DNA methyltransferase expression in the mouse ... - Wiley Online Library

DEVELOPMENTAL DYNAMICS 232:992–1002, 2005

RESEARCH ARTICLE

DNA Methyltransferase Expression in the Mouse Germ Line During Periods of De Novo Methylation Diane 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 required for development, whereas DNMT3L, which has no demonstrable methyltransferase activity, is required for methylation of imprinted genes in the oocyte. We show here that different mechanisms are used to restrict access by these proteins to their targets during germ cell development. Transcriptional control of the Dnmt3l promoter guarantees that message is low or absent except during periods of de novo activity. Use of an alternative promoter at the Dnmt3a locus produces the shorter Dnmt3a2 transcript in the germ line and postimplantation embryo only, whereas alternative splicing of the Dnmt3b transcript ensures that Dnmt3b1 is absent in the male prospermatogonia. Control of subcellular protein localization is a common theme for DNMT3A and DNMT3B, as proteins were seen in the nucleus only when methylation was occurring. These mechanisms converge to ensure that the only time that functional products from each locus are present in the germ cell nuclei is around embryonic day 17.5 in males and after birth in the growing 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 a major epigenetic regulatory mechanism that is of key importance for the transcriptional activity of genes in many different sequence classes, including single-copy imprinted genes and multicopy repeated elements such as endogenous retroviruses. Methylation of certain repeat sequences such as minor satellites also appears to be important for chromosomal stability, as their demethylation results in ab-

normal multiradiate chromosomes (Xu et al., 1999; Okano et al., 1999). DNA methylation undergoes dynamic changes during normal germ line development in the mouse (reviewed in Reik et al., 2001). Here, methylation must be erased from imprinted genes and re-established in the correct sexspecific pattern, otherwise two inactive alleles might be inherited. At the same time, the germ cells need to maintain high levels of methylation of repetitive elements and satellite re-

1

peats. A major demethylation event occurs once the primordial germ cells (PGCs), the precursors of eggs and sperm, have migrated into the gonadal ridges, as many sequences appear to lose methylation at this stage and 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-established during gametogenesis ac-

Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, United Kingdom Department of Ophthalmology, Queen’s University of Belfast, Northern Ireland, United Kingdom 3 Department of Public Health and Cell Biology, University of Rome “Tor Vergata”, Rome, Italy Grant 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] 2

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

© 2005 Wiley-Liss, Inc.

METHYLTRANSFERASE EXPRESSION IN GERM CELLS 993

cording to the sex of the new organism and the class of sequence being methylated. In the male germ line, repeat sequences are only partially demethylated upon PGC entry into the gonad and become completely remethylated in prospermatogonia between embryonic day (e) 15.5 and e17.5 (Hajkova et al., 2002; Lane et al., 2003; Lees-Murdock et al., 2003). Meanwhile methylation is wiped from imprinted genes shortly after entry into the gonadal environment and males begin remethylation by e15.5, but the process takes longer than for repeats and is not complete until after birth (Davis et al., 1999, 2000; Li et al., 2004). In the female germ line, methylation of imprinted genes begins at the postnatal growing oocyte stage (Lucifero et al., 2002; Obata and Kono, 2002). Methylation of repeats is also assumed to occur at this time in oocytes, as they show low levels of methylation before birth but appear fully methylated by the time of ovulation (Walsh et al., 1998; Bourc⬘his et al., 2001). Currently, there are three known families of DNA methyltransferase (DNMT) enzymes. DNMT1 converts hemimethylated DNA to fully methylated duplexes with high efficiency and is believed to function primarily as a maintenance methyltransferase (Yoder et al., 1997; Lyko et al., 1999; Robert et al., 2003). This enzyme has widespread expression, being localized to the replication foci in all mitotically dividing cells (Leonhardt et al., 1992) and can also be detected at the leptotene/zygotene stage of meiosis during sperm maturation (Jue et al., 1995). The function of DNMT2 is not clear, but targeted disruption of the Dnmt2 gene in embryonic stem (ES) cells resulted in no observable effect on genomic methylation patterns or the ability of the cells to methylate newly integrated retroviral DNA (Okano et al., 1998b). DNMT3A and DNMT3B appear to carry out most de novo methylation, which is the addition of a methyl group to previously unmodified DNA (Okano et al., 1998a). They have been shown to be expressed in postimplantation mouse embryos, when de novo methylation of somatic tissues is occurring (Okano et al., 1998a, 1999) and their disruption results in failure to increase methylation levels from the low levels

