Hum. Reprod. Advance Access originally published online on October 23, 2006
Human Reproduction 2007 22(2):457-467; doi:10.1093/humrep/del379
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Sex-specific promoters regulate Dnmt3L expression in mouse germ cells
1 Stem Cells and Epigenetics Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster, Coleraine, UK 2 Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, New York, NY, USA and 3 Montreal Children’s Hospital Research Institute and Departments of Pediatrics, Human Genetics, Pharmacology & Therapeutics, McGill University, Montreal, QC, Canada
4 Present address: The Wellcome Trust/Cancer Research UK Gurdon Institute, The Henry Wellcome Building of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK
5 Present address: INSERM U741, Institut Jacques Monod, 2 Place Jussieu, 75251 Paris, CEDEX 05, France
6 To whom correspondence should be addressed at: Stem Cells and Epigenetics Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, UK. E-mail: cp.walsh{at}ulster.ac.uk
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Abstract |
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BACKGROUND: Dnmt3L, a member of the DNA methyltransferase 3 family, lacks enzymatic activity but is required for de-novo methylation of imprinted genes in oocytes and for transposon repression in male germ cells. METHODS: We used northern blots, RT–PCR, 5' rapid amplification of complementary DNA (cDNA) ends (RACE), RNase H mapping, real-time/quantitative RT–PCR and in situ hybridization to identify and characterize Dnmt3L transcripts produced during germ cell development. RESULTS: Mouse Dnmt3L uses three sex-specific promoters, not the single promoter previously thought. A promoter active in prospermatogonia drives transcription of an mRNA encoding the full-length protein in perinatal testis, where de-novo methylation occurs. Late pachytene spermatocytes activate a second promoter in intron 9 of the Dnmt3L gene. After this stage, the predominant transcripts are three truncated mRNAs, which appear to be non-coding. We could also detect similar adult testis transcripts in humans. In the mouse ovary, an oocyte-specific promoter located in an intron of the neighbouring autoimmune regulator (Aire) gene produces a transcript with the full open reading frame (ORF). This is the only Dnmt3L transcript found in growing oocytes and is absent in the oocytes of Dnmt3L–/– females. CONCLUSIONS: Sex-specific promoters control Dnmt3L expression in the mouse germ line, mirroring the situation at the Dnmt1 and Dnmt3A loci.
Key words: Dnmt3L/imprinting/oocyte/promoter/testis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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DNA methylation is a heritable epigenetic modification which plays an important role in transcriptional repression of imprinted genes (Li et al., 1993
; Bourc’his et al., 2001; Kaneda et al., 2004), transposons (Yoder et al., 1997; Walsh et al., 1998; Bourc’his and Bestor, 2004; Webster et al., 2005) and genes on the inactive X chromosome (Panning and Jaenisch, 1996). There is also evidence to suggest that methylation is important for maintaining the stability of pericentromeric satellite repeats, as mutations in the DNMT3B methyltransferase are found in patients suffering from immunodeficiency-centromeric instability-facial anomaly (ICF) syndrome (OMIM 242860
[OMIM]
) and are accompanied by demethylation of classical satellite repeats and the formation of branched and multiradiate chromosomes (Hansen et al., 1999; Okano et al., 1999; Xu et al., 1999). Aberrant DNA methylation is often seen in cloned embryos (Dean et al., 2001; Humpherys et al., 2001), tumours (Jones and Laird, 1999) and disease syndromes involving imprinted genes (Reik and Walter, 2001b).
