      • Jarnik M.
      • Zaret K.S.
      Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4.
      ,
      • Chaya D.
      • Hayamizu T.
      • Bustin M.
      • Zaret K.S.
      Transcription factor FoxA (HNF3) on a nucleosome at an enhancer complex in liver chromatin.
      ]. This HNF3-mediated transcriptional competence is designated as a prerequisite for the onset of liver ontogeny, and more specifically for the developmental activation of genes required for hepatocyte differentiation and function.

      3.2 Effect of HDAC and DNMT inhibition on liver-specific gene expression

      An overview of the most important hepatic genes affected by HDACi and DNMTi in various in vitro models is presented in Table 1. Out of these data, it appears that both the cellular origin and the type, concentration and exposure time of epigenetic modifier used, critically determine the final outcome. In transformed cells, including colon cells [
      • Lea M.A.
      • Ibeh C.
      • Shah N.
      • Moyer M.P.
      Induction of differentiation of colon cancer cells by combined inhibition of kinases and histone deacetylase.
      ,
      • Mayo C.
      • Lloreta J.
      • Real F.X.
      • Mayol X.
      In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression.
      ,
      • Hinnebusch B.F.
      • Meng S.
      • Wu J.T.
      • Archer S.Y.
      • Hodin R.A.
      The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation.
      ], glioma cells [
      • Gensert J.M.
      • Baranova O.V.
      • Weinstein D.E.
      • Ratan R.R.
      CD81, a cell cycle regulator, is a novel target for histone deacetylase inhibition in glioma cells.
      ,
      • Asklund T.
      • Appelskog I.B.
      • Ammerpohl O.
      • Ekström T.J.
      • Almqvist P.M.
      Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells.
      ], pancreatic cells [
      • Kumagai T.
      • Wakimoto N.
      • Yin D.
      • Gery S.
      • Kawamata N.
      • Takai N.
      • et al.
      Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (Vorinostat, SAHA) profoundly inhibits the growth of human pancreatic cancer cells.
      ], breast cells [
      • Munster P.N.
      • Troso-Sandoval T.
      • Rosen N.
      • Rifkind R.
      • Marks P.A.
      • Richon V.M.
      The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces differentiation of human breast cancer cells.
      ] and hepatoma cells [
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
      ], HDACi-mediated growth arrest is frequently, at least in vitro, associated with induction of differentiation. As such, HDAC inhibition upregulates C/EBPα, HNF-1α, HNF-3α, HNF-3β and HNF-4α levels in various hepatoma cells, resulting in increased CYP expression [
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
      ]. Yet, in spite of this beneficial outcome, the contribution of HDACi in long-term in vitro models is hampered by co-occurrence of cell death. Indeed, HDACi, including TSA, butyrate, valproic acid, SAHA, OSU-HDAC42 and ITF2357, induce both in vitro and in vivo apoptosis in hepatoma cells [
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
      ,
      • Lu Y.S.
      • Kashida Y.
      • Kulp S.K.
      • Wang Y.C.
      • Wang D.
      • Hung J.H.
      • et al.
      Efficacy of a novel histone deacetylase inhibitor in murine models of hepatocellular carcinoma.
      ,
      • Herold C.
      • Ganslmayer M.
      • Ocker M.
      • Hermann M.
      • Geerts A.
      • Hahn E.G.
      • et al.
      The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells.
      ,
      • Wakabayashi K.
      • Saito H.
      • Kaneko F.
      • Nakamoto N.
      • Tada S.
      • Hibi T.
      Gene expression associated with the decrease in malignant phenotype of human liver cancer cells following stimulation with a histone deacetylase inhibitor.
      ]. For example, SAHA reduces, dose- and time-dependently, the viability of HepG2 and Huh6 hepatoma cells as a result of concomitant activation of both extrinsic and intrinsic apoptosis signaling cascades [
      • Emanuele S.
      • Lauricella M.
      • Carlisi D.
      • Vassallo B.
      • D’Anneo A.
      • Di Fazio P.
      • et al.
      SAHA induces apoptosis in hepatoma cells and synergistically interacts with the proteasome inhibitor Bortezomib.
      ]. In contrast, primary hepatocytes are relatively well-resistant against HDACi-induced apoptosis [
      • Papeleu P.
      • Loyer P.
      • Vanhaecke T.
      • Elaut G.
      • Geerts A.
      • Guguen-Guillouzo C.
