DNA methyltransferase expression in the mouse germ line during periods of de novo methylation

RESEARCH ARTICLE

DNA Methyltransferase Expression in theMouse Germ Line During Periods of De NovoMethylationDiane J. Lees-Murdock,1 Tanya C. Shovlin,1 Tom Gardiner,2 Massimo De Felici,3

and Colum P. Walsh1*

DNA methyltransferase (DNMT) 3A and DNMT3B are both active de novo DNA methyltransferases requiredfor development, whereas DNMT3L, which has no demonstrable methyltransferase activity, is required formethylation of imprinted genes in the oocyte. We show here that different mechanisms are used to restrictaccess by these proteins to their targets during germ cell development. Transcriptional control of theDnmt3l promoter guarantees that message is low or absent except during periods of de novo activity. Useof an alternative promoter at the Dnmt3a locus produces the shorter Dnmt3a2 transcript in the germ lineand postimplantation embryo only, whereas alternative splicing of the Dnmt3b transcript ensures thatDnmt3b1 is absent in the male prospermatogonia. Control of subcellular protein localization is a commontheme for DNMT3A and DNMT3B, as proteins were seen in the nucleus only when methylation wasoccurring. These mechanisms converge to ensure that the only time that functional products from eachlocus are present in the germ cell nuclei is around embryonic day 17.5 in males and after birth in thegrowing oocytes in females. Developmental Dynamics 232:992–1002, 2005. © 2005 Wiley-Liss, Inc.

Key words: DNA methyltransferase; transcription; alternative splicing; methylation; mouse; primordial germ cells;testis; ovary

Received 9 April 2004; Revised 1 October 2004; Accepted 4 October 2004

INTRODUCTION

Methylation of mammalian DNA is amajor epigenetic regulatory mecha-nism that is of key importance for thetranscriptional activity of genes inmany different sequence classes, in-cluding single-copy imprinted genesand multicopy repeated elements suchas endogenous retroviruses. Methyl-ation of certain repeat sequences suchas minor satellites also appears to beimportant for chromosomal stability,as their demethylation results in ab-

normal multiradiate chromosomes(Xu et al., 1999; Okano et al., 1999).DNA methylation undergoes dynamicchanges during normal germ line de-velopment in the mouse (reviewed inReik et al., 2001). Here, methylationmust be erased from imprinted genesand re-established in the correct sex-specific pattern, otherwise two inac-tive alleles might be inherited. At thesame time, the germ cells need tomaintain high levels of methylation ofrepetitive elements and satellite re-

peats. A major demethylation eventoccurs once the primordial germ cells(PGCs), the precursors of eggs andsperm, have migrated into the go-nadal ridges, as many sequences ap-pear to lose methylation at this stageand the inactive X is reactivated(Chaillet et al., 1991; Brandeis et al.,1993; Tam et al., 1994; Davis et al.,2000; Hajkova et al., 2002; Lee et al.,2002; Lees-Murdock et al., 2003).Methylation patterns are then re-es-tablished during gametogenesis ac-

1Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, United Kingdom2Department of Ophthalmology, Queen’s University of Belfast, Northern Ireland, United Kingdom3Department of Public Health and Cell Biology, University of Rome “Tor Vergata”, Rome, ItalyGrant sponsor: BBSRC; Grant numbers: 102/G12997; BBS/B/07403; Grant sponsor: Northern Ireland HPSS R&D Office; Grant number:RRG 6.7; Grant sponsor: Royal Society; Grant number: RSRG 20735.*Correspondence to: Colum Walsh, Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster,Coleraine BT52 1SA, UK. E-mail: [email protected]

DOI 10.1002/dvdy.20288Published online 28 February 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 232:992–1002, 2005

© 2005 Wiley-Liss, Inc.

cording to the sex of the new organismand the class of sequence being meth-ylated. In the male germ line, repeatsequences are only partially demeth-ylated upon PGC entry into the gonadand become completely remethylatedin prospermatogonia between embry-onic day (e) 15.5 and e17.5 (Hajkova etal., 2002; Lane et al., 2003; Lees-Mur-dock et al., 2003). Meanwhile methyl-ation is wiped from imprinted genesshortly after entry into the gonadalenvironment and males begin rem-ethylation by e15.5, but the processtakes longer than for repeats and isnot complete until after birth (Daviset al., 1999, 2000; Li et al., 2004). Inthe female germ line, methylation ofimprinted genes begins at the postna-tal growing oocyte stage (Lucifero etal., 2002; Obata and Kono, 2002).Methylation of repeats is also as-sumed to occur at this time in oocytes,as they show low levels of methylationbefore birth but appear fully methyl-ated by the time of ovulation (Walsh etal., 1998; Bourc�his et al., 2001).