Methylation undergoes reprogramming in mouse germ cells, which is essential to allow removal of methylation inherited with the parental alleles and establishment of new patterns of modification (Yoder et al., 1997; Reik et al., 2001). Most imprinted genes carry a methylation mark on the maternal allele at the differentially methylated region (DMR), but a small number inherit a mark on the paternal allele (Reik and Walter, 2001a; Bestor and Bourc’his, 2006). Loss of methylation occurs shortly after the germ cells colonize the embryonic gonad (Davis et al., 2000; Hajkova et al., 2002). Establishment of new imprints by de-novo methylation of the DMR occurs in the prospermatogonia in the male germ line and during the oocyte growth phase in the ovary (Davis et al., 2000; Ueda et al., 2000; Lucifero et al., 2004; Li et al., 2004). Methylation of intracisternal A particles (IAP), LINE1 elements (L1) and minor satellite repeats also decreases in post-migratory germ cells but is not wholly removed (Hajkova et al., 2002; Lees-Murdock et al., 2003), which is important for maintaining transcriptional repression of transposons (Bourc’his and Bestor, 2004; Webster et al., 2005) and probably for the stability of the minor satellite DNA; remethylation of any demethylated elements also occurs in the prospermatogonia in males and the growing oocyte in females (Lees-Murdock et al., 2003; Lucifero et al., 2004).
Three enzymatically active DNA methyltransferases have been characterized in mice (Goll and Bestor, 2005). Dnmt1 is thought to maintain methylation by converting newly replicated hemimethylated DNA strands to fully methylated DNA. In germ cells, Dnmt1 produces an oocyte-specific isoform important for maintaining methylation on both paternally and maternally imprinted genes in the early embryo (Howell et al., 2001), whereas in the male germ line it is down-regulated at the pachytene stage of meiosis by a switch in promoter usage which results in the production of an untranslated mRNA (Mertineit et al., 1998). Dnmt3a and Dnmt3b are more important for de-novo methylation (Okano et al., 1999) and both proteins are localized to the germ cell nuclei during periods of de-novo methylation (Lees-Murdock et al., 2005). Deletion of Dnmt3a specifically in the male germ line results in a failure in spermatogenesis, accompanied by a loss of methylation on two of the three paternally methylated imprinted genes examined (Kaneda et al., 2004). In the female germ line, deletion of Dnmt3a prevents methylation of imprinted genes in the oocyte.
Mice with germ line-specific mutations in Dnmt3a show some similarity to mice carrying a deletion in the Dnmt3L gene (Bourc’his et al., 2001; Hata et al., 2002; Bourc’his and Bestor, 2004). Dnmt3L has homology to the other members of the Dnmt3 family but is missing crucial residues in the conserved catalytic motifs and has not been shown to have enzymatic activity in vitro (Aapola et al., 2001; Bourc’his et al., 2001; Hata et al., 2002). Offspring of Dnmt3L–/– females show loss of methylation of imprinted genes in the oocyte and subsequent loss of imprinting, but methylation on other sequences tested was normal (Hata et al., 2002; Bourc’his and Bestor, 2004).
Mutant males lack viable sperm because of failure of spermatogenesis. Methylation of L1 and IAP elements was also decreased, and very high levels of transcription from these elements were seen. Partial demethylation of some imprinted genes was seen, although this was not accompanied by loss of imprinting; methylation of pericentromeric satellites was unchanged (Bourc’his and Bestor, 2004; Webster et al., 2005). Spermatogenetic failure in these mice appears to be the result of arrest in the pachytene stage of meiosis and is accompanied by abnormal chromosome synapsis.
The similarity in phenotypes of the Dnmt3L–/– and Dnmt3a–/– mice, together with the lack of conservation of catalytic motifs in Dnmt3L, suggests that the latter may act as a cofactor for Dnmt3a. Dnmt3L has also been shown to enhance methylation by Dnmt3a in in vitro assays (Chedin et al., 2002; Suetake et al., 2004).