      • et al.
      Trichostatin A induces cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.
      ,
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      ,
      • Papeleu P.
      • Wullaert A.
      • Elaut G.
      • Henkens T.
      • Vinken M.
      • Laus G.
      • et al.
      Inhibition of NF-kappaB activation by the histone deacetylase inhibitor 4-Me2N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytes.
      ,
      • Armeanu S.
      • Pathil A.
      • Venturelli S.
      • Mascagni P.
      • Weiss T.S.
      • Gottlicher M.
      • et al.
      Apoptosis on hepatoma cells but not on primary hepatocytes by histone deacetylase inhibitors valproate and ITF2357.
      ,
      • Papeleu P.
      • Vanhaecke T.
      • Elaut G.
      • Vinken M.
      • Henkens T.
      • Snykers S.
      • et al.
      Differential effects of histone deacetylase inhibitors in tumor and normal cells-what is the toxicological relevance?.
      ]. In this context, exposure of normal primary rat hepatocytes to TSA and 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me2N-BAVAH) upregulates C/EBPα and HNF-4α expression [
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      ], which in turn increases CYP protein and activity levels (Fig. 4, Fig. 5), gap junctional communication and ALB secretion, without any evidence of cell death [
      • Papeleu P.
      • Loyer P.
      • Vanhaecke T.
      • Elaut G.
      • Geerts A.
      • Guguen-Guillouzo C.
      • et al.
      Trichostatin A induces cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.
      ,
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      ,
      • Vinken M.
      • Henkens T.
      • Snykers S.
      • Lukaszuk A.
      • Tourwé D.
      • Rogiers V.
      • et al.
      The novel histone deacetylase inhibitor 4-Me2N-BAVAH differentially affects cell junctions between primary hepatocytes.
      ,
      • Vinken M.
      • Henkens T.
      • Vanhaecke T.
      • Papeleu P.
      • Geerts A.
      • Van Rossen E.
      • et al.
      Trichostatin A enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes.
      ,
      • Henkens T.
      • Vinken M.
      • Lukaszuk A.
      • Tourwé D.
      • Vanhaecke T.
      • Rogiers V.
      Differential effects of hydroxamate histone deacetylase inhibitors on cellular functionality and gap junctions in primary cultures of mitogen-stimulated hepatocytes.
      ,
      • Vanhaecke T.
      • Henkens T.
      • Kass G.E.
      • Rogiers V.
      Effect of the histone deacetylase inhibitor trichostatin A on spontaneous apoptosis in various types of adult rat hepatocyte cultures.
      ]. Moreover, in epidermal growth factor (EGF)-stimulated primary rat hepatocytes, we could demonstrate that TSA and 4-Me2N-BAVAH even delay the onset of spontaneous apoptosis as evidenced by reduced pro-caspase-3 processing, decreased pro-apoptotic Bid and Bax levels and increased anti-apoptotic BclxL expression [
      • Papeleu P.
      • Loyer P.
      • Vanhaecke T.
      • Elaut G.
      • Geerts A.
      • Guguen-Guillouzo C.
      • et al.
      Trichostatin A induces cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.
      ,
      • Papeleu P.
      • Wullaert A.
      • Elaut G.
      • Henkens T.
      • Vinken M.
      • Laus G.
      • et al.
      Inhibition of NF-kappaB activation by the histone deacetylase inhibitor 4-Me2N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytes.
      ].
      Table 1Effects of epigenetic modifiers on the expression of important liver genes in various in vitro models.
      Epigenetic modifierSpeciesModelRegulationaReference
      HDAC inhibition
      Phase I biotransformation
       CYP1A1TSARatPrimary hepatocytes
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      HumanMammary carcinoma MCF-7 cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      HumanHeLa cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      SAHAHumanMammary carcinoma MCF-7 cells
      • Hooven L.A.
      • Mahadevan B.
      • Keshava C.
      • Johns C.
      • Pereira C.
      • Desai D.
      • et al.
      Effects of suberoylanilide hydroxamic acid and trichostatin A on induction of cytochrome P450 enzymes and benzo[a]pyrene DNA adduct formation in human cells.
       CYP1A2TSAHumanMammary carcinoma MCF-7 cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      HumanHeLa cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      MousePrimary hepatocytes
      • Jin B.
      • Ryu D.Y.
      Regulation of CYP1A2 by histone deacetylase inhibitors in mouse hepatocytes.
