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16: 6-21 Genes Dev.Adrian BirdDNA methylation patterns and
epigenetic
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2012 - Published bygenesdev.cshlp.org Downloaded from REVIEWDNA
methylation patternsand epigenetic memoryAdrian Bird1Wellcome Trust
Centre for Cell Biology, University of Edinburgh, Edinburgh EH9
3JR, UKThe character of a cell is defined by its constituent
pro-teins, whicharetheresultofspecificpatternsofgeneexpression.
Crucial determinants of gene expression pat-terns are DNA-binding
transcription factors that choosegenes for transcriptional
activation or repression by
rec-ognizingthesequenceofDNAbasesintheirpromoterregions.
Interactionofthesefactorswiththeircognatesequences triggers a
chainof events, ofteninvolvingchanges in the structure of
chromatin, that leads to theassembly of an active transcription
complex (e.g., Cosmaet al. 1999). But the types of transcription
factors presentin a cell are not alone sufficient to define its
spectrum ofgeneactivity, asthetranscriptional potential of
age-nomecanbecomerestrictedinastablemannerduringdevelopment. The
constraints imposed by developmen-tal history probably account for
the very low efficiencyof cloning animals from the nuclei of
differentiated cells(Rideout et al. 2001; Wakayama and Yanagimachi
2001).A transcription factors only model would predict
thatthegeneexpressionpatternofadifferentiatednucleuswould be
completely reversible upon exposure to a newspectrumoffactors.
Althoughmanyaspectsofexpres-sioncanbereprogrammedinthisway(Gurdon1999),some
marks of differentiation are evidently so stable thatimmersion in
an alien cytoplasmcannot erase thememory.The genomic sequence of a
differentiated cell isthought to be identical in most cases to that
of the
zy-gotefromwhichitisdescended(mammalianBandTcellsbeinganobviousexception).Thismeansthatthemarks
of developmental history are unlikely to becaused by widespread
somatic mutation. Processes lessirrevocable than mutation fall
under the umbrella termepigenetic mechanisms. A current definition
of epige-neticsis:
Thestudyofmitoticallyand/ormeioticallyheritablechangesingenefunctionthat
cannot beex-plained by changes inDNAsequence (Russo et al.1996).
There are two epigenetic systems that affect ani-mal development
and fulfill the criterion of heritability:DNAmethylationand the
Polycomb-trithorax group(Pc-G/trx) protein complexes. (Histone
modification hassome attributes of an epigenetic process, but the
issue ofheritability has yet to be resolved.) This review
concernsDNAmethylation, focusingonthegeneration, inheri-tance,
andbiological significanceof
genomicmethyl-ationpatternsinthedevelopment of mammals. Datawill be
discussed favoring the notion that DNA methyl-ation may only affect
genes that are already silenced byother mechanisms in the embryo.
Embryonic transcrip-tion, on the other hand, may cause the
exclusion of theDNA methylation machinery. The heritability of
meth-ylation states and the secondary nature of the decision
toinvite or exclude methylation support the idea that
DNAmethylationisadaptedfor aspecificcellular memoryfunction in
development. Indeed, the possibility will bediscussed that DNA
methylation and Pc-G/trx may rep-resent alternativesystems of
epigeneticmemorythathave been interchanged over evolutionary time.
AnimalDNA methylation has been the subject of several recentreviews
(Bird and Wolffe 1999; Bestor 2000; Hsieh 2000;Costello and Plass
2001; Jones and Takai 2001). For re-cent reviews of plant and
fungal DNA methylation, seeFinnegan et al. (2000), Martienssen and
Colot (2001), andMatzke et al. (2001).Variable patterns of DNA
methylation in
animalsAprerequisiteforunderstandingthefunctionofDNAmethylationisknowledgeofitsdistributioninthege-nome.
Inanimals, thespectrumof methylationlevelsandpatternsisverybroad.
Atthelowextremeisthenematode worm Caenorhabditis elegans, whose
genomelacks detectablem5Canddoes not encodeaconven-tional
DNAmethyltransferase. Another invertebrate,the insect Drosophila
melanogaster, long thought to bedevoidof methylation,
hasaDNAmethyltransferase-like gene (Hung et al. 1999; Tweedie et
al. 1999) and isreported to contain very low m5C levels (Gowher et
al.2000; Lyko et al. 2000), although mostly in the CpT
di-nucleotide rather than in CpG, which is the major targetfor
methylation in animals. Most other invertebrate ge-nomes have
moderately high levels of methyl-CpG con-centrated in large domains
of methylated DNA separatedby equivalent domains of unmethylated
DNA (Bird et al.1979; Tweedie et al. 1997). This mosaic methylation
pat-tern has been confirmed at higher resolution in the sea1E-MAIL
[email protected]; FAX 0131-650-5379.Article and publication are at
http://www.genesdev.org/cgi/doi/10.1101/gad.947102.6 GENES &
DEVELOPMENT 16:621 2002 by Cold Spring Harbor Laboratory Press ISSN
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Downloaded from squirt, Cionaintestinalis(Simmenetal. 1999).
Attheopposite extreme from C. elegans are the vertebrate
ge-nomes,whichhavethehighestlevelsofm5Cfoundinthe animal kingdom.
Vertebrate methylation is dis-persed over much of the genome, a
pattern referred to asglobal methylation. The variety of animal DNA
methyl-ationpatterns highlights thepossibilitythat
differentdistributions reflect different functions for the
DNAmethylation system (Colot and Rossignol 1999).Mammalian DNA
methylation patterns vary in timeand spaceIn human somatic cells,
m5C accounts for 1% of
totalDNAbasesandthereforeaffects70%80%ofallCpGdinucleotides in the
genome (Ehrlich 1982). This
averagepatternconcealsintriguingtemporal andspatial varia-tion.
Duringadiscretephaseof
earlymousedevelop-ment,methylationlevelsinthemousedeclinesharplyto
30% of the typical somatic level (Monk et al. 1987;Kafri et al.
1992). De novo methylation restores normallevels by the time of
implantation. A much more
limiteddropinmethylationoccursinthefrogXenopuslaevis(Stancheva and
Meehan 2000), and no drop is seen in thezebrafish, Danio rerio
(MacLeod et al. 1999). Even withinvertebrates, therefore,
interspecies variation is seen thatcouldreflect
differencesinthepreciseroleplayedbymethylation in these organisms.
For mice and probablyother mammals, however, the cycle of early
embryonicdemethylation followed by de novo methylation is criti-cal
in determining somatic DNA methylation patterns.A genome-wide
reduction in methylation is also seen inprimordial germ cells (Tada
et al. 1997; Reik et al. 2001)during the proliferative oogonial and
spermatogonialstages.The most striking feature of vertebrate DNA
methyl-ationpatternsisthepresenceof CpGislands, thatis,unmethylated
GC-rich regions that possess high relativedensitiesof
CpGandarepositionedat the5 endsofmany human genes (for review, see
Bird 1987). Compu-tational analysisof
thehumangenomesequencepre-dicts 29,000 CpG islands (Lander et al.
