cytosine methylation at cpg sites in the genomic dna is a well - characterized epigenetic modification involved in gene regulation and transposon silencing in mammals ( 1,2 ) . upon replication of methylated dna , cytosines in the newly synthesized strand
are initially unmethylated ( resulting in a hemimethylated duplex ) , then become methylated by a maintenance - type dna methylase , dnmt1 .
in addition , the de novo dna methylases , dnmt3a and dnmt3b , also have a role in the maintenance methylation for some genomic regions ( 35 ) . due to this template - dependent copying mechanism ,
in contrast , the level of dna methylation changes dynamically during germ - cell development .
first , primordial germ cells ( pgcs ) undergo genome - wide erasure of methylation and then prospermatogonia , a direct derivative of pgcs in the male gonad , undergo extensive de novo methylation ( 6,7 ) ( figure 1a ) .
the prospermatogonia are mitotically arrested , premeiotic cells expressing high levels of the de novo dna methylase dnmt3a and its related protein dnmt3l ( 8,9 ) .
therefore , de novo dna methylation and its maintenance through cell division can be separately observed in these germ cells .
oocytes are also non - dividing cells that undergo extensive de novo methylation ( 6,7 ) , but the methylation marks in most genomic regions are not maintained through cell divisions after fertilization ( 10 ) . 

mitotically arrested prospermatogonia undergo de novo methylation of cpg sites in the fetal testis , and mitosis resumes after birth .
non - cpg methylation at three representative b1 loci detected by bisulfite sequencing in p0 prospermatogonia ( see supplementary figure s1 for other loci ) .
circles and squares represent cytosines in cpg and non - cpg ( cph where h is a , t or c ) sites , respectively .
( c ) alignment of the sequences of the 33 b1 loci to visualize the common sites of methylation .
cytosines methylated over 65% at cpg sites are highlighted in black , and those methylated over 5% at non - cpg sites are highlighted in gray .
positions of a- and b - box in the pol iii promoter and the binding sites for the ahr and slug transcription factors are indicated above .
( d ) methylation levels in different sequence contexts . the data for dinucleotides and trinucleotides are shown .
( e ) individual cpg and non - cpg sites are categorized according to their methylation levels , and counted .
mitotically arrested prospermatogonia undergo de novo methylation of cpg sites in the fetal testis , and mitosis resumes after birth .
non - cpg methylation at three representative b1 loci detected by bisulfite sequencing in p0 prospermatogonia ( see supplementary figure s1 for other loci ) .
circles and squares represent cytosines in cpg and non - cpg ( cph where h is a , t or c ) sites , respectively .
( c ) alignment of the sequences of the 33 b1 loci to visualize the common sites of methylation .
cytosines methylated over 65% at cpg sites are highlighted in black , and those methylated over 5% at non - cpg sites are highlighted in gray .
positions of a- and b - box in the pol iii promoter and the binding sites for the ahr and slug transcription factors are indicated above .
( d ) methylation levels in different sequence contexts . the data for dinucleotides and trinucleotides are shown .
( e ) individual cpg and non - cpg sites are categorized according to their methylation levels , and counted .
short interspersed elements ( sines ) are a class of retrotransposons and are transcribed by rna plolymerase iii ( pol iii ) .
biochemical studies have shown that cpg methylation of the pol iii promoter inhibits transcription of trnas and human alu sine ( 1113 ) .
we previously showed that the bulk of cpg sites in b1 retrotransposons , a family of sines scattered over the mouse genome , become highly methylated in prospermatogonia and stay methylated throughout the later stages of spermatogenesis ( 14 ) .
we also showed that a mutation in dnmt3a or dnmt3l reduces the methylation levels in a locus - specific manner and that the level of b1 methylation is well correlated with the level of b1 expression ( 14 ) . during the course of that study
, we noticed that methylation at non - cpg sites is abundantly present in prospermatogonia .
this is interesting because non - cpg methylation has been so far found in oocytes , embryonic stem cells ( escs ) , induced pluripotent stem cells ( ipscs ) and the brain ( 3,1521 ) .