      ButyrateMousePrimary hepatocytes
      • Jin B.
      • Ryu D.Y.
      Regulation of CYP1A2 by histone deacetylase inhibitors in mouse hepatocytes.
       CYP1B1TSAHumanMammary carcinoma MCF-7 cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      HumanHeLa cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      HumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Chen H.J.
      • Tian H.
      • Edenberg H.J.
      Differential regulation of the alcohol dehydrogenase 1B (ADH1B) and ADH1C genes by DNA methylation and histone deacetylation.
       CYP2B1/2TSARatPrimary hepatocytes
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      ValproateRatPrimary hepatocytes
      • Rogiers V.
      • Akrawi M.
      • Vercruysse A.
      • Phillips I.R.
      • Shephard E.A.
      Effects of the anticonvulsant, valproate, on the expression of components of the cytochrome-P-450-mediated monooxygenase system and glutathione S-transferases.
       CYP3A4TSAHumanHepG2 hepatoma cells
      • Rodrı´guez-Antona C.
      • Bort R.
      • Jover R.
      • Tindberg N.
      • Ingelman-Sundberg M.
      • Gómez-Lechón M.J.
      • et al.
      Transcriptional regulation of human CYP3A4 basal expression by CCAAT enhancer-binding protein alpha and hepatocyte nuclear factor-3 gamma.
      HumanHepG2 hepatoma cells
      • Kim J.Y.
      • Ahn M.R.
      • Kim D.K.
      • Sheen Y.Y.
      Histone deacetylase inhibitor stimulate CYP3A4 proximal promoter activity in HepG2 cells.
       CYP3A2TSARatPrimary hepatocytes
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
       ADH1ATSAHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Chen H.J.
      • Tian H.
      • Edenberg H.J.
      Differential regulation of the alcohol dehydrogenase 1B (ADH1B) and ADH1C genes by DNA methylation and histone deacetylation.
      HeLa cells
       ADH1BTSAHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Chen H.J.
      • Tian H.
      • Edenberg H.J.
      Differential regulation of the alcohol dehydrogenase 1B (ADH1B) and ADH1C genes by DNA methylation and histone deacetylation.
      HeLa cells
       ADH1CTSAHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Chen H.J.
      • Tian H.
      • Edenberg H.J.
      Differential regulation of the alcohol dehydrogenase 1B (ADH1B) and ADH1C genes by DNA methylation and histone deacetylation.
      HeLa cells
       FMO3TSAHumanHepG2 hepatoma cells
      • Klick D.E.
      • Shadley J.D.
      • Hines R.N.
      Differential regulation of human hepatic flavin containing monooxygenase 3 (FMO3) by CCAAT/enhancer-binding protein beta (C/EBPbeta) liver inhibitory and liver activating proteins.
      Phase II biotransformation
       GSTP1TSAHumanHep3B hepatoma cells
      • Bakker J.
      • Lin X.
      • Nelson W.G.
      Methyl-CpG binding domain protein 2 represses transcription from hypermethylated pi-class glutathione S-transferase gene promoters in hepatocellular carcinoma cells.
      ButyrateHumanColon carcinoma HT29 cells
      • Ebert M.N.
      • Klinder A.
      • Peters W.H.
      • Schäferhenrich A.
      • Sendt W.
      • Scheele J.
      • et al.
      Expression of glutathione S-transferases (GSTs) in human colon cells and inducibility of GSTM2 by butyrate.
      ButyrateHumanPrimary colon cells
      • Pool-Zobel B.L.
      • Selvaraju V.
      • Sauer J.
      • Kautenburger T.
      • Kiefer J.
      • Richter K.K.
      • et al.
      Butyrate may enhance toxicological defence in primary, adenoma and tumor human colon cells by favourably modulating expression of glutathione S-transferases genes, an approach in nutrigenomics.
       GSTA1/2ButyrateHumanColon carcinoma HT29 cells
      • Ebert M.N.
      • Klinder A.
      • Peters W.H.
      • Schäferhenrich A.
      • Sendt W.
      • Scheele J.
      • et al.
      Expression of glutathione S-transferases (GSTs) in human colon cells and inducibility of GSTM2 by butyrate.
      HumanPrimary colon cells
      • Pool-Zobel B.L.
      • Selvaraju V.
      • Sauer J.
      • Kautenburger T.
      • Kiefer J.
      • Richter K.K.
      • et al.