2001; Venter et al.2001). Earlier studies estimatedthat 60%of
humangenes areassociatedwithCpGislands, of whichthegreat majority
are unmethylated at all stages of develop-ment and in all tissue
types (Antequera and Bird 1993).Because many CpG islands are
located at genes that havea tissue-restricted expressionpattern, it
follows thatCpGislands canremainmethylation-free
evenwhentheirassociatedgeneissilent.Forexample,thetissue-specifically
expressed human-globin (Bird et al. 1987)and 2(1) collagen (McKeon
et al. 1982) genes have
CpGislandsthatremainunmethylatedinalltestedtissues,regardless of
expression.A small but significant proportion of all CpG
islandsbecome methylated during development, and when thishappens
the associated promoter is stably silent. Devel-opmentally
programmed CpG-island methylation of thiskind is involved in
genomic imprinting and X chromo-some inactivation (see below). The
de novo methylationevents occur in germ cells or the early embryo
(Jaenischet al. 1982), suggesting that de novo methylation is
par-ticularly active at these stages. There is evidence,
how-ever,thatdenovomethylationcanalsooccurinadultsomaticcells.AsignificantfractionofallhumanCpGislandsarepronetoprogressivemethylationincertaintissues
during aging (for review, see Issa 2000), or in ab-normal cells
such as cancers (for review, see Baylin andHerman 2000) and
permanent cell lines (Harris 1982; An-tequera et al. 1990; Jones et
al. 1990). The rate of accu-mulation of methylated CpGs in somatic
cells appears tobeveryslow. For example, denovomethylationof
aprovirus inmurine erythroleukemia cells
tookmanyweekstocomplete(Lorinczetal. 2000). Similarly,
therecoveryof global DNAmethylationlevels followingchronic
treatment of mouse cells with the DNA
meth-ylationinhibitor5-azacytidinerequiredmonths(Flatauet al.
1984).Howdo patterns of methylated and
unmethylatedmammalianDNAarise indevelopment andhowarethey
maintained? Why are CpG islands usually, but notalways,
methylation-free?Whatcausesmethylationofbulknon-CpG-islandDNA?
These burning questionscannot be answered definitively at present,
but there aredistinct hypotheses that have been addressed
experimen-tally. The available data will be conveniently
consideredinthree parts: (1) mechanisms for maintaining
DNAmethylation patterns; (2) mechanisms and consequencesof
methylationgain; and(3) mechanisms andconse-quences of methylation
loss.Maintenance methylationnot so
simpleMaintenancemethylationdescribestheprocessesthatreproduce DNA
methylation patterns between cell gen-erations. The simplest
conceivable mechanism for main-tenance depends on semiconservative
copying of the pa-rental-strand methylation pattern onto the
progenyDNAstrand(HollidayandPugh1975; Riggs1975). Inkeeping with
the model, the methylating
enzymeDNMT1preferstomethylatethosenewCpGswhosepartnersontheparentalstrandalreadycarryamethylgroup
(Bestor 1992; Pradhan et al. 1999). Thus a
patternofmethylatedandnonmethylatedCpGsalongaDNAstrandtendstobecopied,
andthisprovidesawayofpassing epigenetic information between cell
generations.TheideathatmammalianDNAmethylationpatternsareestablishedinearlydevelopmentbydenovometh-yltransferases
DNMT3AandDNMT3B(Okanoet al.1998a, 1999; Hsieh1999b)
andthencopiedtosomaticcells by the maintenance DNA
methyltransferaseDNMT1 is elegant and simple, but, as discussed
below,maynot fullyexplainpersistenceof methylationpat-terns during
cell
proliferation.Experimentsthatfirstshowedreplicationofmethyl-ationpatternsonartificiallymethylatedDNAalsore-vealed
a relatively low fidelity for the process (Pollack etal. 1980;
Wigler et al. 1981). After many cell generations,methylation of the
introduced DNA was retained, but atamuchlowerlevel
thaninthestartingplasmid. TheDNA methylation and epigenetic
memoryGENES & DEVELOPMENT 7Cold Spring Harbor Laboratory Press
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from failureof maintenancewasestimatedtooccurwithafrequency of 5%
per CpG site per cell division. Quan-titative studies of an
endogenous CpGsite broadlyagreed with this figure (Riggs et al.
1998). Cell clones inwhich this site was initially unmethylated
acquiredmethylationandcloneswhereit wasmethylatedlostmethylation.
The rate of change was estimated at
4%percellgeneration.Errorratesofthismagnitudemeanthat a detailed
methylation pattern would eventually be-come indistinct as cells
proliferate. Indeed, dynamicchanges in detailed methylation
patterns have been ob-served in monoclonal lyomyomas (Silva et al.
1993) andat the methylated FMR1 gene (Stger et al. 1997).
Thesestudiesestablishedthat clonal populationsof cellsdonot
havethehomogeneous methylationpatterns thatwould be predicted by
the replication model of mainte-nance methylation. Not only does
DNA methyltransfer-ase fail to complete half-methylated sites at a
significantrate, but also significant de novo methylation occurs
atunmethylated sites.At first sight, these findings appear to
undermine theconcept of maintenance methylation, but this does
notfollow. Although detailed methylation patterns may
notbemaintainedatthelevelofasingleCpGnucleotide,themethylationstatusofDNAdomainsappearstobefaithfullypropagatedduringdevelopment(Pfeiferetal.1990).CpGislands,forexample,keeptheiroverallun-methylated
state (or methylated state) extremely stablythrough multiple cell
generations. DNMT1 is partly re-sponsible for this stability, but
there is likely to be
an-otherasyetunknowncomponenttothemaintenanceprocess. Dramatic
evidence for this alternative mainte-nance mechanism comes from the
finding that CpG-is-land methylation is stably maintained even in
the appar-ent absence of the only known maintenance DNA
meth-yltransferase, DNMT1 (Rhee et al. 2000). Asimilarphenomenon
may account for the maintenance of allele-specific
DNAmethylationimprints under conditionswhere the concentration of
DNMT1 is severely limiting(Jaenisch 1997).De novo DNA methylation
by default?The origin of DNA methylation patterns is a
long-stand-ing mystery in the field. The de novo
methyltransferasesDNMT3A and DNMT3B (Okano et al. 1998a, 1999)
arehighly expressed in early embryonic cells, and it is at
thisstage that most programmed de novo methylation eventsoccur.
What determineswhichregionsof thegenomeshould be methylated? An
extreme possibility is that
denovoDNAmethylationinearlymammaliandevelop-mentisanindiscriminateprocesspotentiallyaffectingall
CpGs. Compatible with the default model is the ap-parent absence of
intrinsically unmethylatable DNA se-quences inmammaliangenomes.
