1、Epigenetics in liver disease:from biologyto therapeuticsTimothy Hardy,1,2Derek A Mann11Fibrosis Laboratories,Instituteof Cellular Medicine,NewcastleUniversity,Newcastle uponTyne,UK2Department ofGastroenterology andHepatology,The Newcastleupon Tyne Hospitals NHSFoundation Trust,Newcastleupon Tyne,UKC
2、orrespondence toProfessor Derek A Mann,Fibrosis Laboratories,Instituteof Cellular Medicine,4th FloorWilliam Leech Bdg.,NewcastleUniversity,Framlington Place,Newcastle upon Tyne NE24HH,UK;derek.mannncl.ac.ukReceived 28 July 2016Revised 29 July 2016Accepted 1 August 2016Published Online First13 Septem
3、ber 2016To cite:Hardy T,Mann DA.Gut 2016;65:18951905.ABSTRACTKnowledge of the fundamental epigenetic mechanismsgoverning gene expression and cellular phenotype aresufficiently advanced that novel insights into theepigenetic control of chronic liver disease are nowemerging.Hepatologists are in the pr
4、ocess of sheddinglight on the roles played by DNA methylation,histone/chromatin modifications and non-coding RNAs in specificliver pathologies.Alongside these discoveries areadvances in the technologies for the detection andquantification of epigenetic biomarkers,either directlyfrom patient tissue o
5、r from body fluids.The premise forthis review is to survey the recent advances in the fieldof liver epigenetics and to explore their potential fortranslation by industry and clinical hepatologists for thedesign of novel therapeutics and diagnostic/prognosticbiomarkers.In particular,we present findin
6、gs in thecontext of hepatocellular carcinoma,fibrosis and non-alcoholic fatty liver disease,where there is urgent unmetneed for new clinical interventions and biomarkers.INTRODUCTIONIrrespective of aetiology,the progression of chronicliver damage and inflammation to cirrhosis and/orcancer is highly
7、variable between individuals.Ifliver damage persists,a large percentage of patientscan expect their disease to advance over their life-time.However,the course and rate of progressionof their disease is often unpredictable using prog-nostic tools currently available in the clinic.As anexample,a recen
8、t study of paired biopsies from108 patients with non-alcoholic fatty liver disease(NAFLD)revealed that 42%patients had fibrosisprogression,40%had no change in fibrosis,while18%had fibrosis regression;9%underwent pro-gressionfromblandsteatosistofibrosing-steatohepatitis.1However,the degree of progres
9、-sion was variable,ranging from one to three stages,and the molecular basis for this variability remainspoorly defined.While genome-wide associationstudies(GWAS)have identified genetic variants thatshow association with fibrosis and/or hepatocellularcarcinoma(HCC),2 3to date their predictive valuewi
10、th regard to outcomes in clinical practice havebeen limited.Furthermore,the biological functionof variants remains challenging to elucidate anddirect patient benefit appears distant.It is wellestablished that there are numerous non-geneticfactors that influence the progression of liverdisease,includ
11、ing patient age,sex,body compos-ition,diet,exercise,microbiome,alcohol consump-tion and their history of smoking.4The study ofepigenetics is,in essence,the investigation of hownon-genetic factors act upon the genome to influ-ence gene expression and phenotype.Epigeneticscan therefore enable us to in
12、terrogate the mechan-isms that underlie disease phenotype,and it ishoped to shed new light on the basis for interpati-ent variability in disease progression.Furthermore,the highly dynamic nature of epigenetic mechan-isms and their regulators in response to environ-mentalcuesoffershopefortheadventofe
13、pigenetic therapies in liver disease,as is nowoccurring in clinical oncology.However,this dyna-mism,coupled with the complexity of epigeneticmechanisms that can operate both locally at thegene level as well as globally across the epigenome,presents significant challenges.Improvements innext generati
14、on sequencing technologies and theirability to generate genome-wide quantitative dataare helping to meet this challenge.As an example,DNA methylation can now be quantified in asequence-specific manner across the entire genometo generate a methylome map,and there is poten-tial to carry this out on ei
15、ther single cells or circu-lating free DNA.56Similarly,emerging omicsapproaches to the study of histone modificationswill offer unparralled insights into the functionalassociations of alterations in the chromatin land-scape of cells and disease processes.In this review,we will describe the basic epi
16、genetic players andmechanisms before discussing recent important dis-coveries in liver epigenetics.EPIGENETICS AND THE CASE FOR ITS STUDYIN LIVER DISEASEConrad Waddington established the field of epigen-etics in 1942 when he proposed an uncoupling ofgenotype and phenotype,implying that regulatorypro
17、cesses linked the two.7The first conclusive evi-dencethatcellularphenotypeisdictatedbymechanisms other than that encoded within theDNA sequence came from classic experiments infrogs,forwhichSirJohnGurdonwaslaterawarded the Nobel prize.He demonstrated thattransplantation of a nucleus from a fully dif