      Butyrate may enhance toxicological defence in primary, adenoma and tumor human colon cells by favourably modulating expression of glutathione S-transferases genes, an approach in nutrigenomics.
       GSTA4TSA, MS-275, VPAMouseMC3T3-E1 preosteoblasts
      • Schroeder T.M.
      • Nair A.K.
      • Staggs R.
      • Lamblin A.F.
      • Westendorf J.J.
      Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors.
       GSTM2ButyrateHumanColon carcinoma HT29 cells
      • Ebert M.N.
      • Klinder A.
      • Peters W.H.
      • Schäferhenrich A.
      • Sendt W.
      • Scheele J.
      • et al.
      Expression of glutathione S-transferases (GSTs) in human colon cells and inducibility of GSTM2 by butyrate.
       GSTT1/2ButyrateHumanColon carcinoma HT29 cells
      • Ebert M.N.
      • Klinder A.
      • Peters W.H.
      • Schäferhenrich A.
      • Sendt W.
      • Scheele J.
      • et al.
      Expression of glutathione S-transferases (GSTs) in human colon cells and inducibility of GSTM2 by butyrate.
      HumanPrimary colon cells
      • Pool-Zobel B.L.
      • Selvaraju V.
      • Sauer J.
      • Kautenburger T.
      • Kiefer J.
      • Richter K.K.
      • et al.
      Butyrate may enhance toxicological defence in primary, adenoma and tumor human colon cells by favourably modulating expression of glutathione S-transferases genes, an approach in nutrigenomics.
       UGT2B7VPAHumanProstate carcinoma LNCaP cells
      • Valentini A.
      • Biancolella M.
      • Amati F.
      • Gravina P.
      • Miano R.
      • Chillemi G.
      • et al.
      Valproic acid induces neuroendocrine differentiation and UGT2B7 up-regulation in human prostate carcinoma cell line.
       UGT2B11VPAHumanProstate carcinoma LNCaP cells
      • Valentini A.
      • Biancolella M.
      • Amati F.
      • Gravina P.
      • Miano R.
      • Chillemi G.
      • et al.
      Valproic acid induces neuroendocrine differentiation and UGT2B7 up-regulation in human prostate carcinoma cell line.
       SULT2B1TSAHumanHaCaT keratinocytes
      • Lee Y.C.
      • Higashi Y.
      • Luu C.
      • Shimizu C.
      • Strott C.A.
      Sp1 elements in SULT2B1b promoter and 5′-untranslated region of mRNA: Sp1/Sp2 induction and augmentation by histone deacetylase inhibition.
      Ammonia removalTSAHumanHepG2 and Huh-7 hepatoma cells
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
      Albumin synthesis/secretionTSAHumanHepG2 and Huh-7 hepatoma cells
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
      RatPrimary hepatocytes
      • Papeleu P.
      • Loyer P.
      • Vanhaecke T.
      • Elaut G.
      • Geerts A.
      • Guguen-Guillouzo C.
      • et al.
      Trichostatin A induces cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.
      ,
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      Gap junctional intercellular communication
       Cx32TSAHumanHuh-7 hepatoma cells
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Tanaka S.
      • Shirabe K.
      • Ijima H.
      • et al.
      cDNA microarray analysis in hepatocyte differentiation in Huh7 cells.
      HumanNeural progenitor cells
      • Khan Z.
      • Akhtar M.
      • Asklund T.
      • Juliusson B.
      • Almqvist P.M.
      • Ekström T.J.
      HDAC inhibition amplifies gap junction communication in neural progenitors: potential for cell-mediated enzyme prodrug therapy.
      HumankB nasopharyngeal tumor cells
      • Hattori Y.
      • Fukushima M.
      • Maitani Y.
      Non-viral delivery of the connexin 43 gene with histone deacetylase inhibitor to human nasopharyngeal tumor cells enhances gene expression and inhibits in vivo tumor growth.
      HumanProstate carcinoma cells
      • Hernandez M.
      • Shao Q.
      • Yang X.J.
      • Luh S.P.
      • Kandouz M.
      • Batist G.
      • et al.
      A histone deacetylation-dependent mechanism for transcriptional repression of the gap junction gene cx43 in prostate cancer cells.
      HumanNormal prostate epithelial cells
      • Hernandez M.
      • Shao Q.
      • Yang X.J.
      • Luh S.P.
      • Kandouz M.
      • Batist G.
      • et al.