EvenCpGislands,most of which are unmethylated at all times in
normalcells, canacquiremethylationunder special
develop-mentalcircumstancesorinabnormalcells(permanentcell lines or
cancer cells). It is clear, however, that not allregionsof
thegenomeareequallyaccessibletoDNAmethyltransferases. DNMT3B in
particular is known tobe required for de novo methylation of
specific genomicregions, as mice or human patients with DNMT3B
mu-tationsaredeficientinmethylationofpericentromericrepetitiveDNAsequencesandat
CpGislandsontheinactive X chromosome (Miniou et al. 1994; Okano et
al.1998b; Hansen et al. 2000; Kondo et al. 2000). DNMT3Bmay
therefore be adapted to methylate regions of
silentchromatin.Evidence that accessory factors are also needed to
en-sure appropriate methylation came initially from
plants,wheretheSNF2-likeproteinDDM1wasshowntobeessential for full
methylation of the Arabidopsisthalianagenome(Jeddelohetal. 1999).
Anequivalentdependence is seen in animals, as mutations in
humanATRX (Gibbons et al. 2000) and mouse Lsh2 genes (Den-nisetal.
2001), bothof whichencoderelativesof thechromatin-remodeling
protein SNF2, have significant ef-fects on global DNA methylation
patterns. Loss of LSH2protein, inparticular, matches the phenotype
of theDDM1 mutation in Arabidopsis, for both mutants
losemethylation of highly repetitive DNA sequences, but
re-tainsomemethylationelsewhereinthegenome. Per-haps efficient
global methylation of the genome requiresperturbation of chromatin
structure by these chromatin-remodeling proteins so that DNMTs can
gain access totheDNA.
CollaborationbetweenDNMTsandfactorsthatallowthemaccesstospecializedchromosomalre-gions
may be particularly important in regions that areheterochromatic
and inaccessible. Although the net re-sult of these processes is
apparently global genomicmethylation, the evidence for selectivity
means that theword default is probably not appropriate.Targeting de
novo methylation to preferredDNA sequencesAnother hypothesis to
explain global methylation is thattheDNAmethylationmachineryis
preferentiallyat-tractedbycertainDNAsequencesinthemammaliangenome(Turker
1999). Thepresenceof highlevelsofmethylation in DNA outside such a
DNA methylationcenter couldbe explainedbyspreading intothe
sur-rounding DNA. Barriers to spreading would lead to
theformationof CpGislands. Ahypothetical trigger
forDNAmethylationisDNAsequencerepetition, whichcan promote de novo
methylation in filamentous fungiandplants under
certaincircumstances (Selker 1999;Martienssen and Colot 2001). The
most suggestive evi-dence in mammals concerns manipulation of
transgenecopy number at a single locus in the mouse genome us-ing
cre-lox technology (Garrick et al. 1998). High
levelsoftransgenerepetitionwerefoundtocausesignificanttransgenesilencingandconcomitantmethylation.
Theefficiencyofexpressionincreasedascopynumberwasreducedatthelocus,
andthelevel of methylationde-creased. Whether repetition caused
methylation directly,or indirectly as a consequence of some other
event (e.g.,transcriptional silencing; see below), is not known.The
clearest definition of a DNA methylation centerBird8 GENES &
DEVELOPMENTCold Spring Harbor Laboratory Press on February 14, 2012
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comesfromthefungusNeurospora, whereshortTpA-rich segments of DNA
were found to induce methylation(Miao et al. 2000). Identification
of a mammalian
DNAmethylationcenterlocatedupstreamofthemousead-enine
phophoribosyltransferase (APRT) gene has been re-ported (Mummaneni
et al. 1993; Yates et al. 1999). Theregion contains B1 repetitive
elements and attracts highlevels of de novo methylation upon
transfection into em-bryonic cells, althoughthe effect is relative,
becausemanyDNAsequencesaresubjecttodenovomethyl-ationinthesecells.TheAPRTmethylationcenterbe-comesmethylatedinDNMT1-deficient
EScells, sup-porting the idea that it corresponds to a region that
is afavorable substrate for de novo methylation (Yates et
al.1999).Because the evidence suggests that replication
ofmethylation patterns by DNMT1 is only partly respon-sible for
maintenance methylation (see above), an attrac-tive possibility is
that the features of a DNA domain
thathelpmaintainitsmethylatedstatusarethesamefea-tures that promote
its de novo methylation. Imprintingboxes, for example, whose
differential methylation is as-sociated with genomic imprinting
(Tremblay et al. 1997;Birger et al. 1999; Shemer et al. 2000), tend
to retain theirmethylation levels tenaciously even when the amount
ofthemaintenanceenzymeDNMT1isreduced(Beardetal. 1995). The de novo
methylases DNMT3A andDNMT3B(Okanoetal. 1998a, 1999)
maybeattracteddisproportionatelytothesesequences,
andthisattrac-tion may also underlie the decision to methylate the
boxinthefirstplace.Inotherwords,denovomethylationmay not occur once
at a discrete and perhaps rather in-accessible stage of germ-cell
development, but may hap-pen repeatedly (assisted by DNMT1) as
embryonic cellsdivide.Unusual DNA structures and RNAi as
triggersfor de novo methylationStudiesof purifiedDNMT1revealedthat
theenzymepreferstomethylateunusual DNAstructuresinvitro(Smith et
al. 1991; Laayoun and Smith 1995). This led tothe idea that such
structures might be generated duringrecombinationbetweenrepetitive
elements or duringtransposition events and directly trigger de novo
meth-ylation(BestorandTycko1996). Subsequentevidence,however, does
not support a role for DNMT1 in de novomethylationinvivo(Lykoet al.
1999; Howell et al.2001), andtherefore the biological significance
of itspredilectionfor deformedDNAisuncertain. Thereisevidence for
transfer of methylation from one copy of
asequencetoasecondpreviouslyunmethylatedcopyofthe same sequence in
the fungus Ascobolus (Colot et al.1996).
Theprocessmightusemechanismsinvolvedinhomologous DNA recombination
and may therefore in-volve deformationof DNA. Howidentical
sequencessenseoneanotherandtransferepigeneticinformationremains
unknown, however.Exciting recent developments inthe DNAmethyl-ation
field have arisen through molecular genetic
studiesofposttranscriptionalgenesilencinginplants.Double-stranded
RNA directs the destruction of transcripts con-taining the same
sequence, but there is compelling evi-dence that it can also direct
de novo methylation of ho-mologous genomic DNA (Wassenegger et al.
1994;Bender 2001; Matzke et al. 2001).
Posttranscriptionalgenesilencingbydouble-strandedRNAisprobablyanancient
genome defence systembecause it occurs infungi, plants, and
animals; but DNA methylation is notan obligatory accompaniment, as
silencing is efficient inC. elegansinthecompleteabsenceof
genomicm5C.Even in the fungus Neurospora, where transgene arraysare
often methylated, DNA methylation is not
requiredforposttranscriptional genesilencing(orquelling; Co-goni et
al. 1996). There are also specific features of
RNA-directedDNAmethylationthatmaynotoccurinani-mals; notably the
occurrence of methylation at multiplenon-CpG cytosines in an
affected DNA sequence tract.Although there is evidence for non-CpG
methylation inES cells, most probably owing to DNMT3A,
whichstrongly methylates CpA as well as CpG (Ramsahoye etal. 2000;
GowherandJeltsch2001), non-CpGmethyl-ation is barely detectable in
adult cells (Ramsahoye et al.2000).