Previous work had indicated that the Dnmt3L locus has only one promoter driving transcription of a single long transcript (Aapola et al., 2001, 2004a,b; Bourc’his et al., 2001; Hata et al., 2002). We show here that mouse Dnmt3L produces sex-specific transcripts from three different promoters in germ cells. One transcript that encodes the full-length protein is the predominant form in prospermatogonia but not in spermatocytes: instead, an internal promoter is activated in late pachytene spermatocytes to produce three short transcripts that lack significant open reading frame (ORFs). A third promoter located in an intron of the upstream autoimmune regulator (Aire) gene is activated in growing oocytes and produces a transcript that is capable of coding for a full-length Dnmt3L protein. These results indicate an important role for promoter switching in regulating the production of active Dnmt3L protein in mouse germ cells, in agreement with a requirement for precise programming of methylation events in this lineage.
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Materials and methods |
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Mice
C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) (for the laboratories of T.H.B. and J.M.T.), and Swiss To mice were purchased from Harlan UK Ltd (Oxon, England) (C.P.W. laboratory) and bred in-house. Dnmt3L–/– mice have been previously described (Bourc’his et al., 2001
). Natural matings were used to produce embryos: the day of the vaginal plug was taken as 0.5 days post coitum (dpc) (Hogan et al., 1995). Animal work was carried out according to Institutional regulations and with ethical approval from the relevant local licensing authority.
Isolation of RNA
All tissues used were dissected in phosphate-buffered saline (PBS) according to standard methods (Hogan et al., 1995). RNA was extracted following homogenization of tissue in a Dounce homogenizer and/or syringe using Trizol reagent (Invitrogen) as per the manufacturer’s instructions. The RNA was dissolved in diethylpyrocarbonate-treated water and final concentrations were measured using a spectrophotometer. Human adult testis RNA was purchased from BD Biosciences.
Northern blot analysis
Total RNA was electrophoresed on a 1.2% formaldehyde gel and transferred to Nytran Plus nylon membranes (Schleicher and Schuell). Blots were hybridized using the conditions of Church and Gilbert (1984). Probes specific to the various transcripts were generated using PCR (see Table I for primers). Blots were analysed after exposure to Kodak film.
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5 ' and 3' rapid amplification of complementary DNA ends PCR
5' and 3' rapid amplification of complementary DNA (cDNA) ends (RACE) was performed on total RNA using the FirstChoiceTM RLM-RACE Kit (Ambion), exactly as according to the manufacturer’s protocol, using primers specific for Dnmt3L (see Table I). PCR products were cloned into the TOPO-TA vector (Invitrogen) and sequenced in-house. Novel transcripts have been deposited with Genbank (Round spermatid forms 1–3 as EF051621 [GenBank] -EF051623 [GenBank] respectively; oocyte form as EF051624 [GenBank] ) (accession numbers pending).
PCR, cDNA synthesis and RT–PCR
Primers are as listed in Table I, except for the mouse 
-actin primers, which were as previously described (Obata and Kono, 2002). Synthesis of cDNA from the RNA was carried out using standard procedures (Sambrook et al., 2001). In brief, 5 µg of total RNA was added to a 50 µl reaction containing 10 mM Tris–HCl (pH 8.3), 0.2 µg Oligo(dT)15 primer (Promega), 1.5 mM deoxynucleoside triphosates, 1x Moloney murine leukemia virus (MMLV)-RT buffer and 20 U MMLV-RT (Ambion). PCR was performed in 50 µl containing 2 µl cDNA or 100 ng DNA, 1x Taq buffer, 1.5 mM deoxynucleoside triphosphates, 1.5 mM MgCl2, 0.4 µM each primer and 1.25 U Taq polymerase. The mixture was left for 3 min at 94°C for initial denaturation, followed by 30 (-actin) or 35 (Dnmt3L) cycles consisting of 30 s at 94°C, 1 min at 63°C and 1 min at 72°C, followed by a final extension of 7 min at 72°C. Human cDNA was synthesized from testis total RNA (BD Biosciences) using the Superscript II RT method (Invitrogen). Briefly, 4 µg of total RNA was added to a 20 µl reaction consisting of 0.2 µg Oligo(dT)12–18 primer, 0.5 mM deoxynucleoside triphosphates, 40 U RnaseOUT, 200 U Superscript II RT, 4 µl 5x First-strand buffer and 0.1 M dithiothreitol (DTT) and the reaction carried out according to the manufacturer’s guidelines.