18、feren-tiated somatic cell into a denucleated egg supportedthe development of a tadpole.The conclusion wasthat the genome sequence is stable through devel-opment and is not per se determining phenotype;rather the latter is dictated by developmentally pro-grammed patterns of gene expression that arere
19、sponsive to the cellular microenvironment.8Themechanisms that regulate this flow of genetic infor-mation include the actions of key transcriptionfactors(as demonstrated by the experiments ofShinya Yamanaka,who shared the 2012 Nobelprize with Gurdon)that operate in concert withepigenetic factors to m
20、odulate the rate at whichindividualgenesaretranscribedintoRNA.Critically,theactivitiesofepigeneticandHardy T,Mann DA.Gut 2016;65:18951905.doi:10.1136/gutjnl-2015-3112921895Recent advances in basic sciencetranscription factors are plastic and highly dynamic,being influ-enced by the metabolic state of
21、 the cell and its ever changingextracellular environment.In turn,even the most differentiatedpopulation of nucleated cells will display phenotypic heterogen-eity and have the potential for behavioural reprogramming.Thenormal healthy functions of the liver,as with any tissue,are crit-ically dependent
22、 upon robust maintenance of the behaviouralproperties of its constituent cells.Unhealthy perturbation ofliver function reflects a loss of homeostasis,whereby the con-stituent liver cells fail to maintain their distinct phenotypiccharacteristics in the face of challenges in the local microenvir-onmen
23、t induced by metabolic,xenobiotic,immune or microbialtriggers.Hence,by illuminating the epigenetic modificationsthat are associated with specific pathologies such as cirrhosis orcancer we can better understand the molecular basis for loss ofcellular homeostasis in chronic liver disease(box 1).THE MA
24、JOR EPIGENETIC MECHANISMSDNA methylationDNA can be covalently modified and the best-known modifica-tion is methylation of cytosine at its 5th carbon ring,which ismainly,although not exclusively,found at cytosines within CpGdinucleotides.The most well-established role of the me-CpGmark is when it occ
25、urs at high density within the so-called CpGislands which predominantly traverse 50promoter regions;methylation at these regions results in strong transcriptionalrepression.Importantly,methyl-CpG is a signal for recognitionof DNA by specific proteins containing a so-called methylbinding domain(MBD).
26、The MBD family(MBD1,MBD2,MeCP2 and MBD4)mediate transcriptional repression at CpGislands.9The majority of methylated CpG islands are develop-mentally established and become stable in the adult.However incancer,methylation can be acquired at normally unmethylatedCpG islands and is usually accompanied
27、 by a second repressiveepigenetic mark,methylation of histone H3 at its lysine 27residue,which compacts chromatin and inhibits transcription.10Genome-wide,single-base resolution mapping of DNA methyla-tion has revealed that there is considerable dynamic turnoveroutside of CpG islands and suggests th
28、at the occupancy of tran-scription factors at these CpG sites is associated with loss ofmethylation.11 12Despite this knowledge,there is at presentinconclusive evidence that loss of methylation outside CpGislands instructs transcriptional information at a local level.Further insights may emerge now
29、that the enzymes that instructDNAmethylation(DNAmethyltransferases:DNMT1,DNMT3a and DNMT3b)and demethylation(ten-eleven trans-location enzymes TET13)are beginning to be functionallydefined.DNMT1 is a maintenance methylase that during celldivision recognises a hemimethylated site on a new DNA stranda
30、nd regenerates the bimethylated state.13In this way,CpGBox 1An epigenetic glossary5-hydroxymethylcytosine:the oxidation of 5-methylcytosine modified DNA,by the Tet family of enzymes.5-methylcytosine:an epigenetic modification of DNA occurring at CpG dinucleotides.Bisulfite conversion:the selective d
31、eamination of unmethylated cytosine bases to uracil by treatment with sodium bisulfite;used formethylation analysis.Chromatin:the formed complex of DNA and histones required for nuclear compaction.CpG islands:regions of DNA enriched for CpG dinucleotides;CpG islands are 200 bp long,located at transc
32、riptional start sites,andpredominantly unmethylated.Differentially methylated region:regions of DNA in an organisms genome that is methylated differentially between disease phenotypes.DNA methylation:an epigenetic modification in which a methyl group is covalently bonded to the 5th carbon of the cyt
33、osine pyrimidinering in a CpG dinucleotide,frequently(but not exclusively)associated with gene silencing.DNA methyltransferases:a group of enzymes that catalyse the addition of a methyl group to DNA.Members include DNMT1 required formaintenance of DNA methylation and DNMT 3a/b involved in de novo me