PlantshaveaCpGmethylationsystem, but itdoesnotappeartobeessential
forRNA-directedgenesilencing (for reviews, see Wassenegger et al.
1994;Bender 2001; Matzke et al. 2001). Optimism that
RNA-directeddenovomethylationwillalsoapplyinmam-mals is tempered by
this sequence disparity, and by theabsence so far of a clear
demonstration that mammaliandouble-stranded RNA leads to DNA
methylation-medi-ated gene silencing.Transcriptionally silent
chromatin as a de novomethylation targetSeveral lines of evidence
suggest that DNA
methylationdoesnotintervenetosilenceactivepromoters, butaf-fects
genes that are already silent. It was reported manyyears ago that
retroviral transcription is repressed in
em-bryoniccellsat2dafterinfection, whereasdenovomethylation is
delayed until 15 d (Gautsch and Wilson1983; Niwa et al. 1983). De
novo methylation of
proviralsequencesinembryocellsdependsonDNMT3AandDNMT3B (Okano et
al. 1999), but initial retroviral shut-downoccursasusual
evenwhenboththesedenovomethyltransferases are absent (Pannell et
al. 2000).Clearly, de novo methylation is not required for
silenc-ing in the first instance, reinforcing the view that
meth-ylation is a secondary event.Methylationof genesthat
arealreadysilent isalsoobserved during X chromosome inactivation in
themammalianembryo. Kineticstudies showedthat
thephosphoglyceratekinasegeneissilentonthemamma-lianinactiveXchromosomebeforemethylationof
itsCpG-islandpromotersoccurs(Locketal.1987).Subse-quent studies of
the mouse, in which the process is bestunderstood,
haveestablishedthatexpressionofanon-codingchromosomal
RNAfromtheXist geneontheinactive X chromosome triggers the
inactivation processDNA methylation and epigenetic memoryGENES
& DEVELOPMENT 9Cold Spring Harbor Laboratory Press on February
14, 2012 - Published bygenesdev.cshlp.org Downloaded from in cis.
Specifically, activation of the Xist gene and onsetof its late
replication precede CpG-island methylation byseveral days(Keohaneet
al. 1996; WutzandJaenisch2000). In other words, methylation affects
the X chromo-someonwhichgenesarealreadyshutdownbyothermechanisms.
Istranscriptional
inertiaduringembryo-genesisthetriggerfordenovomethylation?Studiesoftheoriginof
methylation-freeCpGislandsoffersomesupport for this idea. The
coincidence between CpG is-lands andpromoters is
striking(Bird1987), andfoot-printing shows that the 5 extremity of
CpG islands of-ten corresponds to the region occupied by
transcriptionfactorsinvivo(Cuadradoetal.2001).EvenwhenCpGislands
areidentifiedinunusual locations,
theyhaveturnedouttocorrespondtopromoters. Forexample,
aCpGislandlocatedinintron2of theIgf2rgeneisanactive promoter (Wutz
et al. 1997; Lyle et al. 2000), as isaCpGislandthatcoversexon2of
theclassII
majorhistocompatibilitygene(MacLeodetal.1998).Thepo-tential
importance of promoter function in the genesis ofCpG islands is
highlighted by studies in transgenic
mice.CpG-island-containingtransgenesnormallyfaithfulre-producetheirmethylation-freecharacter,buttheirim-munitytomethylationislost
if promoter functionisimpaired(Brandeis et al. 1994; MacLeodet al.
1994).Similarly, viral DNA integrated into ES cell genomes
byhomologousrecombinationbecomesmethylatedwhenthe promoter is
weakened by absence of an enhancer, butexcludes methylation when an
enhancer is present(Hertz et al. 1999). A parsimonious
interpretation of theresults is that failure to transcribe invites
de novo meth-ylation(seeFig. 2below),
althoughotherpotentialex-planations (Brandeis et al. 1994;
Mummaneni et al. 1998)cannot be discounted.Thesignal for this
putativegenesilence-relateddenovomethylationisunknown,butthepossibilitythatchromatin
states inform the DNA methylation machin-ery is attractive (Selker
1990). The acetylation andmethylation state of nucleosomal histones
is tightly cor-related with transcriptional activity (Jenuwein
andAllis2001) andcouldbereadbythemethylationma-chinery, leading it
to either methylate or fail to methyl-ate a particular domain.
Indeed, recent work on Neuros-pora(TamaruandSelker2001)
hasshownanintimatelinkbetweenhistone
methylationandDNAmethyl-ationinthatfungus,
asmutationofahistonemethyl-transferasethat methylatesLys9of
histoneH3abol-ishedgenomicmethylation. Inmammalianandyeastsystems,
histone H3Lys 9methylationis
associatedwithtranscriptionallyrepressedheterochromatin(Ban-nisteretal.
2001; Nakayamaetal. 2001; Nomaetal.2001; ZhangandReinberg2001). If
thedependenceofDNAmethylationonpriorhistonemethylationturnsout
tobeapplicabletomammals, this
wouldfurtherstrengthentheargumentthatDNAmethylationistar-geted to
genes that are already silent. The nature of themolecular cues that
trigger transfer of methyl groups
tounmethylatedDNAshouldbeilluminatedbyongoingstudies of
multiproteincomplexes that containDNAmethyltransferases (Fuks et
al. 2000, 2001; Robertson etal. 2000; Bachman et al. 2001) and the
identification ofgenes that modify DNA methylation patterns (Weng
etal. 1995).Consequences of methylation gain: stabletranscriptional
silencing of genesWhy methylate genes that are already silent? A
plausibleanswer is: to silence themirrevocably.
Methylationclearlycontributestothestabilityof inactivation,
be-cause both X inactivation (Mohandas et al. 1981a;Graves 1982;
Venolia et al. 1982) and retroviral silencing(Stewart et al. 1982;
Jaenisch et al. 1985) can be relievedby treatment of somatic cells
with demethylatingagents. Individuals
wholackDNMT3Bshowreducedmethylation of some CpG islands on the
inactive X chro-mosome and also silence X-linked genes
imperfectly(Miniou et al. 1994; Hansen et al. 2000). The
implicationthat irreversibilityinvolvesDNAmethylationissup-ported
by the frequent reactivation of an X-linked trans-gene in mouse
embryo cells and in cultured somatic cellswhen DNMT1 is absent or
inhibited (Sado et al. 2000).This view is sustained by differences
in the stability ofinactivity states pre- and postmethylation. For
example,X inactivation caused by expression of an Xist
transgeneinembryonicstemcellsisinitiallyreversedwhentheXistgeneisshutdown,
butafter3d, inactivationbe-comesirreversibleandindependentof
Xist(WutzandJaenisch2000).Irreversibilitymayreflectthearrivalofpromoter
methylation.In artificial systems, DNA methylation represses
tran-scription in a manner that depends on the location
anddensityof themethyl-CpGs
relativetothepromoter(BoyesandBird1992;Hsieh1994;Kassetal.1997a,b).But
what genes are affected by DNA methylation-medi-ated gene
silencing? Early studies relied on the use of thedemethylating drug
5-azacytidine (Jones and
Taylor1980),whichwasshowntoactivategenesontheinac-tiveXinrodenthumancell
hybrids(Mohandasetal.1981b; Graves1982). Morerecently,
miceandmurinecell lines lacking DNMT1 (Li et al. 1992) have
clarifiedtheeffectsofDNAmethylationongeneexpression.Inplacental
mammals, repressionof X-linkedgenes fol-lows expression of Xist,
which sets in train the inactiva-tion process, culminating in
widespread methylation ofCpGislands. TheactiveXchromosome,
ontheotherhand, must be protected from silencing, and this
requiresrepressionof Xist andagaindepends
onmethylation(PanningandJaenisch1996). Anintact
DNAmethyl-ationsystemisalsoessential
forgenomicimprinting,becausedeletionof Dnmt1leadstodisruptionof
themonoallelic expression of several imprinted genes (Li etal.
