DNA methylation in amphioxus: from ancestral functions to new

14 Pages·2012·1.28 MB·English

BRIEFINGS IN FUNCTIONAL GENOMICS. VOL11.NO 2.142^155 doi:10.1093/bfgp/els009
DNA methylation in amphioxus: from
ancestral functions to new roles in
vertebrates
RicardAlbalat,JosepMart|¤-SolansandCristianCan‹estro
AdvanceAccesspublicationdate 2March2012
Abstract
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In vertebrates,DNA methylation is an epigenetic mechanism thatmodulates gene transcription, andplays crucial w
n
roles during development, cell fate maintenance, germ cell pluripotency and inheritable genome imprinting.DNA lo
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methylation might also play a role as a genome defense mechanism against the mutational activity derived from e
d
transposonmobility.Incontrast to theheavilymethylatedgenomesinvertebrates,mostgenomesininvertebrates fro
m
arepoorlyorjustmoderatelymethylated,andthefunctionofDNAmethylationremainsunclear.Here,wereview h
the DNA methylation system in the cephalochordate amphioxus, which belongs to the most basally divergent ttps
group ofourownphylum, the chordates.First, surveys of the amphioxusgenome databasereveal thepresence of ://a
c
a
theDNAmethylationmachinery,DNAmethyltransferasesandmethyl-CpG-bindingdomainproteins.Second,com- d
e
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parativegenomics andanalysesofconservedsyntenybetweenamphioxus andvertebratesproviderobustevidence
ic
that the DNA methylation machinery of amphioxus represents the ancestral toolkit of chordates, and that its .ou
p
expansioninvertebrateswasoriginatedbythetworoundsofwhole-genomeduplicationthatoccurredinstemver- .c
o
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tebrates.Third,insilicoanalysisofCpGo/eratiosthroughouttheamphioxusgenomesuggestsabimodaldistribution /b
of DNAmethylation, consistentwith a mosaicpatterncomprisingdomains ofmethylated DNAinterspersedwith fg
/a
domainsofunmethylatedDNA,similar to thesituationdescribedinascidians,butradicallydifferentto theglobally rtic
le
methylated vertebrate genomes.Finally, we discuss potential roles of the DNA methylation system in amphioxus -a
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inthecontextofchordategenomeevolutionandtheoriginofvertebrates. s
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Keywords:DNAmethylation;amphioxus;chordateandvertebrategenomeevolution;transposableelements;conserved t/1
1
genomicsynteny;DnmtandMbd /2
/1
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2
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INTRODUCTION inherited cell to cell, and play important roles main- 35
6
DNA methylation is an epigenetic mechanism that taining differential gene expression profiles between 7 b
y
has been mainly associated with regulation of different cell lineages during embryo development, g
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gene expression. DNA methylation marks can be states of cell differentiation in adult tissues or es
t o
n
1
4
Correspondingauthor.CristianCan˜estro,DepartamentdeGene`tica,FacultatdeBiologia,UniversitatdeBarcelona,EdificiPrevosti, A
p
Av. Diagonal, 643, E-08028 Barcelona, Spain. Tel:þ34 93 403 5304;Fax: þ34 93403 4420; E-mail: [email protected] ril 2
RicardAlbalat,PhD(UniversitatdeBarcelona),Postdoc(SkirballInstitute;NewYorkUniversity)isAssociateProfessorofGenetics 01
9
attheUniversitatdeBarcelona.Hiscurrentresearchisfocusedontheevolutionoftheretinoidandsteroidsignalingpathwaysduring
chordate evolution. He is also interested in the impact of transposable elements and the changes of epigenetics mechanisms in the
evolution of chordate genomes.
JosepMart|¤-SolansisaMasterstudentintheDevelopmental BiologyandGeneticsprogramattheDepartmentofGeneticsinthe
University of Barcelona. His research project focuses on understanding mechanisms of gene losses and gene gains in the retinoid
signaling pathway of nonvertebrate chordates (e.g. amphioxus and Oikopleura) that might help to understand the evolution of stem
chordates and theorigin of functional innovations in stemvertebrates.
CristianCan‹estroisanAssistantProfessorattheDepartmentofGenetics,intheUniversityofBarcelona,Spain,since2009.Hemade
his postdoctoral research in the Institute of Neuroscience at the University of Oregon (2002–09). He received his PhD from the
DepartmentofGenetics,UniversityofBarcelona,Spain(2001).HeisinterestedinGenomics,FunctionalEvo-DevoandEpigenetics,
and his research focus on the impact of gene losses on developmental programs. His projects aim to understand the evolution of
chordates and the origin of vertebrate innovations. Amphioxus, ascidians, larvaceans and zebrafish are the main model organisms in
which hestudies hisfavorite signaling molecule, theretinoic acid andits epigenetic role on embryodevelopment.
