title: Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells.
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.
authors.affiliation: Theme of Genetic Disorders, Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Ip
authors.forename: Paul
authors.initials: P
authors.lastname: Canham
authors.forename: K H Andy
authors.initials: KH
authors.lastname: Choo
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Inaba
authors.forename: Shelley A
authors.initials: SA
authors.lastname: Jacobs
authors.forename: Paul
authors.initials: P
authors.lastname: Kalitsis
authors.forename: Deidre M
authors.initials: DM
authors.lastname: Mattiske
authors.forename: Jane
authors.initials: J
authors.lastname: Ng
authors.forename: Richard
authors.initials: R
authors.lastname: Saffery
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Wong
authors.forename: Lee H
authors.initials: LH
authors.lastname: Wong
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Mann
mesh_terms: D000818:Animals; D053487:DEAD-box RNA Helicases / Q000235:genetics* / Q000378:metabolism; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D053595:Embryonic Stem Cells / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130 / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism
chemical_list: D035683:MicroRNAs; C493838:Rbl2 protein, mouse; D050717:Retinoblastoma-Like Protein p130; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases