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Coordinate regulation of DNA methyltransferase expression during oogenesis.

Lucifero D, La Salle S, Bourc'his D, Martel J, Bestor TH, Trasler JM - BMC Dev. Biol. (2007)

Bottom Line: Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the de novo methyltransferase Dnmt3b, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases.Together these results provide a better understanding of the developmental regulation of Dnmt3a, Dnmt3b and Dnmt3L at the time of de novo methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line.This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Montreal Children's Hospital Research Institute and Department of Pediatrics, McGill University, Montreal, Quebec, Canada. diana.lucifero@bbsrc.ac.uk <diana.lucifero@bbsrc.ac.uk>

ABSTRACT

Background: Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of Dnmt3a and Dnmt3b, as well as a regulator of DNA methylation, Dnmt3L, in isolated female germ cells.

Results: Our results indicate that these enzymes are coordinately regulated and that their expression peaks during the stage of postnatal oocyte development when maternal methylation imprints are established. We find that Dnmt3a, Dnmt3b, Dnmt3L and Dnmt1o transcript accumulation is related to oocyte diameter. Furthermore, DNMT3L deficient 15 dpp oocytes have aberrantly methylated Snrpn, Peg3 and Igf2r DMRs, but normal IAP and LINE-1 methylation levels, thereby highlighting a male germ cell specific role for DNMT3L in the establishment of DNA methylation at repeat elements. Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the de novo methyltransferase Dnmt3b, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases.

Conclusion: Together these results provide a better understanding of the developmental regulation of Dnmt3a, Dnmt3b and Dnmt3L at the time of de novo methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line. This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.

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Dnmt3a, Dnmt3b and Dnmt3L expression in DNMT3L deficient 15 dpp oocytes and in DNMT1o deficient 25 dpp GV oocytes. a) QRT-PCR was used to analyze the expression of the de novo DNMT enzymes in Dnmt3L heterozygous (dark grey bars) and homozygous (cross hatch bars) oocytes at 15 dpp. Results for Dnmt3a and Dnmt3b are shown in a) while relative expression of Dnmt3L is illustrated in b). Dnmt3a and Dnmt3b transcripts are up-regulated in DNMT3L depleted growing oocytes. The differences observed for Dnmt3b and Dnmt3L were statistically significant (p < 0.01). c) QRT-PCR was used to analyze the expression of the DNMT enzymes in Dnmt1o heterozygous (dark grey bars) and homozygous (cross hatch bars) 25 dpp GV stage oocytes. While the relative expression of Dnmt3a and Dnmt3b was up-regulated in DNMT1o depleted GV oocytes, Dnmt3L expression remained unchanged. Samples were analyzed in triplicate and relative expression values obtained were normalized to the level of rabbit α-globin expression for each sample and were calibrated to the expression in heterozygous oocytes for Dnmt3a, Dnmt3b and Dnmt3L. For Dnmt3L analysis, expression was calibrated to the expression in homozygous oocytes. Results for one experiment are presented as mean ± SD.
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Figure 4: Dnmt3a, Dnmt3b and Dnmt3L expression in DNMT3L deficient 15 dpp oocytes and in DNMT1o deficient 25 dpp GV oocytes. a) QRT-PCR was used to analyze the expression of the de novo DNMT enzymes in Dnmt3L heterozygous (dark grey bars) and homozygous (cross hatch bars) oocytes at 15 dpp. Results for Dnmt3a and Dnmt3b are shown in a) while relative expression of Dnmt3L is illustrated in b). Dnmt3a and Dnmt3b transcripts are up-regulated in DNMT3L depleted growing oocytes. The differences observed for Dnmt3b and Dnmt3L were statistically significant (p < 0.01). c) QRT-PCR was used to analyze the expression of the DNMT enzymes in Dnmt1o heterozygous (dark grey bars) and homozygous (cross hatch bars) 25 dpp GV stage oocytes. While the relative expression of Dnmt3a and Dnmt3b was up-regulated in DNMT1o depleted GV oocytes, Dnmt3L expression remained unchanged. Samples were analyzed in triplicate and relative expression values obtained were normalized to the level of rabbit α-globin expression for each sample and were calibrated to the expression in heterozygous oocytes for Dnmt3a, Dnmt3b and Dnmt3L. For Dnmt3L analysis, expression was calibrated to the expression in homozygous oocytes. Results for one experiment are presented as mean ± SD.