present in the blastocyst or to methylate newly introduced proviral DNA (Okano et al., 1999). DNMT3B specifically methylates the centromeric minor satellite repeats in ES cells and developing mouse embryos (Okano et al., 1999) and mutations in the human DNMT3B gene have been shown to be responsible for demethylation of classic satellite sequences and subsequent abnormalities such as multiradiate chromosomes observed in ICF patients (Hansen et al., 1999; Okano et al., 1999; Xu et al., 1999). DNMT3A and DNMT3B, therefore, are good candidates for carrying out de novo methylation in the mouse germ line as well and recently, an essential role for DNMT3A in methylating the H19 and Gtl2 imprinted genes in the male germ line has been demonstrated by using targeted deletion of this gene in germ cells, but no effect of the mutation was seen on IAP sequences (Kaneda et al., 2004). Their expression pattern in the germ cell lineage has not been well characterized, however. At least two DNMT3A isoforms and six DNMT3B isoforms have been identified so far (Okano et al., 1998a; Hansen et al., 1999; Robertson et al., 1999; Xie et al., 1999; Chen et al., 2002). DNMT3A and DNMT3A2 are produced from different promoters, the Dnmt3a2 promoter being located in an intron downstream of the Dnmt3a promoter (Chen et al., 2002). All known isoforms of DNMT3B are produced by means of alternative splicing of exons 10, 21, or 22 in various combinations. It has been demonstrated that the full-length DNMT3B1 isoform and DNMT3B2, in which exon 10 is spliced out, can both methylate DNA in vitro (Okano et al., 1998a; Aoki et al., 2001). However, the remaining four isoforms lack exon 22 or both exon 21 and 22, and thus are missing essential conserved motifs in the C-terminal catalytic domain required for enzymatic activity (Robertson et al., 1999; Chen et al., 2003). Recently DNMT3L, a new member of the DNMT3 family, has been described which has no detectable DNA methyltransferase activity and is required for methylation of maternally imprinted genes, but not repeated elements, in the oocyte (Bourc⬘his et al., 2001; Hata et al., 2002). In the male

germ line, DNMT3L appears to be required for methylation of L1 and IAP elements, but not satellite repeats or imprinted genes (Bourc⬘his and Bestor, 2004). Although DNMT3L has no enzymatic activity itself it is believed to regulate methylation through its interaction with DNMT3A and DNMT3B (Chedin et al., 2002; Hata et al., 2002). It is still unclear which particular isoforms of the known DNA methyltransferases are involved in establishing and maintaining methylation patterns in the embryonic germ cells and how the presence or absence of the proteins is controlled. We have investigated the expression of mouse de novo DNA methyltransferase proteins DNMT3A and DNMT3B and transcription of the methylation regulator Dnmt3l during the periods of mouse development where methylation imprints are thought to be established. We demonstrate that DNMT3A and DNMT3B undergo posttranscriptional regulation in the germ line and that the only time that all three proteins are found to be present in the nuclei of germ cells is during those brief periods when de novo methylation is known to occur.

RESULTS Transcript Levels of DNA Methyltransferases in the Germ Line To determine the transcription levels of Dnmt3a, Dnmt3b, and Dnmt3l in developing germ cells, we carried out reverse transcriptase-polymerase chain reaction (RT-PCR) on gonads at different stages of development. Germ cells were purified from testis and ovary at e12.5, when methylation levels are low in both sexes; at e17.5, when de novo methylation is occurring in males but not females; and from adult whole gonad, where de novo methylation is occurring in ovary but not in testis. Total RNA was extracted for RT-PCR analysis using standard techniques. We used e8.5 embryo as a positive control, as somatic expression of the genes is strong at this time point and lung as a negative control, as it has been reported that expression is low in this tissue. A multiplex approach was adopted to ensure that

994 LEES-MURDOCK ET AL.

Fig. 1. Total transcription levels of the DNA methyltransferases in gonads. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) to detect transcripts 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 (third panel). 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 been reported to have lower levels of Dnmt3a and Dnmt3b. Only Dnmt3l showed a pattern of transcription that closely matches the timing of de novo methylation 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 regular cycle intervals, samples were run on a gel, and digital images were subjected to image densitometry analysis as described in the Experimental Procedures section. Values for each band were plotted against cycle number. The reactions can be seen to plateau after 35 cycles. Similar results were obtained 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 Levels Relative to ␤-Actin by RT-PCRa Relative Intensity Tissue