34、thylation.Euchromatin:unpacked chromatin,allowing access for transcription factors and gene expression.Heterochromatin:compacted chromatin,inaccessible to transcription factors,containing poorly expressed genes.Histone acetyltransferase:enzyme that acetylates histones at specific lysine residues.His
35、tone deacetylase:enzyme that remove acetyl groups from N(6)-acetyl-lysine residues on a histone.Histone:the core protein around which DNA is wound tight,ordering DNA into nucleosomes.Core histones include H2A,H2B,H3 and H4.Histone modification:post-translational modifications of histones including t
36、he addition or removal of methylation,acetylation,phosphorylation,ubiquitination,sumoylation and marks.Histone variants:variant proteins that can be inserted into nucleosomes,and may have intrinsic gene regulatory functions.Long non-coding RNA:non-protein-coding RNA200 nucleotides with gene regulato
37、ry properties.Examples include Xist,HOX transcriptantisense intergenic RNA,highly upregulated in liver cancer and high expression in hepatocellular carcinoma.microRNA:Small non-protein coding RNA(22 nucleotides)that regulate cellular processes by controlling transcription and translationof mRNA.Non-
38、coding RNA(ncRNA):the majority of the genome is transcribed into non-protein encoding RNA,involved in many cellular processes.Examples include microRNAs,small interfering RNAs(siRNAs),Piwi-interacting RNAs,long non-coding RNAs(lncRNAs)and longintergenic RNAs.Nucleosome:the basic structural unit of c
39、hromatin,allowing compaction;one nucleosome is comprised of 147 bp of DNA wrappedaround a histone octamer including two molecules each of the core histones H2A/B,H3 and H4.1896Hardy T,Mann DA.Gut 2016;65:18951905.doi:10.1136/gutjnl-2015-311292Recent advances in basic sciencemethylation patterns are
40、faithfully maintained in daughter cells.However,de novo methylation does occur in the absence of celldivision and is regulated by DNMT3a and DNMT3b.14TheTET enzymes catalyse the step-wise oxidation of methyl groupson DNA leading to the eventual restoration of the unmodifiedcytosine residue.15Experim
41、ental deletion of the TET enzymesresults in increased methylation at gene enhancers and subtlealterations in the expression of genes linked with these enhancerregions.16 17Turnover of DNA methylation may therefore be anongoing process in most cell types and has potential to bemodulated via changes i
42、n the relative expression of DNMT andTET proteins.The discovery that vitamin C can enhance TETactivity in cells supports this idea and indicates the existence ofmechanisms for modulating DNA methylation in response toenvironmental cues.18Histone modifications and chromatin remodellingIn order to ach
43、ieve the feat of compaction required for 2 m ofDNA to be condensed into a human nucleus,DNA interactswith specialised proteins to form tightly packed chromatin;forchromosomal processes to occur such as gene transcription,thismust be iteratively unpacked and repacked in a regulatedmanner,providing an
44、 opportunity for dynamic gene regulation.The most basic level of chromatin packing is known as thenucleosome,each core particle consisting of 147 bp of doublestranded DNA wrapped around a complex of eight histone pro-teins(two copies each of H2A,H2B,H3 and H4)seen underan electron microscope as bead
45、s on a string;linker DNA beingthe string and the beads representing the nucleosome core par-ticle.Each of the core histones has an unstructured N-terminalamino acid tail extension that can be subject to a plethora ofcovalent,post-translational modifications that control aspects ofchromatin structure
46、 and function,either directly affecting chro-matin structure or comprising signals to be recognised byprotein effectors.19Histones can be acetylated,methylated onlysine and arginine,phosphorylated on serine,ubiquitinated,sumoylated and ADP-ribosylated.Histone acetylation loosenschromatin to transcri
47、ptionally active euchromatin.By contrast,trimethylation oflysine9 of histone 3(H3K9me3)andH3K27me3 are associated with condensed,transcriptionallysilent heterochromatin.However,histone lysine methylation canalso promote transcription depending on which lysine is modi-fied for example,H3K4me3 and H3K
48、36me2/3 are generallyassociated with euchromatin.Variants of the core histones(except H4)can be inserted by ATP-dependent chromatin-remodelling complexes and regulate nucleosome structure.Asan example,exchange of H2 for H2A.Z is important for geneexpression,20while exchange for macroH2A is associate
49、d withtranscriptional repression.21Histone modifications are highlydynamic,and regulated by writer and eraser enzymes thatadd or remove post-translational modifications,respectively.They can serve as marks for recruitment of ATP-dependentchromatin remodelling complexes such as switch/sucrose non-fer
50、mentable(SWI/SNF)that remodel nucleosome and chromatinstructure allowing access to gene regulatory proteins;mamma-lian SWI/SNF can slide nucleosomes on DNA or can exchangeor extrude histones,promoting gene activation.10In contrast,repressive chromatin remodellers act on nucleosomes to formdensely pa