(cid:2)TheAuthor2012.PublishedbyOxfordUniversityPress.Allrightsreserved.Forpermissions,pleaseemail:[email protected]
DNA methylationinamphioxus 143
pluripotency in germ cells, as well as preserving the genome [25]. Additional Mbd proteins have also
chromosomal imprinting [1–4]. DNA methylation been described (e.g. Mbd5 and Mbd6), and in con-
consists in the addition of a methyl group trast to the rest of the MBD proteins, they do not
covalently bound to the 5-carbon of cytosine of a bind methylated DNA, but might play a role in the
CpG dinucleotide. In animals, DNA methylation is formation of heterochromatin and epigenetic repro-
mediatedbyDNAmethyltransferases(Dnmt),which gramming after fertilization [26].
are evolutionarily conserved enzymes that have been Despite that the DNA methylation is present
classified into three groups, Dnmt1, Dnmt2 and throughout the phylogenetic ladder, substantial
Dnmt3 [5]. Dnmt3 enzymes are responsible for differences in the multiplicity of Dnmt and Mbd
de novo methylation during development [6, 7]. genes, and in the amount and genomic distribution
Dnmt1 enzymes are important in maintaining of methylated cytosines are found among different
methylationpatternsafterDNAreplicationonhemi- taxa. Thus, vertebrates have more Dnmt and
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methylated symmetric motifs [8]. Dnmt2 (also Mbd genes, and higher methylated genomes than w
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termed Trdmt1) enzymes show a robust methyl- non-vertebrates. Vertebrates have at least one lo
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transferase activity of tRNA molecules, and only Dnmt1, one Dnmt2, one to five Dnmt3 genes and at e
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present low activity against DNA [9, 10]. least six Mbd genes in their DNA methylation tool- fro
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TheadditionofmethylgroupstotheDNAregu- kit, whereas invertebrates have a variable lower h
lates gene expression in two ways, either by direct number of Dnmt and Mbd genes ([5, 22, 27–32] ttps
interferencewiththebindingofproteinsthatinteract and Figure 1). Caenorhabditis elegans, for instance, ://a
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with DNA elements [11–13], or indirectly, through lacks Dnmt genes, whereas related nematodes pre- d
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recruitment of proteins that contain a methyl-CpG- serve one Dnmt2-related gene [33]. Among insects, ic
binding domain (MBD), which are called Mbd1, fruit flies and mosquitoes only have Dnmt2, the silk .ou
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Mbd2, Mbd3, Mbd3L, Mbd4 and Mecp2 (methyl moth has both Dmnt1 andDmnt2, and the honeybee .c
o
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CpG-binding protein 2) [14, 15]. Most Mbd pro- possesses the full set of Dmnt genes [34, 35]. The /b
teins specifically recognize and bind to methylated protostome Mbd complement also varies depending fg/a
DNA, and it is thought that they can associate on the species analyzed. Most lophotrochozoan and rtic
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with histone deacetylases (HDACs) and other chro- ecdysozoan species have both Mbd1/2/3 (formerly -a
b
matin remodeling proteins, contributing to the tran- Mbd2/3) and Mbd4/MeCP2 (formerly Mbd4) genes, stra
scriptional repression of promoters ([16, 17] although some ecdisozoans have a truncated or ct/1
reviewed in [18]). Each Mbd protein has, however, even no Mbd4/MeCP2 genes ([28, 31, 33, 36] and 1/2
its own features. MeCP2 was the first discovered Figure 1). Outside bilaterians, a full complement of /14
2
Mbdproteinwithmethyl-CpGDNA-bindingactiv- Dnmt is present in cnidarians and sponges, but so /2
1
3
ity,andithasbeenrelatedtotheHDACcomplexin far a sole Dnmt2 has been found in placozoans [31, 5
6
7
the maintenance of transcriptional silencing [17]. 37, 38]. Mbd1/2/3 and Mbd4/MeCP2 have been also b
y
Mbd1 is the only methyl-CpG-binding protein identified in cnidarians as well as in placozoans [31]. g
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that is capable of repressing transcription from both In addition to the diversity of Dnmt and Mbd genes, s
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methylated and unmethylated promoters, at least in the extent of CpG methylation and its genomic dis- n
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cell culture [19–21]. The DNA binding activity of tribution also differ among non-vertebrate species, 4 A
Mbd2andMbd3proteinsismediatedbytheirMBD, from undetectable or very low levels to mosaic pat- pril 2
andalthoughsuchactivityhasbeenlostinthemam- terns of substantial methylation in different 0
1
malian Mbd3, it is still preserved in amphibians and non-vertebrate genomes, although never reaching 9
fishes[22].Inmammals,Mbd2repressestranscription the high levels of global methylation of vertebrates
in a methylation density-dependent fashion [23]. [39–43].