Dnmt3a

Dnmt3b

Dnmt3l

e12.5 testis e17.5 testis Adult testis e12.5 ovary e17.5 ovary Adult ovary Adult lung e8.5 embryo

0.53 0.57 0.44 0.5 0.57 0.31 0.24 0.59

0.23 0.33 0.30 0.2 0.38 0.38 0.1 0.48

0.04 1.01 0.49 0.06 0.22 0.9 0.25 0.37

Relative intensity was obtained by normalization to the ␤-actin band present in the 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 cells undergoing de novo methylation can be found (e17.5 testis and adult ovary). e, embryonic day; RT-PCR, reverse transcriptase-polymerase chain reaction.

a

the PCR was working in each tube and to provide a rough estimate of levels of expression (Fig. 1A). PCR primers were designed to match the 3⬘-untranslated regions (UTRs) of the transcripts to pick up all the different splice variants of Dnmt3a, Dnmt3b, and Dnmt3l at once (as there are no splice variants in this region), while

minimizing the risk of cross-hybridization. The second primer set used in each multiplex reaction was for ␤-actin, which produces a transcript of 480 bp, allowing it to be distinguished from Dnmt3a (383 bp), Dnmt3b (451 bp), and Dnmt3l (315 bp). RT-PCR reactions with different cycle numbers were carried out to assess the optimal

number of cycles to use for determination of methyltransferase transcript levels (Fig. 1B). The RT-PCR reaction was not saturated and was well within the linear range at 30 cycles; therefore, subsequent RT-PCR reactions were carried out at this cycle number. Strong expression of all the methyltransferases was detected in the e8.5 positive control, and expression was low in lung, as expected. Dnmt3a and Dnmt3b transcripts were detected at uniform levels at all stages of germ cell development, with Dnmt3a being expressed at a slightly higher level throughout than Dnmt3b. Dnmt3l on the other hand showed a clear peak in transcription at e17.5 in male and postnatally in female. This finding was confirmed by comparing the relative intensity of the methyltransferase bands in each lane with that of the control ␤-actin band in the same lane using image densitometry analysis of the digital images, which provided a semiquantitative value for expression (Table 1). Whole-mount in situ on prenatal gonads also confirmed that Dnmt3l transcripts are present at high levels in e17.5 male


Fig. 2. Promoter usage at the Dnmt3a locus. A: Structure of the Dnmt3a locus (adapted from Chen et al., 2002). Dnmt3a2 was amplified using a primer (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 reverse primer R5 is in a common downstream exon. The positions of primers F1 and R1 in the 3⬘-untranslated region common to Dnmt3a and Dnmt3a2 and used for Figure 1 are also indicated. B: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of Dnmt3a and Dnmt3a2 transcription in purified germ cells from embryonic stages, whole gonads, and somatic tissues. Total RNA was extracted, and RT-PCR analysis was carried out as for Figure 1A. Transcription of Dnmt3a2 is low in somatic tissues except for embryonic day 8.5 embryo, where Dnmt3a was undetectable. Both transcripts 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.

gonads, but not in female at this stage (data not shown). Although we cannot rule out contributions by the small percentage of somatic cells present in embryonic germ cell preparations, these results suggested that the amount of DNMT3L protein, but not of DNMT3A or DNMT3B, could be controlled at the level of gene transcription. DNMT3L is not an active methyltransferase enzyme, and de novo methylation does not occur at all stages of germ cell development where we detect transcripts for DNMT3A and DNMT3B, so we looked to see what alternative mechanisms could

also be operating to regulate levels of the latter two proteins.

Promoter Usage at the Dnmt3a Locus It has been suggested that the DNMT3A2 isoform, which is produced from a downstream intronic promoter at the Dnmt3a locus (Fig. 2A), is more important for de novo methylation than DNMT3A (Chen et al., 2002). Dnmt3a and Dnmt3a2 transcripts were amplified in a multiplex reaction using one forward primer unique to Dnmt3a (F9) and one unique to