RNase H mapping
Oligonucleotides used for the RNase H mapping are listed in Table I (exon 10R and small exon 9cR). The oligonucleotides (250 pmol) were annealed to 20 µg of total RNA then incubated with RNase H, which cleaves RNA in the primer:mRNA duplex. The cleaved products were fractionated on an agarose/formaldehyde gel, transferred to a nylon membrane and probed to detect the fragments corresponding to the 5' ends of transcripts. RNase H treatment, electrophoresis, blotting and probing were exactly as previously described (Yoder et al., 1996).
In situ hybridization
Probes for in situ hybridization were generated using PCR, cloned into the TOPO-TA cloning vector (Invitrogen) and sequenced to confirm their identity. Primers for Dnmt3L are given in Table I and primers for Mvh were as described (Toyooka et al., 2000). RNA probes were transcribed in vitro using the DIG RNA labelling kit (Roche). Cryosections were prepared and in situ hybridization carried out as described (Laufer et al., 1997) using anti-DIG antibody from Roche for detection.
Real-time/quantitative RT–PCR (QRT–PCR)
Germ cells were collected on two separate occasions and fractions enriched in specific cell populations isolated as previously described (Trasler et al., 1992). RNA was isolated from these using the RNeasy extraction kit with DNaseI treatment and further concentrated using the RNA MinElute kit (both Qiagen Inc.). Samples were diluted to 10 ng/µl, aliquoted in single-use volumes and stored at –80°C. QRT–PCR was carried out using the Quantitech SYBR Green RT–PCR kit (Qiagen) on an Mx4000 QPCR system (Stratagene) as previously described (La Salle et al., 2004). In brief, 10 ng or 100 pg of total RNA were used for the transcripts and 18S, respectively, and one-step QRT–PCR reactions were performed in a 25 µl volume for 40 cycles. The standard curve for each transcript was generated using total RNA from 70 days post-partum (dpp) testes. In all cases, reactions were performed in triplicate on the same sample of germ cell RNA. Specificity was assessed with the melting curve analysis and confirmed on a 3% agarose gel after each QRT–PCR experiment. Results were normalized to their corresponding 18S rRNA content and calibrated accordingly to the lowest consistently expressing time point (residual bodies). Analysis of expression in the two batches of enriched germ cells gave almost identical results. Primer sequences are indicated in Table II except for 18S, which were as previously described (La Salle et al., 2004).
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Alternative transcripts of Dnmt3L are found in the post-natal testis
Alternative transcripts of the Dnmt3L gene were identified in the adult testis by carrying out 5' and 3' RACE with primers to all the known exons of the gene. This approach recovered several clones corresponding to shorter transcripts from the Dnmt3L locus specifically in adult mouse testis. Using the BLAT alignment tool (http://genome.ucsc.edu/cgi-bin/hgBlat) to compare the sequences to the mouse genome, we found that these clones all originated from a novel exon located between exons 9 and 10, which we have designated exon 9b (Figure 1A). Alternative splicing of transcripts arising from this promoter gives rise to three transcripts, which we have designated forms 1, 2 and 3. Form 1 contains exon 9b, together with a second novel exon, designated exon 9c. Form 2 also contains exons 9b and 9c, but use of a second splice acceptor site in 9c results in a shorter version of this exon (9cS) being included in the final transcript. Form 3 only contains exon 9b.