1993).BothXinactivationandgenomicimprintinginvolvesilencing of one
allele only, leaving the other unaffected.An unusual set of genes
that are active in the germ line,most of which are X-linked,
appears to use
methylationforcompletesilencinginsomaticcells(DeSmetetal.1996,
1999). Several of thehumanandmurineMAGEgenes, for example, have
CpG-island promoters that areBird10 GENES & DEVELOPMENTCold
Spring Harbor Laboratory Press on February 14, 2012 - Published
bygenesdev.cshlp.org Downloaded from methylation-freeingermcells,
but aremethylatedinsomatic cells of the adult. The genes were
discovered asnovelantigensintumors,
wheregenomicmethylationlevelsareoftenlowandMAGE-geneCpGislandsareundermethylated.MAGEexpressioncanbeinducedbytreating
nonexpressing cells with demethylating agents,supportingtheideathat
methylationis
animportantcomponentoftherepressionofthesegenesinsomaticcells.Transposable
element silencing as a consequenceof DNA
methylationAnotherwell-documentedconsequenceofDNAmeth-ylationdeficiencyistheactivationoftransposableele-ment-derived
promoters. Like much of the mammaliangenome, transposable
element-related sequences
areheavilymethylatedandtranscriptionallysilent inso-matic cells.
Mouse cells, for example, normally represstranscription of
intracisternal A particle (IAP)
elements,whichconstituteahomogeneousandtranspositionallyactive
family of elements. In embryos lacking
DNMT1,transcriptionofIAPelementsismassivelyinduced,ar-guing that
methylation is normally responsible for theirrepression(Walshet al.
1998). Derepressionof LINE(Woodcocketal. 1997) andSINE(Liuetal.
1994) pro-motersinthehumangenomealsooccurswhenDNAmethylation is
reduced. The most abundant SINE in thehuman genome is the Alu
family, which consists of sev-eral hundred thousand elements (Smit
1999). Only a tinyminority of elements are capable of transposition
(95% of animal species (Tweedie et al. 1997).Colonization of the
genome by transposable elementscan only occur in the germ-cell
lineage because
somatictranspositioneventsleavenoheritabletrace.Paradoxi-cally,
transposableelementsareoftentranscriptionallyactive and
unmethylated in germ cells and totipotent EScells(forreview,
seeBird1997). IAPelements, forex-ample,
becomeunmethylatedduringthegonial prolif-erationphase,
whenprimordial germcell number in-creases from 75 to 25,000 (Walsh
et al. 1998). The fre-quent absence of DNA methylation in germ
cells, whentranspositioncando long-termdamage (Maliket al.1999),
contrasts with its repressive presence in somaticcells, where
transposition would be an evolutionary deadend. It is too early to
discount the possibility that trans-posonpromoters, mostof
whichbelongtodegenerateelementsthat areincapableof transposition,
must besilenced to suppress transcriptional noise.Mechanisms of DNA
methylation-mediatedtranscriptional repressionWhydoes
DNAmethylationinterferewithtranscrip-tion? Two modes of repression
can be envisaged, and it islikely that both are biologically
relevant. The first modeinvolves direct interference of the methyl
group in bind-ingof aproteintoitscognateDNAsequence(Fig.
1).Manyfactors are knowntobindCpG-containing se-quences, and some
of these fail to bind when the CpG ismethylated. Strong evidence
for involvement of thismechanism in gene regulation comes from
studies of therole of the CTCF protein in imprinting at the
H19/Igf2locus in mice (Bell and Felsenfeld 2000; Hark et al.
2000;Szabo et al. 2000; Holmgren et al. 2001). CTCF is asso-ciated
with transcriptional domain boundaries (Bell et al.1999) and can
insulate a promoter from the influence ofremoteenhancers.
ThematernallyderivedcopyoftheIgf2 gene is silent owing to the
binding of CTCF betweenDNA methylation and epigenetic memoryGENES
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promoter and a downstream enhancer. At the pater-nal locus,
however, these CpG-richbinding sites aremethylated,
preventingCTCFbindingandtherebyal-lowing the downstream enhancer to
activate Igf2 expres-sion. Although there is evidence that H19/Igf2
imprint-inginvolvesadditional processes(Ferguson-SmithandSurani
2001), theroleof CTCFrepresentsoneof theclearest examples of
transcriptional regulation by DNAmethylation.The second mode of
repression is opposite to the first,as it involves proteins that
are attracted to, rather thanrepelled by, methyl-CpG (Fig. 1). A
family of five
methyl-CpG-bindingproteinshasbeencharacterizedthateachcontains
aregioncloselyrelatedtothemethyl-CpG-binding domain (MBD) of MeCP2
(Nan et al. 1993, 1997;Cross et al. 1997; Hendrich and Bird 1998).
Four of theseproteinsMBD1, MBD2, MBD3, and MeCP2havebeen implicated
in methylation-dependent repression oftranscription (for review,
see Bird and Wolffe 1999). Anunrelated protein Kaiso has also
recently been shown tobind methylated DNA and bring about
methylation-de-pendent repression in model systems (Prokhortchouk
etal. 2001). In vitro, Kaiso requires a 5 m5CGm5CG motif,and
binding is highly dependent on the presence of meth-ylation.
Thepresenceofmultiplemethyl-CpG-bindingproteins
withrepressiveproperties supports theargu-ment that these maybe
important mediators of themethylationsignal, but their involvement
inspecificprocesses that require transduction of the DNA
methyl-ation signal has yet to be shown. Targeted mutation ofthe
gene for MeCP2 is, however, associated with neuro-logical
dysfunctioninhumans andmice(Amir et al.1999; Chen et al. 2001; Guy
et al. 2001), and mutation ofthe mouse Mbd2 gene leads to a
maternal behavior de-fect (Hendrich et al. 2001).Excluding DNA
methylation by denying
accessTheprecedingdiscussionhasconsideredsomemecha-nistic aspects
of de novo DNA methylation and its bio-logical consequences.