Dnmt3a2 (F5), together with a reverse primer (R5) common to both (Fig. 2A). Transcription of Dnmt3a2 was much higher than Dnmt3a in e8.5 embryos, where de novo methylation is occurring, but lower in adult somatic tissues, as expected (Fig. 2B). In the male germ line, there was little difference in transcription levels between the two isoforms, although Dnmt3a2 transcripts may be slightly more abundant at e15.5 (Fig. 2B). In the female germ line, expression of Dnmt3a2 was somewhat stronger than Dnmt3a at 12 days postpartum (dpp), when there is a postnatal wave


of oocyte growth (Pedersen, 1970) and de novo methylation (Lucifero et al., 2002; Obata and Kono, 2002). Dnmt3a transcripts were more abundant than Dnmt3a2 in adult ovary, probably due to the greater proportion of somatic cells present here (Fig. 2B) and the same is true to a lesser extent in adult testis. Multiplex reactions with ␤-actin and each isoform on its own gave similar results (data not shown), and our RT-PCR results are in good agreement with a recent study using realtime PCR with primers common to both Dnmt3a and Dnmt3a2 (La Salle et al., 2004).

Alternative Splicing of Dnmt3b The mouse Dnmt3b gene contains 23 exons: exons 5, 10, and 21–22 can be spliced out to give rise to several different transcripts (Fig. 3A). The two major functional isoforms of DNMT3B retain exons 21 and 22, which contain vital catalytic motifs but differ in the presence or absence of exon 10. The other enzymatically inactive isoforms all lack both exons 21 and 22. To examine at which developmental stages the functional isoforms were present in the germ line, we used two primer pairs (Fig. 3A), one pair spanning exon 10 (F6, R6) and another pair spanning exons 21 and 22 (F7, R7). Although previous reports suggest that exons 21 and 22 are spliced out together in mouse, we found that exon 21 can also be spliced out independently of exon 22 (Fig. 3B). RT-PCR over the exon 21–22 region was carried out with a second set of primers (F8, R8) and confirmed that the band representing new isoforms that lack exon 21 but retain exon 22 represented a genuine splice variant and not an artefact of PCR (Fig. 3B). Because exon 21 contains part of motif IX, isoforms lacking this exon are also unlikely to be enzymatically active. The major Dnmt3b isoform that can produce a functional protein that is expressed in the male germ line appears to be Dnmt3b2, which retains exons 21–22 but lacks exon 10 (Fig. 3C). Dnmt3b1 is absent at most stages, including those time points when male germ cells are undergoing methylation, but can be detected at 24 dpp when the germ cells start to enter

the pachytene stage of meiosis and in e8.5 embryos. Both Dnmt3b1 and Dnmt3b2 are present in the female germ line at the majority of time points examined, but Dnmt3b1 appears to be absent in the adult ovary, which would contain growing oocytes (Fig. 3D).

Subcellular Localization of DNMT3A and DNMT3B During Germ Cell Development Previous reports have indicated that DNMT3A was undetectable and DNMT3B is excluded from the nucleus in e12.5 germ cells, where extensive demethylation is occurring (Hajkova et al., 2002). We used immunocytochemistry on disaggregated embryonic gonads to examine whether the protein is found in the nuclei of male germ cells at e15.5 and e17.5. Gonads were removed from embryos and disaggregated in collagenase to obtain a single cell suspension. A small aliquot was placed on a polyL-lysine slide and stained for either DNMT3A or DNMT3B. The slides were counterstained with the DNA fluorochrome 4⬘,6-diamidine-2-phenylidole-dihydrochloride (DAPI), which allows germ cells and the oocyte meiotic stages to be distinguished on the basis of their nuclear morphology (McLaren and Southee, 1997; De Felici et al., 1999). At e15.5, some DNMT3A and DNMT3B protein could be weakly detected in prospermatogonia, and by e17.5, expression was strong for both proteins in nuclei and cytoplasm of most germ cells (Fig. 4A). In the female germ line, which is not undergoing extensive methylation at this time, DNMT3A was absent in both e15.5 and e17.5 primary oocytes, whereas DNMT3B was detected only in the subset of primary oocytes at e17.5, which were in the zygotene/ pachytene stage (Fig. 4A). We also wished to determine DNMT3A and DNMT3B protein expression and localization at the time imprints were being set in the female germ line. We examined cryosections of ovaries at 12 dpp, in which some oocytes have initiated growth, and at 1 dpp where there are no growing oocytes present. Neither protein was detected at 1 dpp in nongrowing oocytes

(Fig. 4B, bottom panel, and data not shown), but both DNMT3A (Fig. 4B, top panel) and DNMT3B (Fig. 4B, middle panel) were detected in the nuclei and cytoplasm of early stage growing oocytes at 12 dpp. At later stages of oocyte development, the staining became predominantly cytoplasmic for both proteins (Fig. 4B). Secondary antibody alone gave no staining in all cases. Investigation of protein localization for DNMT3L and DNMT3A2 were prevented by a lack of reliable antibodies for these proteins.