      A histone deacetylation-dependent mechanism for transcriptional repression of the gap junction gene cx43 in prostate cancer cells.
      RatPrimary hepatocytes
      • Vinken M.
      • Henkens T.
      • Vanhaecke T.
      • Papeleu P.
      • Geerts A.
      • Van Rossen E.
      • et al.
      Trichostatin A enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes.
      4-Me2N-BAVAHRatPrimary hepatocytes
      • Vinken M.
      • Henkens T.
      • Snykers S.
      • Lukaszuk A.
      • Tourwé D.
      • Rogiers V.
      • et al.
      The novel histone deacetylase inhibitor 4-Me2N-BAVAH differentially affects cell junctions between primary hepatocytes.
       Cx26TSAHumanHuh-7 hepatoma cells
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Tanaka S.
      • Shirabe K.
      • Ijima H.
      • et al.
      cDNA microarray analysis in hepatocyte differentiation in Huh7 cells.
      RatPrimary hepatocytes
      • Vinken M.
      • Henkens T.
      • Vanhaecke T.
      • Papeleu P.
      • Geerts A.
      • Van Rossen E.
      • et al.
      Trichostatin A enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes.
      4-Me2N-BAVAHRatPrimary hepatocytes
      • Vinken M.
      • Henkens T.
      • Snykers S.
      • Lukaszuk A.
      • Tourwé D.
      • Rogiers V.
      • et al.
      The novel histone deacetylase inhibitor 4-Me2N-BAVAH differentially affects cell junctions between primary hepatocytes.
       Cx43TSAHumanHuh-7 hepatoma cells
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Tanaka S.
      • Shirabe K.
      • Ijima H.
      • et al.
      cDNA microarray analysis in hepatocyte differentiation in Huh7 cells.
      RatPrimary hepatocytes
      • Vinken M.
      • Henkens T.
      • Vanhaecke T.
      • Papeleu P.
      • Geerts A.
      • Van Rossen E.
      • et al.
      Trichostatin A enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes.
      SAHARatRas transformed WB-F344 liver epithelial cells
      • Ogawa T.
      • Hayashi T.
      • Tokunou M.
      • Nakachi K.
      • Trosko J.E.
      • Chang C.C.
      • et al.
      Suberoylanilide hydroxamic acid enhances gap junctional intercellular communication via acetylation of histone containing connexin 43 gene locus.
      HumanPeritoneal mesothelial cells
      • Ogawa T.
      • Hayashi T.
      • Tokunou M.
      • Nakachi K.
      • Trosko J.E.
      • Chang C.C.
      • et al.
      Suberoylanilide hydroxamic acid enhances gap junctional intercellular communication via acetylation of histone containing connexin 43 gene locus.
      RatC6 glioma cells
      • Ammerpohl O.
      • Thormeyer D.
      • Khan Z.
      • Appelskog I.B.
      • Gojkovic Z.
      • Almqvist P.M.
      • et al.
      HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells.
      PhenylbutyrateHumanGlioblastoma cells
      • Asklund T.
      • Appelskog I.B.
      • Ammerpohl O.
      • Ekström T.J.
      • Almqvist P.M.
      Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells.
      HumanGlioblastoma cells
      • Robe P.A.
      • Jolois O.
      • N’Guyen M.
      • Princen F.
      • Malgrange B.
      • Merville M.P.
      • et al.
      Modulation of the HSV-TK/ganciclovir bystander effect by n-butyrate in glioblastoma: correlation with gap-junction intercellular communication.
      HumanNeural progenitor cells
      • Khan Z.
      • Akhtar M.
      • Asklund T.
      • Juliusson B.
      • Almqvist P.M.
      • Ekström T.J.
      HDAC inhibition amplifies gap junction communication in neural progenitors: potential for cell-mediated enzyme prodrug therapy.
      HumankB nasopharyngeal tumor cells
      • Hattori Y.
      • Fukushima M.
      • Maitani Y.
      Non-viral delivery of the connexin 43 gene with histone deacetylase inhibitor to human nasopharyngeal tumor cells enhances gene expression and inhibits in vivo tumor growth.
      RatGlioma cells
      • Robe P.A.
      • Jolois O.
      • N’Guyen M.
      • Princen F.
      • Malgrange B.
      • Merville M.P.
      • et al.
      Modulation of the HSV-TK/ganciclovir bystander effect by n-butyrate in glioblastoma: correlation with gap-junction intercellular communication.