Mbd3L1 is a protein with significant homology to Amphioxus belongs to the subphylum of the
Mbd2 and Mbd3. It lacks, however, the MBD, cephalochordates, the most basally divergent group
although it might interact with Mbd2 as a methyla- in our own phylum, the chordates. The privileged
tion-dependent transcriptional repressor, likely com- position of amphioxus permits the comparison of its
peting with Mbd3 [24]. Finally, Mbd4 is the only methylation system with that of vertebrates, thereby
memberoftheMBDfamilythatdoesnotseemtobe allowing the inference of the methylation condition
involved in transcriptional repression, but it plays a of the last chordate common ancestor. Such evolu-
role in decreasing the mutability of methyl-CpG in tionary framework helps to understand how changes
144 Albalatet al.
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Figure 1: Phylogenetic distribution of Dnmt and Mbd toolkit.The Dnmt groups (Dnmt1, Dnmt2 and Dnmt3, num- /a
beredboxes) and Mbdgenes(Mbd1, Mbd2, Mbd3, Mbd1/2/3, Mbd4, MeCP2 and Mbd4/MeCP2, numberedboxes) are rtic
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showninseveralanimalspecies.Thenumberofboxesrepresents thenumberofgenecopiesfoundineachspecies. -a
b
Partial sequences similar to the glycosylase domain of Mbd4 are detected in Bombyx mori, Acyrthosiphon pisum, s
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PediculushumanusandAmphimedonqueenslandica.The‘?’denotesthatthegenehasnotbeenfoundinincompletegen- c
omesorESTdatabases.ThisfiguresummarizestheDnmtandMbdtoolkitdescribedin[31,38,44],aswellasnewse- t/11
/2
quencesretrievedbyBLASTsearchesfrompublicdatabases:Anophelesgambiae Mbd1/2/3(XP_318432),B.mori Mbd1/ /1
4
2/3 (BGIBMGA001425 in SilkDB) and Mbd4/MeCP2(BGIBMGA012841 in SilkDB), Tribolium castaneum Mbd1/2/3 2
/2
(XP_969537),Nasoniavitripenis (NP_001164526), A.pisum Mbd1/2/3 (NP_001156167)and Mbd4/MeCP2(XP_003244914),P. 1
3
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humanus Mbd1/2/3 (XP_002428735) and Mbd4/MeCP2(XP_002425074), Brugiamalayi Mbd1/2/3 (XP_001896671) and A. 6
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queenslandicaMbd1/2/3(XP_003389039)andMbd4/MeCP2(XP_003386267). b
y
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s
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A
ifnuntchteioDnaNl Ainmnoetvhaytiloatniosnisnystethmemsitgehmt havveertelebdrattoe cvoerntceobmratiteanintnwovitahtiothnes. origin and diversification of pril 20
1
genome.Here,wefirstreviewthemaincomponents 9
of the DNA methylation machinery (i.e. Dnmt and
EVOLUTIONOF THECHORDATE
Mbd genes) in amphioxus, and perform a compara-
tive analysis with those in vertebrates. Second, we DNA METHYLATIONTOOLKIT:
describe the pattern of distribution of the DNA DNMTANDMBDGENES
methylation throughout the amphioxus genome, The amphioxusDNA methylation
comparing it with that reported in other chordates. toolkit
Finally,wediscusstheevolutionoftheDNAmethy- Genomic surveys revealed that amphioxus has three
lation machinery in chordates, in the context of the Dnmt genes, namely Bf_Dnmt1, Bf_Dnmt2 and
major genomic changes related to the two rounds of Bf_Dnmt3, each one ortholog to one of the three
whole-genome duplication (2R-WGD) that were Dnmt groups [31, 37]. Structurally, the C-terminal
DNA methylationinamphioxus 145
region of Dnmt enzymes contains the cytosine Dnmt1 is able to bind different proteins, including
methyltransferase active site, organized into 10 Dnmt3 [49], histone deacetylase 2 (Hdac2), DNA
motifs, of which 6 of them (I, IV, VI, VIII, IX and methyltransferase 1 associated protein 1 (Dmap1)
X) are evolutionary conserved [45–47]. These six [50] and proliferating cell nuclear antigen (Pcna)
motifs are recognizable in the three Bf_Dnmt [51]. Dnmt3 binds zinc fingers and homeoboxes 1
sequences (Figure 2), suggesting the enzymatic con- protein (Zhx1) [52], and Mbd2 and Mbd3 interact
servation of the amphioxus Dnmt proteins. Each with many proteins in the NuRD/Mi-2 complex,
amphioxus Dnmt contains additional domains spe- including Hdac2, retinoblastoma binding protein 4
cific for each Dnmt group (Figure 2), which agrees (Rbbp4), metastasis associated 1 (Mta1) and GATA
with the tree topology obtained by phylogenetic zincfingerdomaincontaining2A(Gatad2a)[53,54].