DISCUSSION De novo DNA methylation occurs during very limited periods of germ cell development in the mouse, suggesting that there must be mechanisms in place to limit expression of the functional methyltransferases DNMT3A and DNMT3B and the methyltransferase regulator DNMT3L to these time points. In this study, we found that each of the three genes uses a different strategy to confine expression to specific developmental periods. Dnmt3l appears to undergo regulation at the transcriptional level. Transcription of the gene is very low or absent in e12.5 gonads when demethylation of repetitive elements and imprinted genes is occurring. In the male germ line, expression is high at e17.5, when extensive methylation is happening, but remains low in the female at this time point, when most sequences remain demethylated. Dnmt3l expression is high, however, in the adult ovary, where growing oocytes are present. These results indicate that production of the wild-type transcript matches that of a lacZ marker knocked into the Dnmt3l locus and driven by the same promoter: LacZ staining was found to be present in prospermatogonia and growing oocytes in these earlier studies (Bourc⬘his et al., 2001; Hata et al., 2002). A different control mechanism, namely alternative promoter usage, is seen at the Dnmt3a locus. A second transcriptional start within a downstream intron of the gene is used to produce the shorter DNMT3A2 protein, which although it does not differ in amino acid sequence from the longer 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, and 22 show alternative splicing. The full-length Dnmt3b1 transcript contains all the exons, whereas Dnmt3b2 has exon 10 spliced out. The transcripts Dnmt3b3-Dnmt3b6 lack exons 21 and 22 and so cannot produce a catalytically active protein. B: A mouse Dnmt3b splice variant was identified that contains exons 10 and 22 but lacks exon 21. This variant, which has been reported previously in humans but not mice, is unlikely to be catalytically active as it lacks part of motif IX. C,D: Reverse transcriptase-polymerase chain reaction analysis of the male and female germ lines, respectively. The corresponding catalytically active isoforms that could be expressed at each time point are indicated at the bottom of each lane. The exon 10 primers were 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: Disaggregated cells from embryonic day (e) 17.5 embryonal gonads. Red staining indicates presence of DNMT3A or DNMT3B as indicated, and blue 4⬘,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining indicates nuclei. Most prospermatogonia stain weakly for DNMT3A and DNMT3B in the nucleus 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 for DNMT3B 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 is found only in zygotene/pachytene cells and disappears from cells that have fully progressed into pachytene. B: Postnatal ovaries. Cryosections from 12 days postpartum (dpp; top and middle panel) and 1 dpp (bottom panel) mice were stained for DNMT3A and DNMT3B, nuclei were counterstained with 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 detected Dnmt3a transcripts at low levels in most somatic tissues, in agreement with previous data (Okano et al., 1998a), but Dnmt3a2 appears to be transcribed at significant levels only in the germ line and in the e8.5 postimplantation embryo, where it is the dominant transcript. In the male germ line, both messages are present in late embryonic and early postnatal stages, when methylation of repeat sequences and paternally methylated imprinted genes is occurring (Davis et al., 2000; Lees-Murdock et al., 2003; Li et al., 2004), and we detect DNMT3A protein in prospermatogonia with the antibody used in this study, which has been previously shown not to cross-react with DNMT3A2 (Chen et al., 2002). Transcripts from both genes can also be detected in the female germ line at 12 dpp during the first and largest wave of oocyte growth, as well as in adult ovary, which would also contain growing oocytes. Although Dnmt3a2 transcripts seem to predominate at 12 dpp, the longer Dnmt3a transcript is also detected at this stage and the cognate protein can be seen at high levels in the growing oocyte. These results would be consistent with a role for either DNMT3A or DNMT3A2 in de novo methylation in germ cells. The recent tissue-specific knockout in this lineage would have affected both proteins (Kaneda et al., 2004), so a definitive experiment to address which of the two isoforms is more important will require promoter-specific deletions. One clue may be that there are a large number of retrotransposed Dnmt3a2 pseudogenes present in mouse, rat, and human, but no Dnmt3a pseudogenes, which would support the idea that Dnmt3a2 is transcribed more frequently in the germ line and has been over evolutionary time (Lees-Murdock et al., 2004). Dnmt3b has its own unique regulatory mechanism, namely alternative splicing. We found that the full-length DNMT3B1 protein is unlikely to be involved in de novo methylation in the male germ line, as the corresponding transcript is not expressed during the period when imprints are being reset or repeat elements become methylated in this germ line. The main Dnmt3b transcript present in the