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To confirm the presence of these transcripts in the adult testis, we carried out northern blotting using total RNA and probes specific for the different transcripts of the gene (Figure 1B). A probe containing exons 3–12 detected a transcript of
1.7 kb in embryonic stem (ES) cells and 17.5 dpc testis corresponding to the full-length transcript. In the adult testis, the 1.7 kb transcript was absent but two smaller, weaker bands were detected (more clearly visible when more input RNA was used) at 1.45 and 1.3 kb. A probe corresponding to exon 9b detected both of these bands, but not the 1.7 kb transcript, whereas a probe for the long form of exon 9c (9cL) detected the larger of these two bands alone, indicating that the top band (1.45 kb) in the adult testis lane corresponds to form 1, whereas the bottom band (1.3 kb) corresponds to form 2. Form 3 was not detected using this assay, suggesting that this transcript is more weakly expressed or less stable. RT–PCR using primers specific for exons 1 and 9a confirmed that this region was present in transcripts produced by ES cells and 17.5 dpc testis but not detectable in adult testis (Figure 1C). Using primers in exons 9b and 13, three transcripts corresponding to adult testis forms 1–3 were detected in the adult but not in ES cells or 17.5 dpc testis, with form 3 consistently giving a weaker signal than forms 1 and 2. These products were isolated from agarose gels, cloned and sequenced and confirmed that adult testis transcripts 1–3 contain exons 10–13 as well as the alternative 5' exons detailed above. The negative controls for each primer set, done in the absence of RT, are shown in Figure 1: these were uniformly negative in all experiments and are omitted from subsequent figures.
RNase H mapping (Figure 1D) with RNA from adult testis confirmed that exon 9b represents the true 5' end of the adult transcripts and that no further 5' exons exist in this tissue. Primers used for protection were in the 3' region of exon 9c and in exon 10 (see Figure 1A), and exon 9b was used as a probe to detect the fragments corresponding to the 5' ends of the 9b-containing transcripts. Two major products were detected, corresponding to the sizes of the transcripts containing either exon 9b and the long form of exon 9c (form 1) or 9b and the short form of 9c (form 2) as determined by 5' RACE and no larger bands were detected, indicating that no further upstream exons exist. No band clearly matching form 3 (exon 9b alone) could be detected in this assay.
Cell-type specificity of transcripts in the testis
Our results above indicated that a switch to the 9b-containing transcripts occurs during testis maturation. We isolated total RNA from testis at different times during development and carried out multiplex RT–PCR using forward primers in exons 1 and 9b and a common reverse primer in exon 13. Figure 2A shows that the 9b-containing transcripts were detected from 25 dpp onwards. The transcript containing exons 1–13 appeared to be strongly expressed in ES and 17.5 dpc testis but was very weak compared with the 9b transcripts in later stages. Germ cells in the testis start to enter meiosis at around 12–14 dpp, suggesting that the appearance of the short adult transcripts of Dnmt3L may coincide with the entry of the germ cells into a particular meiotic stage.
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As antibodies to Dnmt3L were not available, we carried out RNA in situ hybridization using probes specific for exons 1–9a or for exon 9b on cryosections taken from mouse testis at the 17.5 dpc and adult stages to determine which cell types express the various transcripts. As a positive control and to identify the location of the germ cells, we used a probe for mouse Vasa homologue (Mvh), which labels all of the germ cells at 17.5 dpc, but predominantly the spermatogonia and spermatocytes in the adult stages (Figure 2B) (Toyooka et al., 2000
). The probe corresponding to Dnmt3L exons 1–9a showed strong staining in the prospermatogonia at 17.5 dpc and some weak staining in the spermatogonia in the adult. In contrast, the exon 9b probe showed no staining at 17.5 dpc but strongly stained a population of germ cells in the intermediate layer of the testis corresponding to stages from late pachytene to round spermatid in germ cell development. To clarify which cell types expressed the shorter forms in the adult, we fractionated germ cells using unit gravity sedimentation and carried out QRT–PCR using primers specific for each transcript in enriched germ cell populations (Figure 2C). The 9b-containing transcripts were absent in the spermatogonia but were present at high levels in the pachytene and round spermatid fractions. All three of the adult transcripts showed a similar distribution. It is not possible to compare levels of transcription of one message with the other in this experiment; the expression profiles were, however, in good agreement with the data presented in Figure 2A and B.