analysis [31]. Hence, Bf_Dnmt1 contains the typical Homologs of these Dnmt- and Mbd partners have
Dmap1-binding region, the nuclear localization been identified in amphioxus (e.g. Dmap1, Hdac2,
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signal (NLS), the Cys-rich zinc-binding domain Pcna, Rbbp4, Mta1 and Gatad2a) [31], and in silico w
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and the two bromo-adjacent homology (BAH) reconstruction of a putative interaction network in lo
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motifs. Bf_Dnmt2 presents the characteristic amphioxus is therefore feasible (Figure 3). Although e
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Dnmt2 CFTXXYXXY (CFT) sequence between this insilico approximation is only an initial step that fro
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motifs VIII and IX. Bf_Dnmt3 contains the typical requiresexperimentalvalidation,theidentificationin h
PWWP domain and the Cys-rich region in the amphioxusofbonafideDnmtandMbdproteinsand ttps
N-terminal region of the protein. their plausible partners suggests the presence of an ://a
c
Genomic surveys also revealed that amphioxus interaction network similar to that in vertebrates. ad
e
has two Mbd genes. Considering phylogenetic Other chordates such ascidians have also a full mic
reconstructions [22, 31] and analyses of conserved complement of the Dnmt and two Mbd genes .ou
p
synteny (discussed in the next section), here we sug- [22, 29, 31], whereas the larvacean Oikopleuradioica .c
o
gest a new nomenclature that reflects their evolu- has only Dnmt2 and Mbd1/2/3 (Figure 1) [37]. m/b
tionary relationships with vertebrate genes: Bf_Mbd1/ Outside chordates, the identification in sea urchin fg/a
2/3(formerly Bf_Mbd2/3) because it is the co-ortholog of Dnmt1,Dnmt2, Dnmt3,Mbd1/2/3 and Mbd4/MeCP2 rtic
of vertebrate Mbd1, Mbd2 and Mbd3 paralogs, and genes [31, 56] indicates that the basic Dnmt/Mbd le-a
b
Bf_Mbd4/MeCP2 (formerly Bf_Mbd4) because it is the genetic toolkit was already present at least in the stra
co-orthologofvertebrateMbd4andMeCP2paralogs. last common ancestor of deuterostomes, probably ct/1
Structurally, Bf_Mbd1/2/3 contains, in addition to in stem bilaterians [31]. Hence, amphioxus appears 1/2
the MBD, the coiled-coil domain at the C-terminal to have preserved a DNA methylation machinery /14
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region characteristic of vertebrate Mbd2 and Mbd3 thatrepresentstheancestral chordatetoolkit.Incon- /2
1
trast, within urochordates, though some species have 3
[48], but lacks the CXXC motifs and the hydropho- 5
6
also maintained an intact basic methylation toolkit, 7
bic transcriptional repression domain (hTRD) of b
Mbd1 (Figure 2). Bf_Mbd4/MeCP2 concurs with otherssuchaslarvaceanshavelostmostoftheircom- y g
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ponents [31, 37]. e
the structural features of Mbd4 proteins, including s
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the thymine DNA glycosylase domain and the n
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Fas-associated death-domain (FADD) protein bind- Vertebrate expansionof the DNA 4 A
ing region in its C-terminal half [31], but lacks the methylationtoolkit pril 2
highly basic transcriptional repression domain In contrast to non-vertebrate chordates, vertebrate 0
1
9
(bTRD) present in MeCP2 (Figure 2). Domain ana- Dnmt and Mbd gene families have suffered distinct
lysis reveals, therefore, that Bf_Mbd1/2/3 is more degrees of expansions. Dnmt3, for instance, has
similar to vertebrate Mbd2 and 3 than to Mbd1, been duplicated in vertebrates, and analysis of
and Bf_Mbd4/MeCP2 is more similar to Mbd4 the human genome reveals that DNMT3A and
than to MeCP2. These similarities suggest that DNMT3B paralogs are in conserved syntenic chro-
Bf_Mbd1/2/3, Bf_Mbd4/MeCP2 and vertebrate mosomal segments in Hsa2 and Hsa20, arguing in
Mbd2, Mbd3 and Mbd4 might have maintained favor of the hypothesis that both genes likely origi-
ancestral roles, whereas Mbd1 and MeCP2 might nated during the 2R-WGD, rather than via local
have acquired vertebrate-specific novel functions. duplications (Figure 4A). Genomic analysis of con-
Most Dnmt and Mbd protein domains are served synteny also reveals that Mbd gene family
involved in protein–protein interactions. Vertebrate expansion was due to the 2R-WGD, and synteny