Mentions: In addition to our objective of determining the developmental dynamics of the Dnmt3 genes in postnatal oocytes summarized in Figure 6, we also wished to investigate whether the expression of these transcripts changed in response to the depletion of DNMT3L or DNMT1o. We chose to compare expression profiles after depletion of these two DNMTs in particular because the former provides a mouse model where maternal methylation imprints are affected [14], while the latter represents a model where oocyte methylation imprint patterns are unchanged [8]. Again, we used QRT-PCR to look at the expression of Dnmt3a and Dnmt3b in oocytes isolated from heterozygous and homozygous Dnmt3L females at 15 dpp (Figure 4a) and 25 dpp (see Additional file 3). Expression results were calibrated to the value obtained for the heterozygous oocytes and normalized to rabbit α-globin transcript levels. When compared to the heterozygous oocytes transcript levels, Dnmt3a and Dnmt3b relative expression was up-regulated in response to DNMT3L depletion, increasing roughly 2- and 3-fold respectively. The increase in Dnmt3b expression was highly significant (p < 0.001) while the increase in Dnmt3a expression only approached significance but was reproducible. We also analyzed Dnmt3L expression levels and found heterozygous 15 dpp oocytes to have 360 times the transcript levels as homozygous mutant 15 dpp oocytes (p < 0.01), confirming the purity of our samples (Figure 4b). Analysis of Dnmt3a and Dnmt3b expression changes as a result of DNMT3L depletion in 25 dpp GV oocytes also showed up-regulation of Dnmt3a and Dnmt3b, although not to the same degree as that seen at 15 dpp (see Additional file 3).


Coordinate regulation of DNA methyltransferase expression during oogenesis.

Lucifero D, La Salle S, Bourc'his D, Martel J, Bestor TH, Trasler JM - BMC Dev. Biol. (2007)