although non - cpg methylation dramatically decreases upon differentiation of escs and thus could have some relevance to pluripotency ( 3,19 ) , the biological function of non - cpg methylation remains unknown . in this report
, we characterize the non - cpg methylation that we found at b1 retrotransposons in prospermatogonia in detail .
we show accumulation of the non - cpg methylation in the non - dividing prospermatogonia and its gradual loss after the resumption of cell division .
we also provide evidence suggesting the absence of a template - dependent copying mechanism for non - cpg methylation .
epcam - positive germ cells were collected from testes of c57bl/6 j mice at p0 , p2 , p5 and p7 as described previously ( 14 ) . to collect fetal germ cells at e16.5 , green fluorescent protein (
genomic dna preparation , bisulfite conversion and pcr were done as described previously ( 14 ) .
typically , 100200 ng of genomic dna was treated with bisulfite , and an aliquot equivalent to 510 ng of genomic dna was used for pcr with extaq hs ( takara bio , ohtsu , japan ) . to monitor the efficiency of c - to - t conversion ,
 phage dna that had no methylated cytosine ( promega , madison , wi ) was included in the bisulfite reaction .
all sequenced clones were used for the analysis . to validate the purity of germ - cell preparations , methylation of the imprinted lit1 differentially methylated region ( dmr )
was also analysed using the same bisulfite - treated dna . among at least 40 sequenced clones ,
none showed methylation in the lit1 dmr , suggesting a germ - cell purity of > 95% , given that only the maternal lit1 dmr allele is methylated in somatic cells and no allele is methylated in male germ cells .
the sequences of the primers for the dmrs , cpg islands and control  phage dna are shown in supplementary table s1 .
genomic dna ( 100 ng ) was digested with taqi ( new england biolab , ipswich , ma ) and ligated with a synthetic oligonucleotide ( 5'-cgtgaactgaggtcggaagacctcagttca-3 ' ) that forms a hairpin structure .
the ligation product was denatured at 98c for 10 min in 0.3 m naoh , treated with 9.17 m bisulfite ( 22 ) and used for pcr as described ( 14 ) .
the hairpin - bisulfite pcr primers for the three b1 loci are listed in supplementary table s1 .
epcam - positive germ cells were collected from testes of c57bl/6 j mice at p0 , p2 , p5 and p7 as described previously ( 14 ) . to collect fetal germ cells at e16.5 , green fluorescent protein (
genomic dna preparation , bisulfite conversion and pcr were done as described previously ( 14 ) .
typically , 100200 ng of genomic dna was treated with bisulfite , and an aliquot equivalent to 510 ng of genomic dna was used for pcr with extaq hs ( takara bio , ohtsu , japan ) . to monitor the efficiency of c - to - t conversion ,
 phage dna that had no methylated cytosine ( promega , madison , wi ) was included in the bisulfite reaction .
all sequenced clones were used for the analysis . to validate the purity of germ - cell preparations , methylation of the imprinted lit1 differentially methylated region ( dmr )
was also analysed using the same bisulfite - treated dna . among at least 40 sequenced clones ,
none showed methylation in the lit1 dmr , suggesting a germ - cell purity of > 95% , given that only the maternal lit1 dmr allele is methylated in somatic cells and no allele is methylated in male germ cells .
the sequences of the primers for the dmrs , cpg islands and control  phage dna are shown in supplementary table s1 .
genomic dna ( 100 ng ) was digested with taqi ( new england biolab , ipswich , ma ) and ligated with a synthetic oligonucleotide ( 5'-cgtgaactgaggtcggaagacctcagttca-3 ' ) that forms a hairpin structure .
the ligation product was denatured at 98c for 10 min in 0.3 m naoh , treated with 9.17 m bisulfite ( 22 ) and used for pcr as described ( 14 ) .
the hairpin - bisulfite pcr primers for the three b1 loci are listed in supplementary table s1 .
we collected prospermatogonia at postnatal day 0 ( p0 ) and studied dna methylation states of 33 b1 loci by bisulfite sequencing .