146 Albalatet al.
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Figure 2: Structural protein domains of the DNA methylation toolkit. (A) Structural comparison of Dnmt 13
5
enzymes in amphioxus (Bf) and human (Hs). Dnmt enzymes share six highly conserved methyltranferase motifs 6
7
(I, IV, VI, VIII, IX and X) at the C-terminal region, and several group-specific domains, mostly found at the by
N-terminalregion.ADmap1-bindingregion,anuclearlocalizationsignal(NLS),aregioninvolvedintargetingtorep- gu
e
lication foci, a Cys-rich zinc-binding domain, and two bromo-adjacent homology (BAH) motifs are distinctive of st o
the N-terminalregion of Dnmt1enzymes, which is joined to the C-terminal catalytic region by a tract of glycine n
1
andlysineresidues(GK). ACFTXXYXXY(CFT)sequencebetweenmotifsVIIIandIXischaracteristic ofDnmt2. 4
A
A NLS, a PWWP domain and a Cys-rich region are typically found in the N-terminal region of Dnmt3. p
(B)StructuralcomparisonofamphioxusandhumanMbdproteins.Themethyl-CpGbindingdomain(MBD),distinct- ril 2
0
1
ively found at the N-terminalhalf of all theseproteins, is capable torecognize andbind specifically to methylated 9
DNA. In addition to the MBD, amphioxus Mbd1/2/3 contains a coiled-coil domain (CC) at the C-terminal region
characteristicofvertebrateMbd2andMbd3,butlackstheCXXCmotifsandthehydrophobic transcriptionrepres-
sion domains (hTRD) of the human MBD1. Amphioxus Mbd4/MeCP2 includes a thymine DNA glycosylase domain
and a Fas-associated death-domain protein bindingregion (FADD) in its C-terminal half, distinctive of vertebrate
Mbd4,butlacks thebasic transcriptionrepressiondomain(bTRD)ofMECP2.
conservation is observed between the MBD1/ Hsa3 and the MECP2-bearing paralogon in HsaX
MBD2-bearing paralogon in Hsa18 and the (Figure4BandC).Notably,genomicanalysisreveals
MBD3/MBD3L-bearing paralogon in Hsa19, as conserved synteny between the amphioxus Bf_Mbd1/
well as between the MBD4-bearing paralogon in 2/3-bearing paralogon within a 2-Mb fragment in
DNA methylationinamphioxus 147
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Figure 3: Amphioxus network of protein interactions linked to Dnmt and Mbd inferred by STRING v.9.0 [55]. e
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Spheres represent amphioxus proteins connected to interacting partners by solid lines (confidence score > 0.3). ic
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Stronger interactions are represented by thicker lines. Amphioxus protein IDs from JGI and their respective u
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humanpartnerswiththehighestbit-scorevalueinsequencecomparisonsareshown. .co
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scaffold Bf_V2_32 and the MBD1/MBD2 and that these genes might be refractory to copy number -a
b
MBD3/MBD3L paralogons in 10-Mb fragment in variation, a phenomenon observed in ohnologs that stra
Hsa18 and a 20-Mb fragment in Hsa19, respectively have dosage-balance evolutionary constraints [60]. ct/1
(Figure 4D). It is tempting to speculate that 1/2
there might be still unknown evolutionary con- /14
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straintsmaintainingsuchhighdegreeofsyntenycon- DNA METHYLATION /2
1
3
servation during >500 Mya, the time of divergence DISTRIBUTION PATTERN IN 5
6
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between cephalochordates and vertebrates. Cur- AMPHIOXUS b
y
rently, different projects are being developed to The extent of CpG methylation in the amphioxus gu
e
uncover the causes that could explain the mainten- genome was initially studied by comparing electro- st o
ance of macro- and micro-synteny conservation phoretic patterns of genomic DNA digested with n 1
across such distantly related taxa [57]. methylation-sensitive and methylation-insensitive 4 A
tainDednmat1s sainndgleDcnompt2y, gheonwesevdeers,pihtaevethebeveenrtembariante- rthesetsreictdiiognesteionndsonwuecrleeahseysbri[d3i9z]e.dSwoiutthhegrenneb-slpoetscifoicf pril 20
1
2R-WGDevents.Althoughgenomicanalysisreveals probes to assess the methylation status of certain 9
conserved synteny between the DNMT1 region on amphioxus genes, providing an approximate idea of
human chromosome 9 (Hsa9) and chromosome seg- the genomic distribution of this epigenetic mark.