male germ line that can produce a catalytically active protein is, therefore, Dnmt3b2, which lacks exon 10, and the corresponding protein isoform is presumably the one that carries out de novo methylation. Although Dnmt3b1 transcripts are present during more stages of female germ line development, they appear to be absent from adult ovaries, where the only functional isoform is Dnmt3b2. Taken together, these data suggest that the DNMT3B2 protein is more important in both germ lines for de novo methylation. This conclusion is consistent with the findings of Chen et al. (2003), who demonstrated that introduction of Dnmt3b1 cDNA was unable to cause methylation of the imprinted genes H19 and Igf2 in doubly mutant Dnmt3a⫺/⫺ Dnmt3b⫺/⫺ ES cells. In this study, we also detected mouse Dnmt3b transcripts lacking exon 21 but retaining exon 22, which have been described previously in humans (Xu et al., 1999). Transcripts lacking exon 21 are unlikely to produce proteins with any catalytic activity, as they will be missing part of motif IX. A final regulatory mechanism that is common to DNMT3A and DNMT3B is protein localization. While Dnmt3a and Dnmt3b transcripts are present throughout germ cell development, the corresponding proteins are not. Previous studies have found that DNMT3A protein is undetectable in e12.5 germ cells, while DNMT3B is confined to the cytoplasm (Hajkova et al., 2002). We report here that DNMT3A and DNMT3B can be detected at low levels in the nuclei of e15.5 male prospermatogonia, and by e17.5, staining is strong for both proteins in these cells. We and others have shown that paternally methylated imprinted genes lose their methylation imprint at e12.5, but methylation levels have started to climb again by e15.5, with full methylation not being seen until after birth (Davis et al., 1999; Li et al., 2004). Repetitive sequence methylation, however, is still low at e15.5, but by e17.5, no demethylated sequences can be detected (Lees-Murdock et al., 2003). The presence of de novo methyltransferase protein in the nuclei of prospermatogonia at e15.5 when the repeat elements are still unmethylated suggests that an additional regulatory mechanism is

required to trigger methylation of these elements; this conclusion is also suggested from recent studies done on Dnmt3l and Dnmt3a mutant mice showing that methylation of selfish repeats and imprinted genes can be uncoupled (Bourc⬘his et al., 2001; Kaneda et al., 2004; Bourc⬘his and Bestor, 2004). In the female germ line, both DNMT3A and DNMT3B can be detected in the nuclei of growing oocytes at 12 dpp, when de novo methylation of imprinted genes is occurring (Lucifero et al., 2002). DNMT3A was undetectable in germ cells that are not undergoing active methylation (e15.5, e17.5, and 1 dpp female), and its down-regulation may contribute to the genome-wide demethylation observed at e17.5 in the female germ cells (Lucifero et al., 2002; Lees-Murdock et al., 2003; Li et al., 2004). The absence of protein, despite the presence of some transcripts in these cells, suggests that these are not translated. DNMT3B is also absent in e15.5 and 1 dpp female germ cells but is present in the cytoplasm and nuclei of e17.5 oocytes at the zygotene/pachytene stage. This finding could explain why minor satellites retain a higher degree of methylation than other repeats between e12.5 and e17.5 in the mouse primary oocytes (Lees-Murdock et al., 2003), which may be important to ensure stability of chromosomes during crossing-over in pachytene. Of interest, the only time when the main Dnmt3b1 transcript is produced in the male germ line is at 24 dpp, when spermatocytes are also entering pachytene. This particular isoform has been shown to have preference for methylation of minor satellite repeats over major satellites or endogenous Ctype retroviral DNA (Chen et al., 2003). Dnmt3b1, therefore, may be involved in chromosome stability in both germ lines, even though it is not required for methylation of imprinted genes in the male. As the growing oocytes mature, we found that DNMT3A and DNMT3B expression became more cytoplasmic, as was shown to be the case for DNMT1 (Mertineit et al., 1998). We have shown previously that, on progression of fully grown germinal vesicle stage oocytes to metaphase II oocytes, DNMT3A and DNMT3B become localized to a subcortical layer in a manner similar to