the bisulfite sequencing method is unable to distinguish between 5-methylcytosine ( 5mc ) and 5-hydroxylmethylcytosine ( 5hmc ) , but hereinafter we regard bisulfite - resistant cytosines as 5mc , given that 5hmc is present much less than 5mc in cells / tissues examined so far , and almost exclusively at cpg sites in escs ( 23 ) . as shown in figure 1b , c and supplementary figure s1 , significant methylation was found at non - cpg sites , in addition to cpg sites , both within and around the b1 sequences .
this was not due to imcomplete bisulfite conversion , because the failure rate was only 0.20.5% , as determined using the control unmethylated dna (  phage dna ) mixed to the samples . on average ,
the methylation level at non - cpg sites ( total of 2700 ) in p0 prospermatogonia was 4.8% , which was higher than the levels in escs ( 2.6% ) , ipscs ( 3.2% ) , oocytes ( 3.43.8% ) and the brain ( 1.52.2% ) ( 16,20,21 ) .
of all methylated cytosines , 77% were at cpg sites and 23% were at non - cpg sites .
when the methylation level was determined for each dinucleotide sequence , it was 94% , 9.8% , 1.3% and 0.4% at cpg , cpa , cpt and cpc sites , respectively ( figure 1d ) .
about half of cpa sites showed methylation levels of higher than 5% , whereas the vast majority of cpt and cpc sites showed no methylation ( figure 1e ) .
thus , cpa is the most preferred non - cpg sites . among the cpa sites ,
the cpapc ( 15% ) and cpapg sites ( 12% ) were more frequently methylated than the cpapa and cpapt sites ( figure 1d ) . interestingly , despite that the 33 b1 loci that we analysed were from various genomic locations , some non - cpg sites at equivalent positions in the b1 sequences were more preferentially methylated ( figure 1c ) .
a subfamily of b1 sequences , designated as b1-x35s , has been shown to bind the ahr and slug transcription factors , and the binding was involved in the formation of a chromatin boundary ( 24,25 ) . however
, the preferentially methylated non - cpg sites did not overlap with these binding sites or the rna polymerase iii promoter ( a - box and b - box ) ( figure 1c ) . to address whether the non - cpg methylation is specific to b1 sequences or not
, we examined the methylation status of three paternally methylated dmrs ( the h19 , dlk1/meg3 and rasgrf1 dmrs ) in p0 prospermatogonia .
non - cpg methylation was detected in the dmrs with the levels comparable to those in b1 loci ( supplementary figure s2a ) .
we also examined seven cpg islands that are methylated in spermatozoa ( 26 ) . whereas three of them were not methylated even at cpg sites ( data not shown ) , the other four had methylation at non - cpg sites in p0 prospermatogonia ( supplementary figure s2b ) .
it is known that de novo cpg methylation of the dmrs and retrotransposons occurs in prospermatogonia between embryonic day 14.5 ( e14.5 ) and p0 ( 27 ) .
the presence of non - cpg methylation in p0 prospermatogonia suggested their co - occurence with cpg methylation in fetal prospermatogonia .
we selected eight b1 loci that showed high levels of non - cpg methylation in p0 prospermtogonia ( b1_007 , b1_014 , b1_016 , b1_018 , b1_021 , b1_042 , b1_045 and b1_055 ) ( supplementary figure s1 ) and examined their methylation status in prospermatogonia at embryonic day 16.5 ( e16.5 ) ( figure 2a and c ) .
the average cpa methylation level at e16.5 was six times less than that at p0 , indicating that these non - cpg sites become de novo methylated in prospermatogonia during late gestation .
we previously showed that mutation in dnmt3a or dnmt3l affects cpg methylation at b1 loci ( 14 ) .
we therefore analysed p0 prospermatogonia from dnmt3l knockout mice ( 28 ) for the eight loci ( figure 2b ) .
the cpa methylation level was significantly reduced ( > 5-fold , p < 10 ) by the dnmt3l mutation ( figure 2b and c ) , suggesting that the non - cpg methylation in prospermatogonia is dependent on dnmt3l . 

figure 2.de novo methylation of non - cpg sites occurs concomitantly with de novo cpg methylation .
( a ) site - by - site representation of the bisulfite sequencing data for the eight selected b1 loci in e16.5 prospermatogonia .