      Sodium butyrateHumankB nasopharyngeal tumor cells
      • Hattori Y.
      • Fukushima M.
      • Maitani Y.
      Non-viral delivery of the connexin 43 gene with histone deacetylase inhibitor to human nasopharyngeal tumor cells enhances gene expression and inhibits in vivo tumor growth.
      HumanGlioblastoma cells
      • Robe P.A.
      • Jolois O.
      • N’Guyen M.
      • Princen F.
      • Malgrange B.
      • Merville M.P.
      • et al.
      Modulation of the HSV-TK/ganciclovir bystander effect by n-butyrate in glioblastoma: correlation with gap-junction intercellular communication.
      4-Me2N-BAVAHRatPrimary hepatocytes
      • Vinken M.
      • Henkens T.
      • Snykers S.
      • Lukaszuk A.
      • Tourwé D.
      • Rogiers V.
      • et al.
      The novel histone deacetylase inhibitor 4-Me2N-BAVAH differentially affects cell junctions between primary hepatocytes.
      Liver-enriched transcription factors
       C/EBPαTSAHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
      RatPrimary hepatocytes
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      SAHAHumanPancreatic carcinoma PANC-1 cells
      • Kumagai T.
      • Wakimoto N.
      • Yin D.
      • Gery S.
      • Kawamata N.
      • Takai N.
      • et al.
      Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (Vorinostat, SAHA) profoundly inhibits the growth of human pancreatic cancer cells.
       C/EBPβButyrate, TSARatIntestinal epithelial cell line IEC-6
      • Desilets A.
      • Gheorghiu I.
      • Yu S.J.
      • Seidman E.G.
      • Asselin C.
      Inhibition by deacetylase inhibitors of IL-1-dependent induction of haptoglobin involves CCAAT/Enhancer-binding protein isoforms in intestinal epithelial cells.
       C/EBPδButyrate, TSARatIntestinal epithelial cell line IEC-6
      • Desilets A.
      • Gheorghiu I.
      • Yu S.J.
      • Seidman E.G.
      • Asselin C.
      Inhibition by deacetylase inhibitors of IL-1-dependent induction of haptoglobin involves CCAAT/Enhancer-binding protein isoforms in intestinal epithelial cells.
       HNF1αTSARatPrimary hepatocytes
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
       HNF1βDepsipeptideHumanPapillary thyroid cancer cells
      • Xu J.
      • Hershman J.M.
      Histone deacetylase inhibitor depsipeptide represses nicotinamide N-methyltransferase and hepatocyte nuclear factor-1beta gene expression in human papillary thyroid cancer cells.
       HNF4αTSARatPrimary hepatocytes
      • Henkens T.
      • Papeleu P.
      • Elaut G.
      • Vinken M.
      • Rogiers V.
      • Vanhaecke T.
      Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes.
      Other
       Apolipoprotein CIIITSAHumanHepG2 and Huh-7 hepatoma cells
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
       HCFXTSAHumanHepG2 and Huh-7 hepatoma cells
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
       Glutamine synthetaseTSAHumanHepG2 and Huh-7 hepatoma cells
      • Yamashita Y.
      • Shimada M.
      • Harimoto N.
      • Rikimaru T.
      • Shirabe K.
      • Tanaka S.
      • et al.
      Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells.
      DNMT inhibition
      Phase I biotransformation
       CYP1A15-Aza-dCHumanMammary carcinoma MCF-7 cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      HumanHeLa cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
       CYP1A25-Aza-dCHumanMammary carcinoma MCF-7 cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      HumanHeLa cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      MousePrimary hepatocytes
      • Jin B.
      • Park D.W.
      • Nam K.W.
      • Oh G.T.
      • Lee Y.S.
      • Ryu D.Y.
      CpG methylation of the mouse CYP1A2 promoter.
      MouseHepa1c1c7 hepatoma cells
      • Jin B.
      • Park D.W.
      • Nam K.W.
      • Oh G.T.
      • Lee Y.S.
      • Ryu D.Y.
      CpG methylation of the mouse CYP1A2 promoter.
       CYP1B15-Aza-dCHumanMammary carcinoma MCF-7 cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      HumanHeLa cells
      • Nakajima M.
      • Iwanari M.
      • Yokoi T.
      Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.
      5-AzaCHumanHepG2 hepatoma cells
      • Shehin S.E.