ments in Hsa1 and Hsa19, as well as between the These early studies led to the conclusion that there
DNMT2 region on chromosome Hsa10 and chro- are two roughly comparable fractions of methylated
mosome segments in Hsa2 and Hsa17 (CC, unpub- and unmethylated DNA in the amphioxus genome,
lished data), none of the DNMT1 or DNMT2 with genes distributed in both fractions [39].
ohnologs (i.e. paralogs originated by genome dupli- SequencingofthewholegenomeofBranchiostoma
cation) survived in their respective paralogous floridae has allowed an estimate of the DNA
chromosomal segments. Dnmt1 and Dnmt2 have re- methylation pattern throughout the amphioxus
mained as single copy genes in chordates, suggesting genome. The distribution of DNA methylation in
148 Albalatet al.
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Figure 4: Conserved syntenies between DNMT (A) and MBD (B and C) bearing paralogons in human chromo-
somes provide evidence that the expansion of the DNA methylation toolkit was originated by the two rounds
of whole-genome duplication (2R-WGD) that occurred in stem vertebrates. Interestingly, synteny for the MBD
bearing paralogons has been preserved between human and amphioxus chromosomes for >500 Mya (D), which
may suggest that selective evolutionary constrains might preserved the structural organization of this genomic
regionbecauseofapotentialfunctionalrole.HsaN:humanchromosomeN;Bfl_v2_N,B.floridae scaffoldN.Theana-
lysis of syntenyconservationisinferredfrom the Synteny Database[58] using a100-gene sliding window asprevi-
ouslydescribed[59].
DNA methylationinamphioxus 149
the genome can be inferred from the ratio between curve), whereas the other distribution may represent
the observed and expected CpG content, a metric thehypo-methylated fraction (average CpGo/e ratio
termedCpGo/eratioorCpGscore[41,61].Ingen- (cid:2)0.8, right curve). Interestingly, the relative weight
eral, unmethylated regions show a CpGo/e ratios of each fraction shows small differences among scaf-
close to 1, whereas hiper-methylated regions tend folds, but globally the methylated fraction tends to
to contain few CpG sites due to the mutability of be slightly smaller than the unmethylated one
5-methylcytosine, therefore showing CpGo/e ratios (Figure 5H). Future experimental analyses are
farsmallerthan1(e.g.<0.5asinhumans).Usingthis needed to test these CpGo/e-based inferences that
indirect approach, Okamura et al. [42] analyzed suggests that amphioxus follows an overall methyla-
one genomic region of 2Mb, and concluded that tion pattern similar to that in ascidians, in which
amphioxus genome has a mosaic pattern of DNA about half of the genome is heavily methylated,
methylation. The mean length of methylated and whereas the other half is poorly methylated [41,
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unmethylated regions was estimated 4.8kb 42]. These non-vertebrate patterns are in sharp w
n
(SD ¼ 5.0kb) and 7.7kb (SD ¼ 11.1kb), respect- contrast with those of globally methylated vertebrate lo
a
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ively, comparable to those of other non-vertebrate genomes, in which 80% of the CpGs are e
d
deuterostome genomes (e.g. 3.6 and 6.6kb for sea methylated [4]. fro
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urchin,and5.3and6.8kbforascidians,respectively), In mammals and plants, DNA methylation has h
but distinct from the global methylated genomes of been associated not only to gene silencing, but also ttps
mammals (e.g. 25.5 and 2.6kb for human, respect- to transcriptional repression of transposable elements ://a
c
a
ively). This indirect approach has been experimen- (TEs), decreasing their mobility, and thereby func- d
e
m
tally validated in ascidian and human genomes [42], tioning as a mechanism of genome defense against ic
but no experimental analysis has been reported in their mutational activity [66–68]. The methylation .ou
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amphioxus. To validate whether or not CpGo/e status of the amphioxus transposons remains mostly .c