Currently, there are three knownfamilies of DNA methyltransferase(DNMT) enzymes. DNMT1 convertshemimethylated DNA to fully methyl-ated duplexes with high efficiency andis believed to function primarily as amaintenance methyltransferase (Yo-der et al., 1997; Lyko et al., 1999; Rob-ert et al., 2003). This enzyme haswidespread expression, being local-ized to the replication foci in all mitot-ically dividing cells (Leonhardt et al.,1992) and can also be detected at theleptotene/zygotene stage of meiosisduring sperm maturation (Jue et al.,1995). The function of DNMT2 is notclear, but targeted disruption of theDnmt2 gene in embryonic stem (ES)cells resulted in no observable effecton genomic methylation patterns orthe ability of the cells to methylatenewly integrated retroviral DNA(Okano et al., 1998b). DNMT3A andDNMT3B appear to carry out most denovo methylation, which is the addi-tion of a methyl group to previouslyunmodified DNA (Okano et al.,1998a). They have been shown to beexpressed in postimplantation mouseembryos, when de novo methylation ofsomatic tissues is occurring (Okano etal., 1998a, 1999) and their disruptionresults in failure to increase methyl-ation levels from the low levels

present in the blastocyst or to methy-late newly introduced proviral DNA(Okano et al., 1999). DNMT3B specif-ically methylates the centromeric mi-nor satellite repeats in ES cells anddeveloping mouse embryos (Okano etal., 1999) and mutations in the humanDNMT3B gene have been shown to beresponsible for demethylation of clas-sic satellite sequences and subsequentabnormalities such as multiradiatechromosomes observed in ICF pa-tients (Hansen et al., 1999; Okano etal., 1999; Xu et al., 1999). DNMT3Aand DNMT3B, therefore, are goodcandidates for carrying out de novomethylation in the mouse germ line aswell and recently, an essential role forDNMT3A in methylating the H19 andGtl2 imprinted genes in the malegerm line has been demonstrated byusing targeted deletion of this gene ingerm cells, but no effect of the muta-tion was seen on IAP sequences(Kaneda et al., 2004). Their expres-sion pattern in the germ cell lineagehas not been well characterized, how-ever.

At least two DNMT3A isoforms andsix DNMT3B isoforms have been iden-tified so far (Okano et al., 1998a; Han-sen et al., 1999; Robertson et al., 1999;Xie et al., 1999; Chen et al., 2002).DNMT3A and DNMT3A2 are pro-duced from different promoters, theDnmt3a2 promoter being located in anintron downstream of the Dnmt3apromoter (Chen et al., 2002). Allknown isoforms of DNMT3B are pro-duced by means of alternative splicingof exons 10, 21, or 22 in various com-binations. It has been demonstratedthat the full-length DNMT3B1 iso-form and DNMT3B2, in which exon 10is spliced out, can both methylateDNA in vitro (Okano et al., 1998a;Aoki et al., 2001). However, the re-maining four isoforms lack exon 22 orboth exon 21 and 22, and thus aremissing essential conserved motifs inthe C-terminal catalytic domain re-quired for enzymatic activity (Robert-son et al., 1999; Chen et al., 2003).

Recently DNMT3L, a new memberof the DNMT3 family, has been de-scribed which has no detectable DNAmethyltransferase activity and is re-quired for methylation of maternallyimprinted genes, but not repeated el-ements, in the oocyte (Bourc�his et al.,2001; Hata et al., 2002). In the male

germ line, DNMT3L appears to be re-quired for methylation of L1 and IAPelements, but not satellite repeats orimprinted genes (Bourc�his and Be-stor, 2004). Although DNMT3L has noenzymatic activity itself it is believedto regulate methylation through itsinteraction with DNMT3A andDNMT3B (Chedin et al., 2002; Hata etal., 2002).

It is still unclear which particularisoforms of the known DNA methyl-transferases are involved in establish-ing and maintaining methylation pat-terns in the embryonic germ cells andhow the presence or absence of theproteins is controlled. We have inves-tigated the expression of mouse denovo DNA methyltransferase proteinsDNMT3A and DNMT3B and tran-scription of the methylation regulatorDnmt3l during the periods of mousedevelopment where methylation im-prints are thought to be established.We demonstrate that DNMT3A andDNMT3B undergo posttranscriptionalregulation in the germ line and thatthe only time that all three proteinsare found to be present in the nuclei ofgerm cells is during those brief peri-ods when de novo methylation isknown to occur.

RESULTS

Transcript Levels of DNAMethyltransferases in theGerm Line

To determine the transcription levelsof Dnmt3a, Dnmt3b, and Dnmt3l indeveloping germ cells, we carriedout reverse transcriptase-polymerasechain reaction (RT-PCR) on gonads atdifferent stages of development. Germcells were purified from testis andovary at e12.5, when methylation lev-els are low in both sexes; at e17.5,when de novo methylation is occurringin males but not females; and fromadult whole gonad, where de novomethylation is occurring in ovary butnot in testis. Total RNA was extractedfor RT-PCR analysis using standardtechniques. We used e8.5 embryo as apositive control, as somatic expressionof the genes is strong at this timepoint and lung as a negative control,as it has been reported that expres-sion is low in this tissue. A multiplexapproach was adopted to ensure that