( b ) methylation status of the same b1 loci in dnmt3l knockout ( ko ) p0 prospermatogonia .
de novo methylation of non - cpg sites occurs concomitantly with de novo cpg methylation .
( a ) site - by - site representation of the bisulfite sequencing data for the eight selected b1 loci in e16.5 prospermatogonia .
( b ) methylation status of the same b1 loci in dnmt3l knockout ( ko ) p0 prospermatogonia .
it is known that higher plants have methylation at both cphpg and cphph sites ( where h = a , c or t ) ( 2 ) . at cphpg sites ,
the two cytosines on the different dna strands are symmetrically methylated ( 29,30 ) , and the chromo - methylase cmt3 is responsible for this methylation ( 31,32 ) .
a previous study in human escs and ipscs showed that the levels of methylation at given cphpg sites differ between the two strands ( 19 ) , consistent with the fact that no cmt3-like methylase is found in mammals .
however , in that study , the bisulfite reads were from different dna duplexes and the read numbers were small .
therefore , whether the cphpg sites are indeed hemimethylated in the same duplex has been an open question . to address this issue
, we used the hairpin - bisulfite sequencing method ( 33 ) with some modifications ( figure 3a ) .
briefly , genomic dna was digested with taqi , ligated to synthetic hairpin forming dna , and subjected to bisulfite reaction followed by pcr .
the pcr primers were designed so that the amplicon consisted of the top- and bottom - strand sequences from a single duplex separated by the hairpin dna sequence .
the results obtained from three b1 loci showed that 87.5% of cpg sites were symmetrically methylated in p0 prospermatogonia ( figure 3b and c ) .
in contrast , we detected no symmetric methylation at any cag or ctg sites ( the cumulative number of tested sites was 69 ) ( figure 3b and c ) .
this suggests that a template - dependent copying mechanism does not operate in methylation at cphpg sites .
thus , most or all of non - cpg methylation is ascribed to de novo methylation .
the dinucleotide preference observed at the non - cpg sites ( cpa > cpt > cpc ) in prospermatogonia is well correlated with the target preference of the recombinant dnmt3a protein ( 34,35 ) .
indeed , in human and mouse escs and mouse oocytes , a depletion of the de novo methylases resulted in greatly reduced non - cpg methylation ( 3,17,18,20 , k. shirane et al .
the numbers of sites with symmetric or asymmetric methylation are shown for cag ( and ctg ) and cg sites .
the numbers of sites with symmetric or asymmetric methylation are shown for cag ( and ctg ) and cg sites .
next , we studied the fate of non - cpg methylation after birth , during the transition from the prospermatogonia to spermatogonia .
proliferating spermatogonia at p7 showed very low levels of methylation at all three non - cpg dinucleotides ( 0.20.4% ) ( figure 1d ) , which were comparable to the error rate of bisulfite conversion ( 0.20.5% ) .
we then studied the time course of the loss of non - cpg methylation between p0 and p7 . whereas the cpg methylation levels in the 33 b1 loci stayed around 95% at all time points
, the non - cpg methylation levels declined gradually ( figure 4a and supplementary figure s1 ) .
for example , the average cpa methylation levels were 9.8 , 7.4 , 1.5 and 0.4% at p0 , p2 , p5 and p7 , respectively ( figure 4b ) .
these results suggest that the de novo methylation activity is not high in postnatal germ cells and that the lack of a copying mechanism for non - cpg methylation results in a gradual loss of this methylation after every dna replication .
the dnmt3a and dnmt3l expression levels are high in prospermatogonia but become low after birth , and the dnmt3b expression level is relatively low in prospermatogonia ( 8,9 ) . 

figure 4.loss of non - cpg methylation after the resumption of mitosis in postnatal development .
( a ) gradual loss of methylation at a representative b1 locus in the transition period from prospermatogonia to spermatogonia ( p0 , p2 , p5 and p7 ) .
cpg sites are shown in circles and non - cpg sites are shown in squares , with open and filled ( black or gray ) symbols representing unmethylated and methylated cytosines , respectively .
( b ) methylation levels of the 33 b1 loci at cpg sites and cpa sites at p0 , p2 , p5 and p7 .
the boxes indicate 75 and 25 percentiles , with the thick line inside being the median .