o
m
ratio is a good estimate of the DNA methylation unknown. Experimental data have been provided /b
status in amphioxus, we have examined here the only for one type of TE, in which analysis of fg/a
CpGo/e ratios of 18 amphioxus genes that had methylation-sensitive DNA digestion revealed that rtic
le
been experimentally verified to be either methy- thenon-LTR retrotransposon Bf-CR1 waspredom- -a
b
lated (8 genes) or unmethylated (10 genes). inantly free of methylation [69, 70]. Important cav- stra
Our analysis shows that the CpGo/e ratios of eats need to be considered if CpGo/e ratios are used ct/1
17 out of the 18 genes analyzed correlate with to infer the methylation status of TEs due to their 1/2
their methylation status (Figure 5A), supporting peculiar behavior. For instance, populations of TEs /14
2
Okamura’s analysis [42]. may suffer sequence bias due to bottlenecks derived /2
1
3
To test if the inferences about the methylation from the burst of mobilization of a reduced number 5
6
7
pattern made by Okamura and collaborators from of TEs, and thereby the CpGo/e ratios of the new b
y
the analysis of 2Mb could be extended to other TEs could reflect the CpG ratio of the original gen- g
u
e
genomic regions, here we have analyzed the omichostingregion,ratherthanabiasedratiodueto s
t o
CpGo/e ratio across 17Mb from three arbitrarily TE-specific targeted methylation. Another aspect to n
1
selected scaffolds of the amphioxus genome be considered is the possibility of horizontal transfers 4 A
(Figure 5B–D). According to the CpGo/e values, ofTEsbetweendifferentphyla,inwhichCpGratios pril 2
the distribution of DNA methylation across different of recently acquired TEs might still reflect the 0
1
scaffolds shows a mosaic pattern, with methylated methylation status of the donor genome rather than 9
segments interspersed with similar segments of that of the host genome. However, considering that
unmethylated DNA. Consistent with Okamura’s TE horizontal transfers between different phyla are
analysis, the plot of the distribution of the frequen- rare,andthattheTEsofamphioxusdonotappearto
cies of the CpGo/e ratios reveals statistically signifi- have suffered significant recent expansions [71], it is
cant bimodal distributions in all regions analyzed plausible to assume that most of the current amphi-
(G2 over 5% significance level >5.99; left and right oxus TEs might be ancient and vertically inherited.
curves in Figure 5E–G). The bimodal distributions We argue, therefore, that if TEs had been major
have very similar means among different scaffolds. targets for DNA methylation over a long evolution-
One of the distributions might represent the methy- arytimeintheamphioxuslineage,theCpGo/eratios
lated fraction (average CpGo/e ratio (cid:2)0.4, left of most TEs should be biased toward low values.
150 Albalatet al.
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Figure 5: Mosaic pattern of DNA methylation in the amphioxus genome. (A) Histograms ordered by
increasingvaluesoftheCpGo/eratiosof18amphioxusgenesofknownmethylationstatus.Methylatedgenesinclude
(continued)
DNA methylationinamphioxus 151
Totestthispossibility,wehaveanalyzedtheCpGo/e amphioxus also have a mosaic methylation pattern,
ratios of all the 623 TE families provided in the DNA methylation is targeted to ‘gene bodies’ rather
amphioxus genome database (http://genome.jgi- than to intergenic regions or TEs [41]. It has been
psf.org/Brafl1/Brafl1.home.html), and the results proposed that DNA methylation in gene bodies
do not show any obvious bias toward low CpGo/e might prevent aberrant transcription from cryptic
ratios. In fact, only (cid:2)7% of TE sequences showed initiation sites, which may interfere with the
CpGo/e ratios <0.5 (0.4 is the average of the normal transcription machinery [73]. Zilberman
methylatedgenomicfractioninamphioxus),whereas etal. [73] have hypothesized that a large number of
>62% of TEs have CpGo/e ratios >0.7 (0.8 is the elongating RNA polymerases might prevent the use