Dnmt3a, Dnmt3b and Dnmt3L expression in DNMT3L deficient 15 dpp oocytes and in DNMT1o deficient 25 dpp GV oocytes. a) QRT-PCR was used to analyze the expression of the de novo DNMT enzymes in Dnmt3L heterozygous (dark grey bars) and homozygous (cross hatch bars) oocytes at 15 dpp. Results for Dnmt3a and Dnmt3b are shown in a) while relative expression of Dnmt3L is illustrated in b). Dnmt3a and Dnmt3b transcripts are up-regulated in DNMT3L depleted growing oocytes. The differences observed for Dnmt3b and Dnmt3L were statistically significant (p < 0.01). c) QRT-PCR was used to analyze the expression of the DNMT enzymes in Dnmt1o heterozygous (dark grey bars) and homozygous (cross hatch bars) 25 dpp GV stage oocytes. While the relative expression of Dnmt3a and Dnmt3b was up-regulated in DNMT1o depleted GV oocytes, Dnmt3L expression remained unchanged. Samples were analyzed in triplicate and relative expression values obtained were normalized to the level of rabbit α-globin expression for each sample and were calibrated to the expression in heterozygous oocytes for Dnmt3a, Dnmt3b and Dnmt3L. For Dnmt3L analysis, expression was calibrated to the expression in homozygous oocytes. Results for one experiment are presented as mean ± SD.
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Figure 4: Dnmt3a, Dnmt3b and Dnmt3L expression in DNMT3L deficient 15 dpp oocytes and in DNMT1o deficient 25 dpp GV oocytes. a) QRT-PCR was used to analyze the expression of the de novo DNMT enzymes in Dnmt3L heterozygous (dark grey bars) and homozygous (cross hatch bars) oocytes at 15 dpp. Results for Dnmt3a and Dnmt3b are shown in a) while relative expression of Dnmt3L is illustrated in b). Dnmt3a and Dnmt3b transcripts are up-regulated in DNMT3L depleted growing oocytes. The differences observed for Dnmt3b and Dnmt3L were statistically significant (p < 0.01). c) QRT-PCR was used to analyze the expression of the DNMT enzymes in Dnmt1o heterozygous (dark grey bars) and homozygous (cross hatch bars) 25 dpp GV stage oocytes. While the relative expression of Dnmt3a and Dnmt3b was up-regulated in DNMT1o depleted GV oocytes, Dnmt3L expression remained unchanged. Samples were analyzed in triplicate and relative expression values obtained were normalized to the level of rabbit α-globin expression for each sample and were calibrated to the expression in heterozygous oocytes for Dnmt3a, Dnmt3b and Dnmt3L. For Dnmt3L analysis, expression was calibrated to the expression in homozygous oocytes. Results for one experiment are presented as mean ± SD.
Mentions: In addition to our objective of determining the developmental dynamics of the Dnmt3 genes in postnatal oocytes summarized in Figure 6, we also wished to investigate whether the expression of these transcripts changed in response to the depletion of DNMT3L or DNMT1o. We chose to compare expression profiles after depletion of these two DNMTs in particular because the former provides a mouse model where maternal methylation imprints are affected [14], while the latter represents a model where oocyte methylation imprint patterns are unchanged [8]. Again, we used QRT-PCR to look at the expression of Dnmt3a and Dnmt3b in oocytes isolated from heterozygous and homozygous Dnmt3L females at 15 dpp (Figure 4a) and 25 dpp (see Additional file 3). Expression results were calibrated to the value obtained for the heterozygous oocytes and normalized to rabbit α-globin transcript levels. When compared to the heterozygous oocytes transcript levels, Dnmt3a and Dnmt3b relative expression was up-regulated in response to DNMT3L depletion, increasing roughly 2- and 3-fold respectively. The increase in Dnmt3b expression was highly significant (p < 0.001) while the increase in Dnmt3a expression only approached significance but was reproducible. We also analyzed Dnmt3L expression levels and found heterozygous 15 dpp oocytes to have 360 times the transcript levels as homozygous mutant 15 dpp oocytes (p < 0.01), confirming the purity of our samples (Figure 4b). Analysis of Dnmt3a and Dnmt3b expression changes as a result of DNMT3L depletion in 25 dpp GV oocytes also showed up-regulation of Dnmt3a and Dnmt3b, although not to the same degree as that seen at 15 dpp (see Additional file 3).

Bottom Line: Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the de novo methyltransferase Dnmt3b, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases.Together these results provide a better understanding of the developmental regulation of Dnmt3a, Dnmt3b and Dnmt3L at the time of de novo methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line.This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Montreal Children's Hospital Research Institute and Department of Pediatrics, McGill University, Montreal, Quebec, Canada. diana.lucifero@bbsrc.ac.uk <diana.lucifero@bbsrc.ac.uk>

ABSTRACT

Background: Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of Dnmt3a and Dnmt3b, as well as a regulator of DNA methylation, Dnmt3L, in isolated female germ cells.

Results: Our results indicate that these enzymes are coordinately regulated and that their expression peaks during the stage of postnatal oocyte development when maternal methylation imprints are established. We find that Dnmt3a, Dnmt3b, Dnmt3L and Dnmt1o transcript accumulation is related to oocyte diameter. Furthermore, DNMT3L deficient 15 dpp oocytes have aberrantly methylated Snrpn, Peg3 and Igf2r DMRs, but normal IAP and LINE-1 methylation levels, thereby highlighting a male germ cell specific role for DNMT3L in the establishment of DNA methylation at repeat elements. Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the de novo methyltransferase Dnmt3b, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases.

Conclusion: Together these results provide a better understanding of the developmental regulation of Dnmt3a, Dnmt3b and Dnmt3L at the time of de novo methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line. This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.

Show MeSH
Related in: MedlinePlus