Although methylation affectsmost of the mammalian genome, it is
conspicuously ab-sent fromcertainregions. Ways
inwhichthesenon-methylated domains may arise will now be
considered.Asimplemechanismforcreatinganonmethylateddo-main within
an otherwise densely methylated genome isto mask a stretch of DNA
by protein binding. The DNA-binding protein would accomplish this
passive demeth-ylationby, for example, stericallyexcluding
DNMTs(Bird 1986). The feasibility of this mechanism has
beenverifiedusinganartificiallymethylatedepisomecon-taining EBNA1
or lac repressor binding sites (Hsieh1999a; Linetal. 2000).
TheideathatCpGislandsareentirely attributable to exclusion of this
kind is in doubt,however, as in vivo footprinting and nuclease
accessibil-itystudiesshowCpGislandstobemoreaccessibletoproteins(nucleases)
thanbulkgenomicDNA,
notless(TaziandBird1990).Ofcourse,itispossiblethatpro-tection is
only present at the transient embryonic stagewhen mammalian de novo
methylation occurs and hastherefore escaped detection. A protein
that is reported tobind unmethylated CpGs might be a candidate
CpG-is-land protector (Voo et al. 2000).Immunity to DNA methylation
causedby transcriptionally active chromatin:the origin of
unmethylated CpG
islandsManyoftheknownbiologicaleffectsofDNAmethyl-ation are
associated with CpG islands. It has been arguedabove that their
methylation in the early embryo followsFigure1.
Mechanismsoftranscriptionalrepression by DNA methylation. A
stretchof nucleosomal DNAis shownwithallCpGsmethylated(redcircles).
Belowthediagram is a transcription factor that is
un-abletobinditsrecognitionsitewhenamethylatedCpGiswithinit.Manytran-scriptionfactorsarerepelledbymethyl-ation,
including the boundary elementprotein CTCF (see text). Above the
line areprotein complexes that can be attracted bymethylation,
includingthemethyl-CpG-binding proteinMeCP2(plus the Sin3Ahistone
deacetylase complex), the MeCP1complex comprising MBD2 plus
theNuRDcorepressorcomplex, andtheun-characterized MBD1 and Kaiso
complexes.MeCP2 and MBD1 are chromosome-boundproteins,
whereasMeCP1maybeless tightly bound. Kaiso has not yet beenshown to
associate with methylated sitesin vivo.Bird12 GENES &
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that are likely to be DNA methylation-independent. If
transcriptional silence indeedtriggersDNAmethylation,
thenthecorollaryisthatpromoteractivityearlyindevelopmentshouldcreateamethyl-ation-free
CpG island (Fig. 2). In other words, unmethyl-ated CpG islands
might be footprints of embryonic pro-moteractivity.
AnobviouspredictionofthismodelisthatallunmethylatedCpGislands,
includingthoseatpromotersofhighlytissue-specificallyexpressedgenes,should
contain promoters that function during early de-velopment whenthe
methylationmemorysystemismost active. Although very limited, the
data so far favorthis theory, because a CpG-island promoter whose
prod-uctRNAisnotexpectedtooccurintheearlyembryo(-globin) is
nevertheless expressed, whereas transcriptsfroma CpG-deficient
promoter (-globin) are not de-tected (Daniels et al. 1997).
Similarly, expression of the68kneurofilamentgene,
whichhasaCpG-islandpro-moter, wasdetectedinEScells,
butopsinandcaseingenes, whichareCpG-deficient genes,
appearedtobesilent (MacLeod et al.
1998).Whyshouldactivepromoterregionsescapedenovomethylation? CpG
islands often colocalize with originsof DNA replication (Delgado et
al. 1998), and, accordingto one speculation, an early replication
intermediate cre-ates the DNA methylation-free footprint (Antequera
andBird1999). Amoredirect(butnotmutuallyexclusive)mechanismwould
involve the sensing of
chromatinstatesbythedenovomethylationsystemasdiscussedabove.
WhereashistoneH3tailsmodifiedbymethyl-ationonLys9mightrecruitDNAmethyltransferases(Tamaru
and Selker 2001), modifications associated withactivechromatin,
suchasacetylationof H3or H4ormethylation of Lys 4 of histone H3,
may actively excludethese enzymes. Biochemical evidence addressing
this is-sue is eagerly awaited.Active demethylation of
DNAProtectionagainstdenovomethylationbyboundpro-teinsorchromatincanensurethatDNAmethylationneverreachesaDNAsequencedomain.UnmethylatedFigure
2. A hypothetical scenario relating embryonic transcriptional
activity to DNA methylation status in mammals. Starting froma
notional transcription ground state, embryonic demethylation leads
to substitution of methylated sites (red circles) by nonmethyl-ated
sites (yellow circles). Two alternative fates are then envisaged:
either transcription persists leading to restoration of the
unmeth-ylated CpG island (bracket) flanked by methylated
non-island-flanking DNA (pink arrows); or transcription is
extinguished by othermechanisms in the embryo and this invites de
novo methylation of the CpG island and its flanks. In this way the
activity of embryonicpromoters is imprinted for the duration of
that somatic lifetime.DNA methylation and epigenetic memoryGENES
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could also arise by actively removing the modi-fication from DNA.
This so-called active demethylationcould be accomplished either by
the thermodynamicallyunfavorablebreakageof
thecarboncarbonbondthatlinks the pyrimidine to its methyl group, or
by a repair-likeprocessthat excisesthem5Cbaseor
nucleoside,leadingtoitsreplacement withC(Kresset al. 2001).Several
laboratories have striven to isolate
demethylaseenzymes(forreview,seeWolffeetal.1999).Themostimpressivecatalyticactivitywasshownbyafractionderived
from human cells (Ramchandani et al. 1999)
thatwassubsequentlyidentifiedasMBD2(Bhattacharyaetal. 1999).
Theexpressedproteinreportedlyshowedro-bust demethylation in vitro
in the absence of added co-factors and released methanol as a
by-product.
AttemptstoobservethispropertyofMBD2inotherlaboratorieshave not been
successful.Acellextractshowingdemethylaseactivitywasde-tectedinratmyoblastcells(Weissetal.
1996). Initialindications that the reaction was RNA-dependent
werenotsustaineduponfurtherenrichmentof theactivity(Swisher et al.
1998). An RNA-containing demethylatingcomplex was, however,
reported in chicken cells (Jost etal. 1997, 1999). These
investigators searched for
proteinswithm5C-DNAglycosylaseactivityandidentifiedthepreviously
known thymine DNA glycosylase TDG,which can remove the pyrimidine
base from T:G or U:Gmismatches (Zhuet al. 2000b). MBD4,
anunrelatedDNA glycosylase with similar properties, was also
foundtobeactiveagainstm5C:Gpairs(Zhuetal.2000a).Astheefficiencyofthesereactionswasmuchlowerthanthat
seenwiththecognatemismatchedsubstrates,
itmightbearguedthatthem5Cglycosylaseactivityrep-resentsaminor
sidereactionof littleinvivosignifi-cance. Set against this is
evidence that stable
expressionofachickenTDGresultsinsignificantactivationandconcomitant
demethylation of a reporter gene driven bya methylated
ecdysone-retinoic acid-responsive pro-moter (Zhuet al. 2001).