DNMT1 (Lees-Murdock et al., 2004). It appears that these proteins are all synthesized and stored in the cytoplasm and move to the nucleus to carry out their enzymatic function, moving back out when they are no longer required. In conclusion, it is notable that the only time points at which Dnmt3l and Dnmt3a2 transcripts are present and where DNMT3A and DNMT3B can be detected in the nucleus are at the two stages where the male and female germ cells are undergoing de novo methylation, e15.5 to e17.5 in the male and after birth in the growing oocytes in the female. This finding suggests that, although differing control mechanisms operate at each locus, the combined effect is to restrict de novo methylation to particular limited periods during germ cell development. Our results indicate that DNMT3L, DNMT3A2, and DNMT3B2 may be more important for de novo methylation of imprinted genes and most repeats in the male and female germ line, whereas DNMT3B1 may be more important for methylation of satellite DNA sequences.

EXPERIMENTAL PROCEDURES Mice HsdOla:To mice were purchased for Harlan UK, Ltd. (Oxon, England). Natural matings were used to produce embryos and neonatal mice for the gonad dissection and germ cell isolation. The day the plug was observed was taken as e0.5, and the day of birth was taken as 0 dpp.

Purification of PGCs, Prospermatogonia, and Primary Oocytes Embryos were collected at e12.5, e15.5, and e17.5 and sexed by morphology (Hogan et al., 1986). Germ cells were purified without culture as described previously (Lees-Murdock et al., 2003). Briefly, embryonic gonads with mesonephros were incubated in 0.54 mM ethylenediaminetetraacetic acid, 136 mM NaCl, 2.68 mM KCl, 9.58 mM Na2HPO4, 1.47 mM KH2PO4 for 20 min at room temperature with rocking. Gonads were rinsed in M2 medium and

Total RNA was extracted by using an RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. RNA was resuspended in nuclease-free water (Promega) and stored at ⫺80°C for use in RT-PCR.

CGT GTA GTG AGC. PCR was carried out in a 25-␮l volume containing 5 ␮l of cDNA, 1⫻ Taq buffer, 1.5 mM deoxynucleoside triphosphates (determined to be the optimal amount in pilot experiments), 0.5 pmol of each primer and 2 U Taq (Sigma). Samples were initially deannealed at a temperature of 94°C for 3 min, followed by 30 cycles of 94°C for 30 sec, 65°C for 1 min, and 68°C for 1 min, followed by a final elongation step of 7 min at 72°C. PCR products were then separated on 3% agarose gels, and images were captured by using a Kodak digital camera and saved as TIFF files for subsequent image analysis.

Semiquantitative RT-PCR

Image Densitometry Analysis

First-strand cDNA was synthesized in a 50-␮l reaction mixture containing 10 mM Tris HCl (pH 8.3), 0.2 ␮g of oligo (dT)15 primer (Promega), 1.5 mM deoxynucleoside triphosphates (Sigma), 1⫻ AMV-RT buffer, 1␮g of RNA, and 20 U AMV reverse transcriptase (Promega). Primers from the 3⬘-UTR region were initially used to simultaneously amplify all transcripts of Dnmt3a (F1, CGC CTG GCC CTC TGT GCA AAG; R1, GGT CAC TTT CCC TCA CTC TGG), Dnmt3b (F2, GGA AGG GTG GGT GGA GTG GC; R2, GCC CAC ACC TTG CGA GTT AC), and Dnmt3L (F3, CTG AAG AGC AAG CAT GCG CC; R3, GCT GCA CAG AGG CAT CCT GG). ␤-actin primers (Obata and Kono, 2002) were included as an internal control (F4, GCT GTG CTA TGT TGC TCT AGA CTT C; R4,CTC AGT AAC AGT CCG CCT AGA AGC). For further investigation of individual isoforms, Dnmt3a and Dnmt3a2 were was amplified by using specific forward primers (F9, CAG CGA CCC ATG CCA AGA CTC; F5, AGG GGC TGC ACC TGG CCTT, respectively) and a common reverse primer (R5, TCC CCC ACA CCA GCT CTC C). F5 and R5 have been described elsewhere (Chen et al., 2002). Primers that span exon 10 and exons 21–22 of Dnmt3b were as follows: exon 10: F6, TGG GAT CGA GGG CCT CAA AC; and R6, TTC CAC AGG ACA AAC AGC GG and exon 21–22: F7, CGA TGC CAT CAA GGT GTC; and R7, CAC TGG CTC TGA CCG AGA GC; or F8, TGC GCG ACA ACC GTC CAT TCT TC; and R8, GGA CAC GTC