METHYLTRANSFERASE EXPRESSION IN GERM CELLS 993

the PCR was working in each tube andto provide a rough estimate of levels ofexpression (Fig. 1A). PCR primerswere designed to match the 3�-un-translated regions (UTRs) of the tran-scripts to pick up all the differentsplice variants of Dnmt3a, Dnmt3b,and Dnmt3l at once (as there are nosplice variants in this region), while

minimizing the risk of cross-hybrid-ization. The second primer set used ineach multiplex reaction was for �-ac-tin, which produces a transcript of 480bp, allowing it to be distinguishedfrom Dnmt3a (383 bp), Dnmt3b (451bp), and Dnmt3l (315 bp). RT-PCR re-actions with different cycle numberswere carried out to assess the optimal

number of cycles to use for determina-tion of methyltransferase transcriptlevels (Fig. 1B). The RT-PCR reactionwas not saturated and was well withinthe linear range at 30 cycles; there-fore, subsequent RT-PCR reactionswere carried out at this cycle number.

Strong expression of all the methyl-transferases was detected in the e8.5positive control, and expression waslow in lung, as expected. Dnmt3a andDnmt3b transcripts were detected atuniform levels at all stages of germcell development, with Dnmt3a beingexpressed at a slightly higher levelthroughout than Dnmt3b. Dnmt3l onthe other hand showed a clear peak intranscription at e17.5 in male andpostnatally in female. This findingwas confirmed by comparing the rela-tive intensity of the methyltrans-ferase bands in each lane with that ofthe control �-actin band in the samelane using image densitometry analy-sis of the digital images, which pro-vided a semiquantitative value for ex-pression (Table 1). Whole-mount insitu on prenatal gonads also con-firmed that Dnmt3l transcripts arepresent at high levels in e17.5 male

Fig. 1. Total transcription levels of the DNA methyltransferases in gonads. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) to detecttranscripts in gonads. Total RNA from purified germ cells (embryonic day [e] 12.5, e17.5) or gonads (adult) were incubated in RT buffer with or without(RT Neg panel) reverse transcriptase before carrying out multiplex RT-PCR for �-actin and Dnmt3a (top panel), Dnmt3b (second panel), or Dnmt3l (thirdpanel). Primers were designed to pick up all known isoforms at once. RNA from e8.5 embryos was used as a positive control, whereas lung has beenreported to have lower levels of Dnmt3a and Dnmt3b. Only Dnmt3l showed a pattern of transcription that closely matches the timing of de novomethylation in the male and female germ lines. B: Control to ensure RT-PCR is in the linear range. Samples of RT-PCR reactions were taken at regularcycle intervals, samples were run on a gel, and digital images were subjected to image densitometry analysis as described in the ExperimentalProcedures section. Values for each band were plotted against cycle number. The reactions can be seen to plateau after 35 cycles. Similar results wereobtained for the other multiplex reactions. RT-PCR reactions for quantitative estimations (Table 1), therefore, were carried out at 30 cycles. Diamonds,Dnmt3a; squares, �-actin.

TABLE 1. Methyltransferase Transcription LevelsRelative to �-Actin by RT-PCRa

Tissue

Relative Intensity

Dnmt3a Dnmt3b Dnmt3l

e12.5 testis 0.53 0.23 0.04e17.5 testis 0.57 0.33 1.01Adult testis 0.44 0.30 0.49e12.5 ovary 0.5 0.2 0.06e17.5 ovary 0.57 0.38 0.22Adult ovary 0.31 0.38 0.9Adult lung 0.24 0.1 0.25e8.5 embryo 0.59 0.48 0.37

aRelative intensity was obtained by normalization to the �-actin band present inthe same lane in the analysis gel, which was assigned an arbitrary value of 1.0.Only Dnmt3l shows marked peaks in transcription at time points where cellsundergoing de novo methylation can be found (e17.5 testis and adult ovary). e,embryonic day; RT-PCR, reverse transcriptase-polymerase chain reaction.

994 LEES-MURDOCK ET AL.

gonads, but not in female at this stage(data not shown). Although we cannotrule out contributions by the smallpercentage of somatic cells present inembryonic germ cell preparations,these results suggested that theamount of DNMT3L protein, but notof DNMT3A or DNMT3B, could becontrolled at the level of gene tran-scription. DNMT3L is not an activemethyltransferase enzyme, and denovo methylation does not occur at allstages of germ cell development wherewe detect transcripts for DNMT3Aand DNMT3B, so we looked to seewhat alternative mechanisms could

also be operating to regulate levels ofthe latter two proteins.