Thenormallysilent reportercould also be activated by demethylation
with 5-azacyti-dine, but generalized demethylation of the genome
wasnot observed in TDG transfected cells. Previous
studiesshowedanassociationbetweenretinoidreceptorsandTDG, and
implicated TDG in transcriptional activation(Umet al. 1998).
Timewill tell if
thestimulationofretinoid-responsivepromotersbyTDGdependsonitsdemethylating
activity.Theneedtoisolatedemethylatingenzymeshasbe-come more acute
with the finding that the paternal
ge-nomeissubjecttoactivedemethylationsoonafterfer-tilization (Mayer
et al. 2000; Oswald et al. 2000). Similarprocesses have been
reported in pig and bovine embryos(Bourchis et al. 2001; Kang et
al. 2001a,b). This dramaticillustration of methylation loss in the
absence of
DNAreplicationraisesquestionsabouttheprevalenceofde-methylationbythismechanism.Interestingly,thema-ternal
genome, whichalsodemethylates duringearlymouse development, does so
by a different
mechanism:passivefailuretomethylateprogenystands(Rougieretal.
1998). Whyshouldmaternal andpaternal genomeschoose such different
routes to the same end? An intrigu-ing possibility is that the
parental struggle over maternalresources for the embryo that is
thought to underlie ge-nomicimprinting(MooreandHaig1991)
maybein-volved. The oocyte may be equipped to directly
disarmthespermgenomeofmethylationimprintsthatmightoverexploit
maternal resources (Reik and Walter 2001). Itisevenpossiblethat
thepaternal genome, indelayedretaliation, may organize a campaign
of interferencewiththemaintenancemethylation(e.g.,
byexportingmaternal DNMTs to the cytoplasm). The extraordinaryneed
for an oocyte variant of DNMT1 to translocate intothenucleusfor
onlyonecleavagecycle(thedoublingfrom8to16cells; Howelletal. 2001)
couldrepresentmaternal measures to compensate for interference of
thiskind.Consequences of methylation loss: gene activationduring
developmentInterest in DNA methylation has long been fueled by
thenotion that strategic loss of methyl groups during devel-opment
could lead to activation of specific genes in theappropriate
lineage. As has been emphasized (Walsh
andBestor1999),muchoftheevidenceforthisscenarioisinconclusive, but
recent studies have revived the idea. Inthe frog, gene expression
is suppressed from fertilizationuntil the mid-blastula stage (5000
cells), at which timetranscription is activated. Inhibition of
DNMT1 using anantisense strategy caused reduced methylation and
pre-matureactivationof certaingenes, suggestingadirectrole for DNA
methylation in maintaining their early si-lence prior to the
blastula stage (Stancheva and Meehan2000). Deletion of the Dnmt1
gene in cultured
somaticcellsofthemousealsocausedwidespreadgeneactiva-tion
(Jackson-Grusby et al. 2001). About 10% of all genesdetectedusing
microarraytechnologywere activated,whereas only 1%2% were
down-regulated. Some of theup-regulated genes are normally only
expressed in termi-nally differentiated cells. These findings raise
the possi-bility that DNA methylation contributes to silencing
oftissue-specific genes innonexpressing cells, andtheyconfirm DNA
methylation as a global repressor of geneexpression. The scenario
has been modeled using an ar-tificial construct that
containedaDNAsequenceca-pableofexcludingmethylationlocallyduringearlyde-velopment
(Siegfriedet al. 1999). Whenthissequencewas present, the reporter
gene stayed unmethylated dur-ingdevelopment,
andwidespreadexpressionoccurred.Deletion of the element in situ in
the early embryo led tomethylationof thereporter geneandconcomitant
si-lencing in several adult tissues.Asubtlepotential rolefor lossof
methylationat aspecific gene has been reported for the rat tyrosine
ami-notransferase gene (Thomassin et al. 2001). When
amethylatedformofthisgeneisinducedbyglucocorti-coids,delayeddemethylationoccursatspecificsitesinanenhancerandadditionalDNA-associatedfactorsaresubsequentlyrecruited.Demethylation(whetheractiveor
passive is not known) persists after the wave of TATBird14 GENES
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expressionhassubsided, andreinductionof thesilentgene bya further
hormone treatment is significantlystronger as a result. This system
provides a model for aDNAmethylation-mediatedmemoryof thefirst
hor-moneinduction(Kresset al. 2001). Itssignificanceissomewhat less
certain in normal development,
however,becausedemethylationofthesesitesoccursbeforethegene becomes
hormone-inducible.Thereissuggestiveevidencethat
programmedrear-rangementof
theimmunoglobulingenesduringB-celldevelopment mayinvolve
DNAmethylation(Mosto-slavskyetal. 1998). Demethylationof oneof
thetwoparentally derived alleles of the kappa light chain gene
isobserved in small pre-B cells, and there is evidence
thatthisearlylossof methylationpredisposestheaffectedallele to
rearrangement. By precluding rearrangement ofoneallele,
differential
DNAmethylationmayhelptoexplainallelicexclusionatthekappachainlocus.Itisnot
certainwhether transcriptional regulationper seplaysarole,
althoughtheprocessisdependentontheintronic and 3 kappa gene
enhancers.Loss of genome integrity as a consequence of
DNAmethylation loss?Early studies with the DNA-methylation
inhibitor5-azacytidinerevealedbizarrechromosomal rearrange-ments
intreatedculturedcells (Viegas-Pequignot
andDutrillaux1976).Althoughthesefindingsmightbeat-tributed to the
effects of reduced DNA methylation,
theycouldalsobearesultofthechemicalreactivityoftheincorporated base
analog, inparticular, its ability tocross-link proteins to DNA
(Juttermann et al. 1994). Theformer possibility is supported
somewhat by the findingthat mitogen-stimulated lymphocytes
frompatientswith mutations in DNMT3B show very similar
chromo-somerearrangements, involvingcoalescenceofcentro-meric
regions that contain methylation-deficient
repeti-tivesequences(Jeanpierreetal. 1993; Xuetal. 1999).Oddly,
therearrangementsarenotseenincellsofthepatients, despitesimilar
hypomethylationof
thesere-gions.Itseemsthatlossofgenomicintegrityisnotanobligatory
consequence of hypomethylation of juxtacen-tromeric repeat
elements.Atafinerlevel, twolaboratorieshaveexaminedtheeffectsof
greatlyreducedDNAmethylationlevelsonmutationrates
inmouseembryonicstemcells,
withsomewhatdifferingresults.Inonestudy,themutationrate at two
endogenous loci was found to have
increased10-foldcomparedtothesameloci
inwild-typecells(Chenetal.1998),suggestingthatlackofmethylationpredisposed
to aberrant recombination events. A
secondstudyexaminedtransgenesofexogenousoriginusingaselection
system to detect mutations (Chan et al. 2001).This allowed
screening of large numbers of mutations attwoindependentloci,
butneitherpointmutationsnorgenomic rearrangements were
increasedunder
condi-tionsoflimitingDNAmethylation.Infact,mutationsappeared to be