An ovary-specific transcript indicates the presence of a third promoter
The presence of testis-specific transcripts of Dnmt3L led us to examine the ovary for evidence of specific transcripts in this tissue as well. A Dnmt3L expressed sequence tag (EST) (CA599020
[GenBank]
) derived from ovary contained two novel 5' exons (Figure 3A) spliced directly to exon 2. The sequence of 5' RACE products generated from adult mouse ovary RNA using primers in exon 2 matched this EST with slightly more 5' sequence. We confirmed that no further 5' sequence was present using RNase H (data not shown). These novel 5' exons were designated exons 1b and 1c, with the first exon in the prospermatogonia transcripts being exon 1a. Using the BLAT alignment tool, we mapped exon 1b mapped between exons 3 and 4 of the Aire gene, which is proximal to the Dnmt3L locus and transcribed in the opposite direction (Figure 3A).
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To assess whether the transcript containing these novel 5' sequences is only found in the ovary, we carried out RT–PCR using forward primers in exons 1b and 1c and a reverse primer in exon 4 and found that the messenger RNA (mRNA) could be detected in ovary but not in ES cells or testis (Figure 3B). Importantly, we could not detect exon 1a-containing transcripts in ovary (Figure 3C). These results, together with the 5' RACE, show that the ovary contained a novel transcript which originates at a promoter close to exon 1b that is not active in prospermatogonia and ES cells and that this upstream promoter gave rise to all the Dnmt3L transcripts present in ovary.
We isolated total RNA from ovaries at various stages of development and carried out RT–PCR to determine when the oocyte-specific transcript started to appear. Transcripts containing exons 1b and 1c could be detected from 7 dpp, which corresponds to the entry of the first cohort of oocytes into the growth phase (Figure 3D). In situ hybridization with a probe spanning exons 1b and 1c showed that the transcripts were confined to the germ cells and confirmed that mRNA started to accumulate as the unilaminar primary oocyte began to grow, with high levels detected in early secondary follicles (Figure 3E). We also carried out RT–PCR on 35 dpp ovary RNA with reverse primers in downstream exons (Figure 3F), which confirmed that the oocyte-specific transcript contained all the protein-coding exons 2–13 present in the prospermatogonia transcript but lacked the testis-specific exon 9b.
These results suggested that the effect on the female germ line in the Dnmt3L knockout mice may be due to disruption of the transcript which originates from the oocyte-specific promoter. To confirm this, we carried out RT–PCR on RNA from ovaries of wild-type and Dnmt3L–/– mice (Bourc’his et al., 2001), in which exons 3–5 have been replaced with a -geo cassette, using primers in exons 1b and 6. It can be seen from Figure 3G that no oocyte-specific transcripts were detected in the adult knockout mice (–/–), but they were present in their wild-type littermates (+/+). Primers for -actin resulted in amplification in both wild-type and mutant mice (Figure 3G), indicating that intact mRNA was present in all samples. This also suggests that the -galactosidase staining pattern seen in the growing oocyte in mutant mice arises from a transcript which initiates at the oocyte-specific promoter described here (Bourc’his et al., 2001).