Promoter Usage at theDnmt3a Locus

It has been suggested that theDNMT3A2 isoform, which is producedfrom a downstream intronic promoterat the Dnmt3a locus (Fig. 2A), is moreimportant for de novo methylationthan DNMT3A (Chen et al., 2002).Dnmt3a and Dnmt3a2 transcriptswere amplified in a multiplex reactionusing one forward primer unique toDnmt3a (F9) and one unique to

Dnmt3a2 (F5), together with a reverseprimer (R5) common to both (Fig. 2A).Transcription of Dnmt3a2 was muchhigher than Dnmt3a in e8.5 embryos,where de novo methylation is occur-ring, but lower in adult somatic tis-sues, as expected (Fig. 2B). In themale germ line, there was little differ-ence in transcription levels betweenthe two isoforms, although Dnmt3a2transcripts may be slightly moreabundant at e15.5 (Fig. 2B). In thefemale germ line, expression ofDnmt3a2 was somewhat strongerthan Dnmt3a at 12 days postpartum(dpp), when there is a postnatal wave

Fig. 2. Promoter usage at the Dnmt3a locus. A: Structure of the Dnmt3a locus (adapted from Chen et al., 2002). Dnmt3a2 was amplified using aprimer (F5) placed in a 5� noncoding exon unique to Dnmt3a2 and Dnmt3a using a primer (F9) in an upstream exon not present in Dnmt3a2. The reverseprimer R5 is in a common downstream exon. The positions of primers F1 and R1 in the 3�-untranslated region common to Dnmt3a and Dnmt3a2 andused for Figure 1 are also indicated. B: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of Dnmt3a and Dnmt3a2 transcription inpurified germ cells from embryonic stages, whole gonads, and somatic tissues. Total RNA was extracted, and RT-PCR analysis was carried out asfor Figure 1A. Transcription of Dnmt3a2 is low in somatic tissues except for embryonic day 8.5 embryo, where Dnmt3a was undetectable. Bothtranscripts can be detected in germ cells, with relative transcription of Dnmt3a2 slightly higher at 12 days postpartum (dpp) in the female,corresponding to the midgrowth stage for the first wave of maturing oocytes.


of oocyte growth (Pedersen, 1970) andde novo methylation (Lucifero et al.,2002; Obata and Kono, 2002). Dnmt3atranscripts were more abundant thanDnmt3a2 in adult ovary, probably dueto the greater proportion of somaticcells present here (Fig. 2B) and thesame is true to a lesser extent in adulttestis. Multiplex reactions with �-ac-tin and each isoform on its own gavesimilar results (data not shown), andour RT-PCR results are in good agree-ment with a recent study using real-time PCR with primers common toboth Dnmt3a and Dnmt3a2 (La Salleet al., 2004).

Alternative Splicing ofDnmt3b

The mouse Dnmt3b gene contains 23exons: exons 5, 10, and 21–22 can bespliced out to give rise to several dif-ferent transcripts (Fig. 3A). The twomajor functional isoforms of DNMT3Bretain exons 21 and 22, which containvital catalytic motifs but differ in thepresence or absence of exon 10. Theother enzymatically inactive isoformsall lack both exons 21 and 22. To ex-amine at which developmental stagesthe functional isoforms were presentin the germ line, we used two primerpairs (Fig. 3A), one pair spanningexon 10 (F6, R6) and another pairspanning exons 21 and 22 (F7, R7).Although previous reports suggestthat exons 21 and 22 are spliced outtogether in mouse, we found that exon21 can also be spliced out indepen-dently of exon 22 (Fig. 3B). RT-PCRover the exon 21–22 region was car-ried out with a second set of primers(F8, R8) and confirmed that the bandrepresenting new isoforms that lackexon 21 but retain exon 22 repre-sented a genuine splice variant andnot an artefact of PCR (Fig. 3B). Be-cause exon 21 contains part of motifIX, isoforms lacking this exon are alsounlikely to be enzymatically active.The major Dnmt3b isoform that canproduce a functional protein that isexpressed in the male germ line ap-pears to be Dnmt3b2, which retainsexons 21–22 but lacks exon 10 (Fig.3C). Dnmt3b1 is absent at moststages, including those time pointswhen male germ cells are undergoingmethylation, but can be detected at 24dpp when the germ cells start to enter

the pachytene stage of meiosis and ine8.5 embryos. Both Dnmt3b1 andDnmt3b2 are present in the femalegerm line at the majority of timepoints examined, but Dnmt3b1 ap-pears to be absent in the adult ovary,which would contain growing oocytes(Fig. 3D).

Subcellular Localization ofDNMT3A and DNMT3BDuring Germ CellDevelopment

Previous reports have indicated thatDNMT3A was undetectable andDNMT3B is excluded from the nu-cleus in e12.5 germ cells, where exten-sive demethylation is occurring (Ha-jkova et al., 2002). We usedimmunocytochemistry on disaggre-gated embryonic gonads to examinewhether the protein is found in thenuclei of male germ cells at e15.5 ande17.5. Gonads were removed from em-bryos and disaggregated in collage-nase to obtain a single cell suspension.A small aliquot was placed on a poly-L-lysine slide and stained for eitherDNMT3A or DNMT3B. The slideswere counterstained with the DNAfluorochrome 4�,6-diamidine-2-pheny-lidole-dihydrochloride (DAPI), whichallows germ cells and the oocyte mei-otic stages to be distinguished on thebasis of their nuclear morphology(McLaren and Southee, 1997; DeFelici et al., 1999). At e15.5, someDNMT3A and DNMT3B protein couldbe weakly detected in prospermatogo-nia, and by e17.5, expression wasstrong for both proteins in nuclei andcytoplasm of most germ cells (Fig. 4A).In the female germ line, which is notundergoing extensive methylation atthis time, DNMT3A was absent inboth e15.5 and e17.5 primary oocytes,whereas DNMT3B was detected onlyin the subset of primary oocytes ate17.5, which were in the zygotene/pachytene stage (Fig. 4A).