average of the hypo-methylated genomic fraction in ofcrypticsitesinhighlytranscribedgenes,whereasin
amphioxus) (Figure 5I). These theoretical inferences moderately transcribed genes, methylation of gene
suggest that, globally, TEs are not major targets bodies is needed to avoid aberrant transcripts. This D
o
of methylation, a similar condition to that hypothesis predicts that the methylated fraction of a w
n
described in ascidians, in which experimental evi- given genome will be enriched in genes moderately loa
d
dence has proved that most TEs are poorly transcribed, and that this enrichment will be percep- ed
methylated [41, 70, 72]. If future experimental ana- tibleingenomesinwhichDNAmethylationhasnot fro
m
lysis (e.g. bisulfite sequencing) of amphioxus TEs beenrecruitedforotherfunctionssuchassilencingof h
corroborates this inference, we could conclude that TEs. The analyses of DNA methylation of ascidians ttps
the methylation of TEs as a defense mechanism andotherprotostomegenomesagreewiththesepre- ://a
c
a
mightbeanevolutionaryinnovationacquiredinver- dictions, and genes with weak–moderate expression de
tebrates, not present in cephalochordates and m
(e.g. housekeeping genes) belong to the heavily ic
urochordates. .o
methylated fraction, while genes with high tissue- u
p
specific expression belong to the poorly methylated .co
m
fraction [41, 74–76]. Analysis of a small set of genes /b
DNA METHYLATION, FROM in amphioxus shows a tempting correlation between fg/a
ANCESTRALFUNCTIONS housekeeping genes in the methylated fraction [e.g. rtic
le
TONEW ROLES ribosomal proteins P2 (Brf6) and S6 (Brf61), mito- -a
b
s
What functions might DNA methylation be playing chondrial carrier protein (Brf38)], and tissue-specific tra
c
in amphioxus? In ascidians, which similarly to genes in the unmethylated fraction [e.g. Lipase t/1
1
/2
/1
4
2
Figure 5: Continued /2
1
Brf6 (60Sribosomalprotein P2,NW_003101422), Brf16 (NADH dehydrogenase subunit,NW_003101225), Brf32 (uni- 35
6
dentified cDNA, NW_003101419), Brf38 (mitochondrial proton carrier-like protein, NW_003101433), Brf43 (snRNP- 7
b
like protein, NW_003101422), Brf61 (40S ribosomal protein S6, NW_003101524) [39], Adh3 (alcohol dehydrogenase y g
class III, from exons 2 to 6, NW_003101484) [62], and presinilin (AF369890) [63], and unmethylated genes are ue
s
AmphiMLC-alk(alkalimyosinlightchain,NW_003101500),Brf5(unidentifiedcDNA,NW_003101462),Brf21(triacylgly- t o
n
cerol lipase, NW_003101520), Brf37 ((cid:2)-tubulin, NW_003101522), Brf41 (unidentified cDNA, NW_003101534), Brf51 (b- 1
4
actin,NW_003101548), Brf52 (unidentifiedcDNA,NW_003101505), Brf54 (unidentifiedcDNA,NW_003101377), Brf57 A
p
(unidentified cDNA, NW_003101328) and rDNA (AF061796) [39]. (B^D) Pattern of DNA methylation inferred by ril 2
CpGo/e ratios across 17Mb from three arbitrarily selected genomic regions (scaffold_1: 7.2Mb, scaffold_2: 6.5Mb 0
1
9
andscaffold40:3.1Mb)splittedin2kbsegments.Thisplotrevealsamosaicpatternofinterspersedregionsofheavily
methylated andpoorlymethylated fractions across the amphioxusgenome.Estimations of the CpGo/eratio were
performedwithCpGproDalgorithm[64].(E^G)AnalysisofthefrequencydistributionofCpGo/eratiosin0.1inter-
vals revealed statistically significant (G2 over 5% significance level >5.99) bimodal distributions of methylated (left
curve)andunmethylated(rightcurve)fractionsinthethreeregionsanalyzed.Testforuni- orbi-modaldistributions
wasperformedwith the NOCOMsoftware[65].(H)Globalanalysis of the threepreviouslyselectedscaffolds.(I)
Analysis of the frequency distribution of CpGo/e ratios of all the 623 TE sequences provided in the amphioxus
genome database (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html), representing a total of18Mb (Supplementary
Table S1).The distribution of the frequencies of the CpGo/e ratios of all amphioxusTE families does not show a
bimodaldistributionasdoestherestof thegenomeinamphioxus[dashedlinesin(I)],andimportantly,thedistribu-
tion is not biased toward low values, suggesting that most TE might not be main targets of methylation in
amphioxus.