Analysis of the coding potential and conservation of the mRNA
Analysis of the ORFs indicates that it is likely that the ovary transcript uses the same ATG as the prospermatogonia transcript and yields an identical protein. The longest ORF in each round spermatid transcript began at the ATG found in exon 11 and is not in a favourable context for translation initiation (Kozak, 1996), suggesting that these transcripts may be non-coding. In human, five short ESTs (BQ028660
[GenBank]
, AI631758
[GenBank]
, AW237277
[GenBank]
, BF222724
[GenBank]
and AI468252
[GenBank]
) mapped to the intron between exons 9 and 10 of DNMT3L (Aapola et al., 2004b), some of which were spliced to exon 10 and continued to the last exon, suggesting that they represent transcripts from the round spermatid promoter in humans. Three of the five ESTs were from cDNA libraries derived from germ cell sources. To verify the presence of these transcripts in human adult testis, we carried out RT–PCR using a forward primer based on these ESTs and a reverse primer in exon 10 (Figure 4A). This generated a band of 654 bp from the genomic DNA and a 468-bp band from the cDNA, indicating the presence of a spliced transcript originating in exon 9b and splicing into exon 10 in this tissue in humans (Figure 4B). Sequencing indicated that splice donor and acceptor sites match those of ESTs AI631758
[GenBank]
and AW237277
[GenBank]
. Comparison of the mouse, human and predicted rat (Lees-Murdock et al., 2004) Dnmt3L sequences reveals a lack of conservation of the start methionine in adult testis isoforms. These analyses indicate that although the production of short transcripts in adult testis is conserved between mouse and human, indicating they may have some function, these transcripts are unlikely to be protein coding.
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Figure 5 summarizes the data regarding the location of the sex-specific exons and the splicing events occurring at the mouse Dnmt3L locus. Exon 1a is used to drive transcription predominantly in the prospermatogonia in the male, in ES cells and to a much lesser extent in adult spermatogonia. An alternative promoter upstream of exon 1b drives expression in the oocyte, with the transcript including a second unique non-coding exon as well as the coding exons 2–13. In the post-natal testis, there is a decrease in transcription from the prospermatogonia promoter, and as cells enter meiosis, transcription from this promoter ceases. At the end of prophase I of meiosis, a third promoter becomes active, producing truncated transcripts that are likely to be non-coding and contain one or two unique 5' exons. This situation is very similar to that for the Dnmt1 and Dnmt3a loci in mouse, which also produce sex-specific transcripts from unique promoters, and the structure of these loci are shown here for comparison.
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Gene knockout experiments have demonstrated the importance of the Dnmt3L protein in male germ cell development (Bourc’his et al., 2001
; Hata et al., 2002; Bourc’his and Bestor., 2004; Webster et al., 2005). A transcript coding for a full-length version of the protein is initially transcribed in prospermatogonia in the perinatal period, when de-novo methylation occurs. Transcription from this promoter declines as prospermatogonia differentiate into type A spermatogonia during sexual maturation, as we show here and elsewhere (Bourc’his and Bestor, 2004; La Salle et al., 2004), and ceases as the cells progress through spermatogonial proliferation and meiosis. At this point, an alternative promoter is activated and three short transcripts are produced from late pachytene stage onwards from this promoter in the 3' end of the gene. These transcripts are unlikely to produce a functional protein, because the only ORF in-frame with the Dnmt3L protein has an ATG which does not match the Kozak consensus sequence, is preceded by multiple upstream ORFs and is not conserved between mice and humans. There are a number of other genes that utilize an alternative promoter to produce a different transcript during late meiosis or round spermatid stages in male germ cells (see Kleene, 2001 for a recent review). For many of these genes, such as SOD-1 (Gu and Hecht, 1996), Cytochrome C (Hake and Hecht, 1993), Proenkephalin (Rao and Howells, 1993) and Dnmt1 (Trasler et al., 1992; Mertineit et al., 1998), the presence of additional sequences in the 5' untranslated region has been shown to correlate with low or undetectable protein. As in the case of Dnmt3L, production of a pachytene-specific transcript for 
-tubulin lacking any ORF is conserved across species, suggesting that these non-coding transcripts have important functions (Kleene, 2001). Conservation of adult testis transcript production for Dnmt3L between humans and mice strongly suggest that these may play an important role in regulating production of the full-length protein in round spermatids. In the mouse ovary, a third promoter at the Dnmt3L locus, upstream of those used in prospermatogonia and round spermatids, is used to drive transcription of the gene in growing oocytes. This transcript is likely to utilize the same initiation codon as the prospermatogonia transcript and thus produce a full-length Dnmt3L protein containing all of the conserved motifs. This appears to be the only transcript present in the ovary, which would indicate that all of the protein produced in the oocyte must be derived from this transcript.