We also wished to determineDNMT3A and DNMT3B protein ex-pression and localization at the timeimprints were being set in the femalegerm line. We examined cryosectionsof ovaries at 12 dpp, in which someoocytes have initiated growth, and at1 dpp where there are no growing oo-cytes present. Neither protein was de-tected at 1 dpp in nongrowing oocytes

(Fig. 4B, bottom panel, and data notshown), but both DNMT3A (Fig. 4B,top panel) and DNMT3B (Fig. 4B,middle panel) were detected in the nu-clei and cytoplasm of early stage grow-ing oocytes at 12 dpp. At later stagesof oocyte development, the stainingbecame predominantly cytoplasmicfor both proteins (Fig. 4B). Secondaryantibody alone gave no staining in allcases. Investigation of protein local-ization for DNMT3L and DNMT3A2were prevented by a lack of reliableantibodies for these proteins.

DISCUSSION

De novo DNA methylation occurs dur-ing very limited periods of germ celldevelopment in the mouse, suggestingthat there must be mechanisms inplace to limit expression of the func-tional methyltransferases DNMT3Aand DNMT3B and the methyltrans-ferase regulator DNMT3L to thesetime points. In this study, we foundthat each of the three genes uses adifferent strategy to confine expres-sion to specific developmental periods.

Dnmt3l appears to undergo regula-tion at the transcriptional level. Tran-scription of the gene is very low orabsent in e12.5 gonads when demeth-ylation of repetitive elements and im-printed genes is occurring. In the malegerm line, expression is high at e17.5,when extensive methylation is hap-pening, but remains low in the femaleat this time point, when mostsequences remain demethylated.Dnmt3l expression is high, however,in the adult ovary, where growing oo-cytes are present. These results indi-cate that production of the wild-typetranscript matches that of a lacZmarker knocked into the Dnmt3l locusand driven by the same promoter:LacZ staining was found to be presentin prospermatogonia and growing oo-cytes in these earlier studies(Bourc�his et al., 2001; Hata et al.,2002).

A different control mechanism,namely alternative promoter usage, isseen at the Dnmt3a locus. A secondtranscriptional start within a down-stream intron of the gene is used toproduce the shorter DNMT3A2 pro-tein, which although it does not differin amino acid sequence from thelonger DNMT3A, is produced in a


Fig. 3. Dnmt3b transcript usage during mouse development. A: Structure of the Dnmt3b locus (adapted from Xu et al., 1999). Exons 5, 10, 21, and22 show alternative splicing. The full-length Dnmt3b1 transcript contains all the exons, whereas Dnmt3b2 has exon 10 spliced out. The transcriptsDnmt3b3-Dnmt3b6 lack exons 21 and 22 and so cannot produce a catalytically active protein. B: A mouse Dnmt3b splice variant was identified thatcontains exons 10 and 22 but lacks exon 21. This variant, which has been reported previously in humans but not mice, is unlikely to be catalyticallyactive as it lacks part of motif IX. C,D: Reverse transcriptase-polymerase chain reaction analysis of the male and female germ lines, respectively. Thecorresponding catalytically active isoforms that could be expressed at each time point are indicated at the bottom of each lane. The exon 10 primerswere used in combination with �-actin primers to act as an internal control for the polymerase chain reaction. e, embryonic day; dpp, days postpartum.


Fig. 4. Subcellular localization of methyltransferase proteins during known periods of de novo methylation by immunocytochemistry. A: Disaggre-gated cells from embryonic day (e) 17.5 embryonal gonads. Red staining indicates presence of DNMT3A or DNMT3B as indicated, and blue4�,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining indicates nuclei. Most prospermatogonia stain weakly for DNMT3A and DNMT3B in thenucleus at e15.5, but staining becomes more intense and can also be seen in the cytoplasm by e17.5. Some somatic cells also showed staining forDNMT3B in the cytoplasm at e17.5 but not DNMT3A. In e15.5 primary oocytes, both proteins are absent, whereas at e17.5 staining for DNMT3B isfound only in zygotene/pachytene cells and disappears from cells that have fully progressed into pachytene. B: Postnatal ovaries. Cryosections from12 days postpartum (dpp; top and middle panel) and 1 dpp (bottom panel) mice were stained for DNMT3A and DNMT3B, nuclei were counterstainedwith propidium iodide, and confocal images were acquired. DNMT3A and DNMT3B were expressed in the nuclei and cytoplasm of growing oocytes(arrows) but later became confined to the cytoplasm (arrowheads). No staining above background was seen at 1 dpp.


