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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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Onset of DNA methyltransferase expression during mammalian germ cell development. The results presented in this paper as well as observations made for the male germ line [25, 26] are depicted here in relationship to the timing of de novo methylation establishment on imprinted genes and repeats during germ cell development. The figure is adapted from [47]. The top panel illustrates the methylation dynamics of maternally and paternally methylated imprinted genes, depicted by the red and blue lines respectively. During gametogenesis the pattern of non-imprinted gene methylation closely resembles that of imprinted genes. The time at which DNA methyltransferases are expressed during germ cell development are indicated with the transcripts examined in this study highlighted in bold. Although expressed at similar stages of oogenesis, the de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are grouped separately from Dnmt3L which does not have DNA methyltransferase activity. The onset of Dnmt1o expression has been previously shown in early growing oocytes [43]. Our findings show that the expression of Dnmt3a, Dnmt3b and Dnmt3L coincide with the establishment of DNA methylation on imprinted genes and repeat sequences in the female germ line. The progression of methylation imprint acquisition during oogenesis is illustrated in the bottom panel and depicted by the red shading above the female germ cells. PGC = primordial germ cell, Oog = oogonia, PL = preleptotene, L = leptotene, Z = zygotene, P = pachytene, NG = non-growing oocyte G = growing oocyte, MII = metaphase II oocyte.
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Figure 6: Onset of DNA methyltransferase expression during mammalian germ cell development. The results presented in this paper as well as observations made for the male germ line [25, 26] are depicted here in relationship to the timing of de novo methylation establishment on imprinted genes and repeats during germ cell development. The figure is adapted from [47]. The top panel illustrates the methylation dynamics of maternally and paternally methylated imprinted genes, depicted by the red and blue lines respectively. During gametogenesis the pattern of non-imprinted gene methylation closely resembles that of imprinted genes. The time at which DNA methyltransferases are expressed during germ cell development are indicated with the transcripts examined in this study highlighted in bold. Although expressed at similar stages of oogenesis, the de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are grouped separately from Dnmt3L which does not have DNA methyltransferase activity. The onset of Dnmt1o expression has been previously shown in early growing oocytes [43]. Our findings show that the expression of Dnmt3a, Dnmt3b and Dnmt3L coincide with the establishment of DNA methylation on imprinted genes and repeat sequences in the female germ line. The progression of methylation imprint acquisition during oogenesis is illustrated in the bottom panel and depicted by the red shading above the female germ cells. PGC = primordial germ cell, Oog = oogonia, PL = preleptotene, L = leptotene, Z = zygotene, P = pachytene, NG = non-growing oocyte G = growing oocyte, MII = metaphase II oocyte.

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)

Onset of DNA methyltransferase expression during mammalian germ cell development. The results presented in this paper as well as observations made for the male germ line [25, 26] are depicted here in relationship to the timing of de novo methylation establishment on imprinted genes and repeats during germ cell development. The figure is adapted from [47]. The top panel illustrates the methylation dynamics of maternally and paternally methylated imprinted genes, depicted by the red and blue lines respectively. During gametogenesis the pattern of non-imprinted gene methylation closely resembles that of imprinted genes. The time at which DNA methyltransferases are expressed during germ cell development are indicated with the transcripts examined in this study highlighted in bold. Although expressed at similar stages of oogenesis, the de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are grouped separately from Dnmt3L which does not have DNA methyltransferase activity. The onset of Dnmt1o expression has been previously shown in early growing oocytes [43]. Our findings show that the expression of Dnmt3a, Dnmt3b and Dnmt3L coincide with the establishment of DNA methylation on imprinted genes and repeat sequences in the female germ line. The progression of methylation imprint acquisition during oogenesis is illustrated in the bottom panel and depicted by the red shading above the female germ cells. PGC = primordial germ cell, Oog = oogonia, PL = preleptotene, L = leptotene, Z = zygotene, P = pachytene, NG = non-growing oocyte G = growing oocyte, MII = metaphase II oocyte.
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Show All Figures
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Figure 6: Onset of DNA methyltransferase expression during mammalian germ cell development. The results presented in this paper as well as observations made for the male germ line [25, 26] are depicted here in relationship to the timing of de novo methylation establishment on imprinted genes and repeats during germ cell development. The figure is adapted from [47]. The top panel illustrates the methylation dynamics of maternally and paternally methylated imprinted genes, depicted by the red and blue lines respectively. During gametogenesis the pattern of non-imprinted gene methylation closely resembles that of imprinted genes. The time at which DNA methyltransferases are expressed during germ cell development are indicated with the transcripts examined in this study highlighted in bold. Although expressed at similar stages of oogenesis, the de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are grouped separately from Dnmt3L which does not have DNA methyltransferase activity. The onset of Dnmt1o expression has been previously shown in early growing oocytes [43]. Our findings show that the expression of Dnmt3a, Dnmt3b and Dnmt3L coincide with the establishment of DNA methylation on imprinted genes and repeat sequences in the female germ line. The progression of methylation imprint acquisition during oogenesis is illustrated in the bottom panel and depicted by the red shading above the female germ cells. PGC = primordial germ cell, Oog = oogonia, PL = preleptotene, L = leptotene, Z = zygotene, P = pachytene, NG = non-growing oocyte G = growing oocyte, MII = metaphase II oocyte.
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