Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells

Jonathan Ip, Paul Canham, Andy Choo, Yoshima Inaba, Shelley A Jacobs, Paul Kalitsis, Deidrie Mattiske, Jane Ng, Richard Saffery, Nicholas C Wong, Lee Wong, Jeffrey R Mann

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line age, given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1(-/-) ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1(-/-), allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1(-/-) ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway.
Original languageEnglish
Article numbere1002919
Number of pages13
JournalPLoS Genetics
Volume8
Issue number9
DOIs
Publication statusPublished - 2012

Cite this

Ip, Jonathan ; Canham, Paul ; Choo, Andy ; Inaba, Yoshima ; Jacobs, Shelley A ; Kalitsis, Paul ; Mattiske, Deidrie ; Ng, Jane ; Saffery, Richard ; Wong, Nicholas C ; Wong, Lee ; Mann, Jeffrey R. / Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells. In: PLoS Genetics. 2012 ; Vol. 8, No. 9.
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abstract = "Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line age, given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1(-/-) ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1(-/-), allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1(-/-) ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway.",
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Ip, J, Canham, P, Choo, A, Inaba, Y, Jacobs, SA, Kalitsis, P, Mattiske, D, Ng, J, Saffery, R, Wong, NC, Wong, L & Mann, JR 2012, 'Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells' PLoS Genetics, vol. 8, no. 9, e1002919. https://doi.org/10.1371/journal.pgen.1002919

Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells. / Ip, Jonathan; Canham, Paul; Choo, Andy; Inaba, Yoshima; Jacobs, Shelley A; Kalitsis, Paul; Mattiske, Deidrie; Ng, Jane; Saffery, Richard; Wong, Nicholas C; Wong, Lee; Mann, Jeffrey R.

In: PLoS Genetics, Vol. 8, No. 9, e1002919, 2012.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells

AU - Ip, Jonathan

AU - Canham, Paul

AU - Choo, Andy

AU - Inaba, Yoshima

AU - Jacobs, Shelley A

AU - Kalitsis, Paul

AU - Mattiske, Deidrie

AU - Ng, Jane

AU - Saffery, Richard

AU - Wong, Nicholas C

AU - Wong, Lee

AU - Mann, Jeffrey R

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