more tissue-restricted pattern. We de-tected Dnmt3a transcripts at low lev-els in most somatic tissues, in agree-ment with previous data (Okano et al.,1998a), but Dnmt3a2 appears to betranscribed at significant levels onlyin the germ line and in the e8.5postimplantation embryo, where it isthe dominant transcript. In the malegerm line, both messages are presentin late embryonic and early postnatalstages, when methylation of repeat se-quences and paternally methylatedimprinted genes is occurring (Davis etal., 2000; Lees-Murdock et al., 2003;Li et al., 2004), and we detectDNMT3A protein in prospermatogo-nia with the antibody used in thisstudy, which has been previouslyshown not to cross-react withDNMT3A2 (Chen et al., 2002). Tran-scripts from both genes can also bedetected in the female germ line at 12dpp during the first and largest waveof oocyte growth, as well as in adultovary, which would also contain grow-ing oocytes. Although Dnmt3a2 tran-scripts seem to predominate at 12 dpp,the longer Dnmt3a transcript is alsodetected at this stage and the cognateprotein can be seen at high levels inthe growing oocyte. These resultswould be consistent with a role foreither DNMT3A or DNMT3A2 in denovo methylation in germ cells. Therecent tissue-specific knockout in thislineage would have affected both pro-teins (Kaneda et al., 2004), so a defin-itive experiment to address which ofthe two isoforms is more importantwill require promoter-specific dele-tions. One clue may be that there area large number of retrotransposedDnmt3a2 pseudogenes present inmouse, rat, and human, but noDnmt3a pseudogenes, which wouldsupport the idea that Dnmt3a2 istranscribed more frequently in thegerm line and has been over evolution-ary time (Lees-Murdock et al., 2004).

Dnmt3b has its own unique regula-tory mechanism, namely alternativesplicing. We found that the full-lengthDNMT3B1 protein is unlikely to beinvolved in de novo methylation in themale germ line, as the correspondingtranscript is not expressed during theperiod when imprints are being resetor repeat elements become methyl-ated in this germ line. The mainDnmt3b transcript present in the

male germ line that can produce a cat-alytically active protein is, therefore,Dnmt3b2, which lacks exon 10, andthe corresponding protein isoform ispresumably the one that carries out denovo methylation. Although Dnmt3b1transcripts are present during morestages of female germ line develop-ment, they appear to be absent fromadult ovaries, where the only func-tional isoform is Dnmt3b2. Taken to-gether, these data suggest that theDNMT3B2 protein is more importantin both germ lines for de novo methyl-ation. This conclusion is consistentwith the findings of Chen et al. (2003),who demonstrated that introductionof Dnmt3b1 cDNA was unable tocause methylation of the imprintedgenes H19 and Igf2 in doubly mutantDnmt3a�/� Dnmt3b�/� ES cells. Inthis study, we also detected mouseDnmt3b transcripts lacking exon 21but retaining exon 22, which havebeen described previously in humans(Xu et al., 1999). Transcripts lackingexon 21 are unlikely to produce pro-teins with any catalytic activity, asthey will be missing part of motif IX.

A final regulatory mechanism thatis common to DNMT3A and DNMT3Bis protein localization. While Dnmt3aand Dnmt3b transcripts are presentthroughout germ cell development,the corresponding proteins are not.Previous studies have found thatDNMT3A protein is undetectable ine12.5 germ cells, while DNMT3B isconfined to the cytoplasm (Hajkova etal., 2002). We report here thatDNMT3A and DNMT3B can be de-tected at low levels in the nuclei ofe15.5 male prospermatogonia, and bye17.5, staining is strong for both pro-teins in these cells. We and othershave shown that paternally methyl-ated imprinted genes lose their meth-ylation imprint at e12.5, but methyl-ation levels have started to climbagain by e15.5, with full methylationnot being seen until after birth (Daviset al., 1999; Li et al., 2004). Repetitivesequence methylation, however, isstill low at e15.5, but by e17.5, no de-methylated sequences can be detected(Lees-Murdock et al., 2003). The pres-ence of de novo methyltransferase pro-tein in the nuclei of prospermatogoniaat e15.5 when the repeat elements arestill unmethylated suggests that anadditional regulatory mechanism is

required to trigger methylation ofthese elements; this conclusion is alsosuggested from recent studies done onDnmt3l and Dnmt3a mutant miceshowing that methylation of selfish re-peats and imprinted genes can be un-coupled (Bourc�his et al., 2001;Kaneda et al., 2004; Bourc�his and Be-stor, 2004). In the female germ line,both DNMT3A and DNMT3B can bedetected in the nuclei of growing oo-cytes at 12 dpp, when de novo meth-ylation of imprinted genes is occurring(Lucifero et al., 2002). DNMT3A wasundetectable in germ cells that are notundergoing active methylation (e15.5,e17.5, and 1 dpp female), and itsdown-regulation may contribute tothe genome-wide demethylation ob-served at e17.5 in the female germcells (Lucifero et al., 2002; Lees-Mur-dock et al., 2003; Li et al., 2004). Theabsence of protein, despite the pres-ence of some transcripts in these cells,suggests that these are not translated.DNMT3B is also absent in e15.5 and 1dpp female germ cells but is present inthe cytoplasm and nuclei of e17.5 oo-cytes at the zygotene/pachytene stage.This finding could explain why minorsatellites retain a higher degree ofmethylation than other repeats be-tween e12.5 and e17.5 in the mouseprimary oocytes (Lees-Murdock et al.,2003), which may be important to en-sure stability of chromosomes duringcrossing-over in pachytene. Of inter-est, the only time when the mainDnmt3b1 transcript is produced in themale germ line is at 24 dpp, whenspermatocytes are also enteringpachytene. This particular isoformhas been shown to have preference formethylation of minor satellite repeatsover major satellites or endogenous C-type retroviral DNA (Chen et al.,2003). Dnmt3b1, therefore, may be in-volved in chromosome stability in bothgerm lines, even though it is not re-quired for methylation of imprintedgenes in the male. As the growing oo-cytes mature, we found that DNMT3Aand DNMT3B expression becamemore cytoplasmic, as was shown to bethe case for DNMT1 (Mertineit et al.,1998). We have shown previouslythat, on progression of fully growngerminal vesicle stage oocytes tometaphase II oocytes, DNMT3A andDNMT3B become localized to a sub-cortical layer in a manner similar to