Image densitometry analysis was performed on TIFF images using Phoretix 2D software (Nonlinear Dynamics). The ␤-actin band in each lane was assigned an arbitrary value of 1.0 and used to calculate the relative intensity of the methyltransferase band in each lane.

stabbed 25 times with two 25-gauge needles in a microdroplet of M2 medium. Germ cells were collected in the microdroplet, washed, and resuspended in Ca2⫹-Mg2⫹-free phosphate buffered saline (PBS). Purity was uniformly greater than 85% using this method.

RNA Extraction From Mouse Tissues

Disaggregation of Gonads and Immunocytochemistry on Prospermatogonia and Primary Oocytes Whole gonads from e17.5 embryos with the mesonephros removed were incubated in 1 mg/ml collagenase at 37°C for 15 min. Trituration with a yellow tip was carried out to ensure that a single cell suspension was obtained. Cell suspensions were washed in M2 medium before being resuspended in M2 supplemented with 1 mg/ml bovine serum albumin (BSA). A small aliquot was placed on a polylysine slide (approximately 10 ␮l), and cells were allowed to attach for 10 min before addition of 50 ␮l of ice-cold 4% paraformaldehyde. After 5 min, most of the liquid was removed and replaced with a fresh aliquot of 4% paraformaldehyde and incubated at room temperature for 5 min. Slides were subsequently washed three times in M2 supplemented with 1 mg/ml BSA and blocked in this solution for 2–3 hr before permeabilization by incubation with antibody buffer (1% Triton X-100, 10 mg/ml BSA, 0.2% sodium dodecyl sulfate) for 30 min at room temperature. Primary antibody for


DNMT3A or DNMT3B (kind gifts of Dr. En Li) were diluted 1:400 and incubated overnight with samples. After three 5-min washes in antibody buffer, slides were incubated with a 1:400 dilution of donkey anti-rabbit secondary conjugated with rhodamine (Santa Cruz Biotechnology) for 1 hr at room temperature. Slides were washed three times in antibody buffer and incubated with 125 ng/␮l DAPI for 10 min at room temperature. After three PBS washes, slides were mounted with Vectashield (Vector Laboratories), sealed, and viewed on a Nikon Eclipse 600 microscope. Images were photographed on slide film and scanned and edited in Photoshop 7.0 (Adobe).

Collection of Ovaries for Cryosectioning and Immunohistochemistry Ovaries were collected from neonatal mice at 1 dpp and 12 dpp and placed into ice-cold 4% paraformaldehyde. Tissues were incubated at 4°C overnight with rocking and rinsed twice in PBS followed by two 5-min washes in PBS, then incubation overnight in 30% sucrose at 4°C with rocking. Subsequently, ovaries were placed in molds with OCT compound, incubated at 4°C for 1 hr and then placed in dry ice until frozen. Samples were stored at ⫺80°C until sectioning. Sections were 10 ␮m thick and mounted onto SuperFrost Plus slides and stored at ⫺80°C until use. For immunohistochemistry on ovary cryosections, sections were fixed in 4% ice-cold paraformaldehyde for 15 min and washed three times in PBS before blocking in M2 containing 4% goat serum instead of BSA for 2–3 hr at room temperature. Permeabilization was carried out by incubation with antibody buffer as described above, except 4% goat serum was included instead of BSA. Primary and secondary antibodies (goat anti-rabbit Alexa 488, Molecular Probes) were diluted 1:400 and incubated as above. After secondary antibody incubation, slides were washed and incubated with 100 ␮g/ml RNase ONE (Promega) for 10 min and subsequently stained with 25 ␮g/ml propidium iodide for 5 min at room temperature. Slides were washed three times with PBS before mounting in

Vectashield and viewing on a Bio-Rad Olympus confocal microscope. Images were processed in Lasersharp/Confocal Assistant.

ACKNOWLEDGMENTS We thank Dr. En Li for the kind gift of the DNMT3A and DNMT3B antibodies used in this study;J. McDaid, C. Lynch, L. Hipiri, and H. Wheadon for helpful discussions; and R. Black for technical support. C.P.W. was funded by grants from the BBSRC, the Northern Ireland HPSS R&D office, and the Royal Society.

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