( a ) gradual loss of methylation at a representative b1 locus in the transition period from prospermatogonia to spermatogonia ( p0 , p2 , p5 and p7 ) .
cpg sites are shown in circles and non - cpg sites are shown in squares , with open and filled ( black or gray ) symbols representing unmethylated and methylated cytosines , respectively .
( b ) methylation levels of the 33 b1 loci at cpg sites and cpa sites at p0 , p2 , p5 and p7 .
the boxes indicate 75 and 25 percentiles , with the thick line inside being the median .
the dashed line indicates the average . since a high level of non - cpg methylation
was first detected in pluripotent cells such as escs and ipscs , a correlation of non - cpg methylation with pluripotency has been discussed ( 19 ) .
however , the detection of non - cpg methylation in oocytes , brain cells and prospermatogonia suggests that the above may not be the case .
rather , the feature common to these cell types seems relatively high expression levels of the de novo methylase(s ) ( 6,7,36,37 ) .
indeed , in human escs and ipscs , the level of non - cpg methylation is better correlated with the expression levels of the de novo methylases than those of pluripotency factors ( 20 ) . in summary
, we showed that non - cpg methylation accumulates within and around b1 sine sequences , as well as the paternally methylated dmrs and some cpg islands , in the mitotically arrested prospermatogonia during fetal development .
the observed non - cpg methylation level was relatively high compared with other cells , but this does not necessarily mean that prospermatogonia have high levels of non - cpg methylation throughout the genome , as non - cpg methylation is relatively high in the alu sine sequences in human escs ( 20 ) .
our hairpin - bisulfite sequencing revealed that non - cpg sites including symmetrical cphpg sites are hemimethylated .
therefore , it appears that no template - dependent copying mechanism exsists for non - cpg methylation in prospermatogonia .
perhaps , the presence and the level of non - cpg methylation are determined by the balance between the de novo methylation activity and the rate of cell proliferation . in the case of b1 retrotransposons ,
the non - cpg methylation may not have a role in regulation of transcription or boundary function as shown above , but further studies are needed to know whether non - cpg methylation in other genomic regions has a function .
supplementary data are available at nar online : supplementary table 1 and supplementary figures 1 and 2 .
grants - in - aid for scientific research from the ministry of education , culture , sports , science and technology of japan [ 22770009 , 21200037 to k.i .
and 20062010 to h.s . ] ; career center for women researchers hand - in - hand program at kyushu university ( research assistants dispatching program to t.i . ) .
funding for open access charge : grants - in - aid for scientific research from the ministry of education , culture , sports , science and technology of japan [ 20062010 to h.s . ] .
 dna methylation is a well - characterized epigenetic modification involved in gene regulation and transposon silencing in mammals . 
 it mainly occurs on cytosines at cpg sites but methylation at non - cpg sites is frequently observed in embryonic stem cells , induced pluriotent stem cells , oocytes and the brain . 
 the biological significance of non - cpg methylation is unknown . here , we show that non - cpg methylation is also present in male germ cells , within and around b1 retrotransposon sequences interspersed in the mouse genome . 
 it accumulates in mitotically arrested fetal prospermatogonia and reaches the highest level by birth in a dnmt3l - dependent manner . 
 the preferential site of non - cpg methylation is cpa , especially cpapg and cpapc . 
 although cpapg ( and cptpg ) sites contain cytosines at symmetrical positions , hairpin - bisulfite sequencing reveals that they are hemimethylated , suggesting the absence of a template - dependent copying mechanism . 
 indeed , the level of non - cpg methylation decreases after the resumption of mitosis in the neonatal period , whereas that of cpg methylation does not . 
 the cells eventually lose non - cpg methylation by the time they become spermatogonia . 
 our results show that non - cpg methylation accumulates in non - replicating , arrested cells but is not maintained in mitotically dividing cells during male germ - cell development . 
INTRODUCTION
MATERIALS AND METHODS
Cell preparation and bisulfite-PCR sequencing
Hairpin-bisulfite PCR analysis
RESULTS AND DISCUSSION
SUPPLEMENTARY DATA
FUNDING
Supplementary Material