DNMT1 (Lees-Murdock et al., 2004).It appears that these proteins are allsynthesized and stored in the cyto-plasm and move to the nucleus tocarry out their enzymatic function,moving back out when they are nolonger required.

In conclusion, it is notable that theonly time points at which Dnmt3l andDnmt3a2 transcripts are present andwhere DNMT3A and DNMT3B can bedetected in the nucleus are at the twostages where the male and femalegerm cells are undergoing de novomethylation, e15.5 to e17.5 in themale and after birth in the growingoocytes in the female. This findingsuggests that, although differing con-trol mechanisms operate at each lo-cus, the combined effect is to restrictde novo methylation to particular lim-ited periods during germ cell develop-ment. Our results indicate thatDNMT3L, DNMT3A2, and DNMT3B2may be more important for de novomethylation of imprinted genes andmost repeats in the male and femalegerm line, whereas DNMT3B1 may bemore important for methylation of sat-ellite DNA sequences.

EXPERIMENTALPROCEDURES

Mice

HsdOla:To mice were purchased forHarlan UK, Ltd. (Oxon, England).Natural matings were used to produceembryos and neonatal mice for the go-nad dissection and germ cell isolation.The day the plug was observed wastaken as e0.5, and the day of birth wastaken as 0 dpp.

Purification of PGCs,Prospermatogonia, andPrimary Oocytes

Embryos were collected at e12.5, e15.5,and e17.5 and sexed by morphology(Hogan et al., 1986). Germ cells werepurified without culture as describedpreviously (Lees-Murdock et al., 2003).Briefly, embryonic gonads with mesone-phros were incubated in 0.54 mM eth-ylenediaminetetraacetic acid, 136 mMNaCl, 2.68 mM KCl, 9.58 mMNa2HPO4, 1.47 mM KH2PO4 for 20 minat room temperature with rocking. Go-nads were rinsed in M2 medium and

stabbed 25 times with two 25-gaugeneedles in a microdroplet of M2 me-dium. Germ cells were collected in themicrodroplet, washed, and resuspendedin Ca2�-Mg2�-free phosphate bufferedsaline (PBS). Purity was uniformlygreater than 85% using this method.

RNA Extraction From MouseTissues

Total RNA was extracted by using anRNeasy Mini Kit (Qiagen) followingmanufacturer’s instructions. RNAwas resuspended in nuclease-free wa-ter (Promega) and stored at �80°C foruse in RT-PCR.

Semiquantitative RT-PCR

First-strand cDNA was synthesized ina 50-�l reaction mixture containing 10mM Tris HCl (pH 8.3), 0.2 �g of oligo(dT)15 primer (Promega), 1.5 mM de-oxynucleoside triphosphates (Sigma),1� AMV-RT buffer, 1�g of RNA, and20 U AMV reverse transcriptase (Pro-mega). Primers from the 3�-UTR re-gion were initially used to simulta-neously amplify all transcripts ofDnmt3a (F1, CGC CTG GCC CTCTGT GCA AAG; R1, GGT CAC TTTCCC TCA CTC TGG), Dnmt3b (F2,GGA AGG GTG GGT GGA GTG GC;R2, GCC CAC ACC TTG CGA GTTAC), and Dnmt3L (F3, CTG AAG AGCAAG CAT GCG CC; R3, GCT GCACAG AGG CAT CCT GG). �-actinprimers (Obata and Kono, 2002) wereincluded as an internal control (F4,GCT GTG CTA TGT TGC TCT AGACTT C; R4,CTC AGT AAC AGT CCGCCT AGA AGC). For further investi-gation of individual isoforms, Dnmt3aand Dnmt3a2 were was amplified byusing specific forward primers (F9,CAG CGA CCC ATG CCA AGA CTC;F5, AGG GGC TGC ACC TGG CCTT,respectively) and a common reverseprimer (R5, TCC CCC ACA CCA GCTCTC C). F5 and R5 have been de-scribed elsewhere (Chen et al., 2002).Primers that span exon 10 and exons21–22 of Dnmt3b were as follows: exon10: F6, TGG GAT CGA GGG CCTCAA AC; and R6, TTC CAC AGG ACAAAC AGC GG and exon 21–22: F7,CGA TGC CAT CAA GGT GTC; andR7, CAC TGG CTC TGA CCG AGAGC; or F8, TGC GCG ACA ACC GTCCAT TCT TC; and R8, GGA CAC GTC