suppressed by genomic hypomethylation.These inconsistencies raise
questions about the pro-posed relationship between genome integrity
and DNAmethylationthatwill
needtobeaddressedbyfurtherresearch.Developmental memory: DNA
methylationand Polycomb/trithorax complexesas interchangeable
systemsThe foregoing discussion has highlighted features of
theDNAmethylationsysteminmammalsthat
resembleanotherestablishedsystemof cellularmemory: Pc-G/trx. The
final section of the review will compare the twosystems. The
credentials of Pc-G/trx protein complexesas an epigenetic system in
development are compelling(Paroet al. 1998; Pirrotta1999;
FrancisandKingston2001). This multiprotein assembly is targeted to
specificregionsof
thegenomewhereiteffectivelyfreezestheembryonicexpressionstatusof
agene, beitactiveorinactive, and propagates that state stably
through devel-opment. Elegant experiments with model gene
con-structs have shown that brief activation (or inactivation)of a
promoter during early Drosophila development
leadstostableactivity(orinactivity) thereafter(CavalliandParo1998,
1999; Pouxet al. 2001). Attemptstoalterexpression at most other
stages of development were un-successful, indicating that there is
a window of time
dur-ingwhichtranscriptionpatternscanbecommittedtodevelopmental
memory. The Pc-G/trx system is reactiverather than proactive, as
the setting up of
segment-spe-cificpatternsofactivegenesisnotdisruptedbymuta-tions in
Pc-G group genes. Only the capacity to sustainthe patterns is lost
in the mutants. This ability to copyand propagate the expression
patterns without influenc-ing or perturbing them makes this a
subtle and flexiblememorysystem. Littleisknown, however, about
themechanisms responsible for the heritability of Pc-G/trx.What do
Pc-G/trx and DNA methylation in mammalshave in common? First, both
systems are able to represstranscription in a heritable manner.
Second, both appearto be reactive in that they lock in expression
states thatthey played no part in setting up (e.g., DNA
methylationinviralgenomesilencingandCpG-islandmethylationon the X
chromosome). Third, both are activated
prima-rilyduringadiscretewindowoftimeinearlydevelop-ment. Thus,
likePc-G/trx, DNAmethylationhastheproperties of a developmental
memory.What is memorized by DNA methylation? Arguably,its major
role is to stably demarkate by its absence a setof embryonically
active promoters, namely, CpGis-lands, so that they remain
potentially active throughoutdevelopment and adulthood. At the same
time, regionsdevoid of promoter activity in the embryo become
meth-ylatedandcarrythis repressive influence
withthemthroughdevelopment.Thedegreeofrepressionmaybeweak or strong
depending on the density of methylation(BoyesandBird1992;
Hsieh1994). Thus,
CpGislandsthataresilencedbyothermechanismsduringembryo-genesiswouldacquiredensemethylationleadingtoir-reversible
silencing. When, however, the density ofmethylated CpGs is low, as
it is in most of the genome,DNA methylation and epigenetic
memoryGENES & DEVELOPMENT 15Cold Spring Harbor Laboratory Press
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from repression is likely to be weak and may be overcome bythe
presence of strong activators. Weak repression of
tis-sue-specificgenes(e.g., -globin) thatareembeddedinregionsof
low-densitymethylationmaycontributetotheir silence in inappropriate
tissues.It is proposed here that DNA methylation and
Pc-G/trxarealternativesystemsofcellularmemorythatareinterchangeableover
evolutionarytime. InC. elegansand Drosophila, for example, Pc-G
group proteins(Birchleretal.2000)havebeenimplicatedinsilencingof
repetitive-element transcription in somatic cells,whereas DNA
methylation may play this role in mam-mals (Yoder et al. 1997). The
involvement of DNA meth-ylation in genome defence may, therefore,
be to memo-rize the silent state of elements imposed by primary
ge-nomedefencesystems.Themoststrikingevidenceforinterchangeability
is the finding that X chromosome
in-activationinextraembryonictissuesof
themousede-pendsonthepolycombgroupproteinEed. Lossoftheeed gene
leads to reactivation of the inactive X in extra-embryonic tissue,
but has no effect in somatic cell
types(Wangetal.2001).Incontrast,Dnmt1mutationsreac-tivate the
inactive X of the embryo proper, but not theextraembryonic inactive
X (Sado et al. 2000). The findingthat certain CpG islands on the
inactive X chromosomeare methylated in somatic cells but not in
extraembry-onictissues (Iidaet al. 1994) fits
withtheviewthatmethylation replaces Pc-G in somatic tissues.
Therefore,evenwithinasinglespecies, it appearsthat
differenttissuesemployPc-G/trxandDNAmethylationinter-changeably.
From an evolutionary perspective, it is
pos-siblethatvaryingdegreesoffunctionalsubstitutionbyPc-G (or vice
versa) can explain the dramatic differencesbetween DNA methylation
levels across animal species.Concluding remarksOur understanding of
the relationshipbetweenDNAmethylation and transcriptional control
is growing fast,but is still far fromcomplete. Ongoing
biochemicalanalysisof thegrowingnumberof componentsof
theDNAmethylationsystem(andtheirpartners),coupledwithgeneticapproaches,
will strengthenthelinksbe-tween DNAmethylation and
mainstreamtranscrip-tional mechanisms. Regulationof gene
expressioniscomplex (Lemon and Tjian 2000), and the emerging
evi-dence hints that the roles of DNA methylation will betoo. It
maybeunrealistictoexpect that anyunifiedtheory will encompass all
the biological consequences ofDNA methylation.Least understood are
the mechanisms by which
meth-ylationpatternsaregenerated.Followingconsiderationof the
criteria for attracting and repelling DNA methyl-ation, this review
has entertained the possibility that aprimaryfunctionof
denovoDNAmethylationis tomemorize patterns of embryonic gene
activity, creatingCpG islands that are competent for
transcriptionthroughout development, or their antithesis,
regionsthataremethylatedandtranscriptionallyincompetent.The idea
depends on evidence that methylation does notintervene to silence
genes that are actively transcribed,but only affects genes that
have already been shut downby other means. There is reason to
believe that transcrip-tional activity may somehow imprint the
methylation-freestatusof CpGislands. Theinvolvement of
DNAmethylationininactivationof transposable elementscould likewise
be due to its capacity for stabilizing thetranscriptional
shutdownorganizedbyother systems.Parallels betweenthese emerging
attributes of DNAmethylation and the Pc-G system in Drosophila
suggestthatbotharemechanismsforsensingandpropagatingcellular
memory.AcknowledgmentsI amgrateful toEricSelker, BernardRamsahoye,
HelleJr-gensen, Brian Hendrich, and Catherine Millar for comments
onthe manuscript. Research by A.B. is supported by The
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