Targeted disruptions of the Dnmt3L gene have replaced exons 3–5 (Bourc’his et al., 2001) and exons 3–8 (Hata et al., 2002) with a -geo cassette containing a splice acceptor sequence followed by multiple stops in all frames and the gene for a -galactosidase/neomycin resistance fusion protein. Because neither of these mutations disrupt the round-spermatid promoter between exons 9a and 9b, the phenotype seen in the male mice, including formation of aberrant chromosome pairing structures, activation of selfish DNA elements, partial demethylation of H19 and Rasgrf1 and loss of germ cells in the adult, must be due to the absence of the prospermatogonia transcript. Male germ cells do not progress to late pachytene in Dnmt3L–/– mutants and so cannot be tested for the presence of transcripts containing exon 9b. In contrast, because the oocyte-specific transcript described here is the only one produced in the ovary and is absent in the mutant mice, the phenotype seen in existing female knockout mice and their offspring must be due to disruption of this transcript in vivo, supporting the prediction that the ovary-specific transcript codes for a functional protein.
Alternative promoter usage has also been reported for the Dnmt3a (Chen et al., 2002) and Dnmt1 (Mertineit et al., 1998) genes (Figure 5). A second promoter at the Dnmt3a locus produces a shorter, more active form of the protein known as Dnmt3a2 specifically in tissues showing de-novo methylation, including the germ cells of both sexes: however, no evidence has been found so far of any sex-specific transcripts of this gene. There are clearer parallels however between the Dnmt3L and Dnmt1 loci in terms of structure and germ cell expression (see Figure 5). Both genes use an oocyte-specific promoter to drive protein production in the egg and use promoter switching in the male germ line to produce transcripts with low translation potential. The oocyte-specific promoter of Dnmt1 produces a longer transcript but a slightly shorter protein, which is stored in the oocyte and required for maintaining methylation on imprinted genes in the early embryo. Disruption of the Dnmt3L oocyte transcript (Bourc’his et al., 2001; Hata et al., 2002) results in a failure to establish methylation on the same genes: both promoters are also active in growing oocytes, suggesting that they may be controlled in parallel. In the male germ line, the switch from the coding form of Dnmt1 to the non-coding form appears to occur in the early pachytene stage, where both transcripts can be detected (Trasler et al., 1992; Mertineit et al., 1998). By the late pachytene stage only the non-coding form is seen, whereas for Dnmt3L the switch appears to occur in the late pachytene stage, with the non-coding transcripts predominating in round spermatids. Although the function of the non-translated form of Dnmt1 that is present in pachytene spermatocytes and the truncated forms of Dnmt3L that are present in round spermatids are not known, a role in the suppression of the full-length protein by transcriptional or translational interference is likely.
In conclusion, our results show that Dnmt3L production is controlled by three separate promoters, not just one as previously thought. These ensure production of the full-length protein is restricted to times when de-novo methylation is known to occur, i.e. the prospermatogonia and growing oocyte stages.
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We thank G. McKerr, R. Black, V. Hayes, K. Pogue and C. McKeogh for technical assistance and T. Bjourson and J. Yoder for advice on RACE cloning and RNase H mapping, respectively. This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR) to J.M.T., from the NIH to T.H.B. and from the BBSRC (BBS/B/07403 and G12997 [GenBank] ), the Northern Ireland HPSS Cancer RRG (RRG 6.7) and the Royal Society (RSRG 20735) to C.P.W. T.C.S. is the recipient of a Vice-Chancellor’s Research studentship and S.L.S. of a CIHR studentship. J.M.T. is a William Dawson Scholar of McGill University and a Scholar of the Fonds de la recherche en santé du Quebec. Funding to pay the Open Access publication charges for this article was provided by the BBSRC and Cancer RRG.
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Submitted on July 13, 2006; resubmitted on August 22, 2006; accepted on August 31, 2006.
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