CGT GTA GTG AGC. PCR was carriedout in a 25-�l volume containing 5 �lof cDNA, 1� Taq buffer, 1.5 mM de-oxynucleoside triphosphates (deter-mined to be the optimal amount inpilot experiments), 0.5 pmol of eachprimer and 2 U Taq (Sigma). Sampleswere initially deannealed at a temper-ature of 94°C for 3 min, followed by 30cycles of 94°C for 30 sec, 65°C for 1min, and 68°C for 1 min, followed by afinal elongation step of 7 min at 72°C.PCR products were then separated on3% agarose gels, and images were cap-tured by using a Kodak digital cameraand saved as TIFF files for subsequentimage analysis.

Image Densitometry Analysis

Image densitometry analysis was per-formed on TIFF images usingPhoretix 2D software (Nonlinear Dy-namics). The �-actin band in each lanewas assigned an arbitrary value of 1.0and used to calculate the relative in-tensity of the methyltransferase bandin each lane.

Disaggregation of Gonadsand Immunocytochemistryon Prospermatogonia andPrimary Oocytes

Whole gonads from e17.5 embryoswith the mesonephros removed wereincubated in 1 mg/ml collagenase at37°C for 15 min. Trituration with ayellow tip was carried out to ensurethat a single cell suspension was ob-tained. Cell suspensions were washedin M2 medium before being resus-pended in M2 supplemented with 1mg/ml bovine serum albumin (BSA). Asmall aliquot was placed on a poly-lysine slide (approximately 10 �l), andcells were allowed to attach for 10 minbefore addition of 50 �l of ice-cold 4%paraformaldehyde. After 5 min, mostof the liquid was removed and re-placed with a fresh aliquot of 4% para-formaldehyde and incubated at roomtemperature for 5 min. Slides weresubsequently washed three times inM2 supplemented with 1 mg/ml BSAand blocked in this solution for 2–3 hrbefore permeabilization by incubationwith antibody buffer (1% TritonX-100, 10 mg/ml BSA, 0.2% sodiumdodecyl sulfate) for 30 min at roomtemperature. Primary antibody for


DNMT3A or DNMT3B (kind gifts ofDr. En Li) were diluted 1:400 and in-cubated overnight with samples. Afterthree 5-min washes in antibodybuffer, slides were incubated with a1:400 dilution of donkey anti-rabbitsecondary conjugated with rhodamine(Santa Cruz Biotechnology) for 1 hr atroom temperature. Slides werewashed three times in antibody bufferand incubated with 125 ng/�l DAPIfor 10 min at room temperature. Afterthree PBS washes, slides weremounted with Vectashield (VectorLaboratories), sealed, and viewed on aNikon Eclipse 600 microscope. Imageswere photographed on slide film andscanned and edited in Photoshop 7.0(Adobe).

Collection of Ovaries forCryosectioning andImmunohistochemistry

Ovaries were collected from neonatalmice at 1 dpp and 12 dpp and placedinto ice-cold 4% paraformaldehyde.Tissues were incubated at 4°C over-night with rocking and rinsed twice inPBS followed by two 5-min washes inPBS, then incubation overnight in30% sucrose at 4°C with rocking. Sub-sequently, ovaries were placed inmolds with OCT compound, incubatedat 4°C for 1 hr and then placed in dryice until frozen. Samples were storedat �80°C until sectioning. Sectionswere 10 �m thick and mounted ontoSuperFrost Plus slides and stored at�80°C until use. For immunohisto-chemistry on ovary cryosections, sec-tions were fixed in 4% ice-cold para-formaldehyde for 15 min and washedthree times in PBS before blocking inM2 containing 4% goat serum insteadof BSA for 2–3 hr at room tempera-ture. Permeabilization was carriedout by incubation with antibody bufferas described above, except 4% goat se-rum was included instead of BSA. Pri-mary and secondary antibodies (goatanti-rabbit Alexa 488, MolecularProbes) were diluted 1:400 and incu-bated as above. After secondary anti-body incubation, slides were washedand incubated with 100 �g/ml RNaseONE (Promega) for 10 min and subse-quently stained with 25 �g/ml pro-pidium iodide for 5 min at room tem-perature. Slides were washed threetimes with PBS before mounting in

Vectashield and viewing on a Bio-RadOlympus confocal microscope. Imageswere processed in Lasersharp/Confo-cal Assistant.

ACKNOWLEDGMENTSWe thank Dr. En Li for the kind gift ofthe DNMT3A and DNMT3B antibod-ies used in this study;J. McDaid, C.Lynch, L. Hipiri, and H. Wheadon forhelpful discussions; and R. Black fortechnical support. C.P.W. was fundedby grants from the BBSRC, the North-ern Ireland HPSS R&D office, and theRoyal Society.

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DNA methyltransferase expression in the mouse germ line during periods of de novo methylation

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