      • Stephenson R.O.
      • Greenlee W.F.
      Transcriptional regulation of the human CYP1B1 gene. Evidence for involvement of an aryl hydrocarbon receptor response element in constitutive expression.
       CYP3A45-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
       CYP3A55-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
       CYP3A75-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
       FMO35-Aza-dCHumanHepG2 hepatoma cells
      • Klick D.E.
      • Shadley J.D.
      • Hines R.N.
      Differential regulation of human hepatic flavin containing monooxygenase 3 (FMO3) by CCAAT/enhancer-binding protein beta (C/EBPbeta) liver inhibitory and liver activating proteins.
      Phase II biotransformation
       GSTP15-Aza-dCHumanHep3B hepatoma cells
      • Bakker J.
      • Lin X.
      • Nelson W.G.
      Methyl-CpG binding domain protein 2 represses transcription from hypermethylated pi-class glutathione S-transferase gene promoters in hepatocellular carcinoma cells.
      ProcainamideHumanLNCaP prostate cancer cells
      • Lin X.
      • Asgari K.
      • Putzi M.J.
      • Gage W.R.
      • Yu X.
      • Cornblatt B.S.
      • et al.
      Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide.
       UGT1A65-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
       UGT2B155-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
       UGT2B285-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
      Gap junctional intercellular communication
       Cx325-Aza-dCHumanCaki-2 renal cell carcinoma cells
      • Hirai A.
      • Yano T.
      • Nishikawa K.
      • Suzuki K.
      • Asano R.
      • Satoh H.
      • et al.
      Down-regulation of connexin 32 gene expression through DNA methylation in a human renal cell carcinoma cell.
      HumanCaki-2 renal cell carcinoma cells
      • Hagiwara H.
      • Sato H.
      • Ohde Y.
      • Takano Y.
      • Seki T.
      • Ariga T.
      • et al.
      5-Aza-2′-deoxycytidine suppresses human renal carcinoma cell growth in a xenograft model via up-regulation of the connexin 32 gene.
      HumanHK-2 renal tubular cells
      • Hirai A.
      • Yano T.
      • Nishikawa K.
      • Suzuki K.
      • Asano R.
      • Satoh H.
      • et al.
      Down-regulation of connexin 32 gene expression through DNA methylation in a human renal cell carcinoma cell.
       Cx265-Aza-dCHumanMammary carcinoma cells
      • Tan L.W.
      • Bianco T.
      • Dobrovic A.
      Variable promoter region CpG island methylation of the putative tumor suppressor gene Connexin 26 in breast cancer.
      HumanMammary carcinoma cells
      • Singal R.
      • Tu Z.J.
      • Vanwert J.M.
      • Ginder G.D.
      • Kiang D.T.
      Modulation of the connexin26 tumor suppressor gene expression through methylation in human mammary epithelial cell lines.
      HumanLung cancer cells
      • Chen Y.
      • Hühn D.
      • Knösel T.
      • Pacyna-Gengelbach M.
      • Deutschmann N.
      • Peterson I.
      Downregulation of connexin 26 in human lung cancer is related to promoter methylation.
      HumanEsophageal cells
      • Loncarek J.
      • Yamasaki H.
      • Levillain P.
      • Milinkevitch S.
      • Mesnil M.
      The expression of the tumor suppressor gene connexin 26 is not mediated by methylation in human esophageial cancer cells.
       Cx435-Aza-dCHumanEsophageal cancer cells
      • Loncarek J.
      • Yamasaki H.
      • Levillain P.
      • Milinkevitch S.
      • Mesnil M.
      The expression of the tumor suppressor gene connexin 26 is not mediated by methylation in human esophageial cancer cells.
      HumanCervical adenocarcinoma cells
      • King T.J.
      • Fukushima L.H.
      • Donlon T.A.
      • Hieber A.D.
      • Shimabukuro K.A.
      • Bertram J.S.
      Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression.
      HumanCNE-1 nasopharyngeal cancer cells
      • Yi Z.c.
      • Wang H.
      • Zhang G.Y.
      • Xia B.
      Downregulation of connexin 43 in nasopharyngeal carcinoma cells is related to promoter methylation.
      Liver-enriched transcription factors
       C/EBPα5-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
       C/EBPβ5-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.
       C/EBPγ5-Aza-dCHumanHepG2 hepatoma cells
      • Dannenberg L.O.
      • Edenbergh H.J.
      Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation.