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Repression of retrotransposal elements in mouse embryonic stem cells is primarily mediated by a DNA methylation-independent mechanism.

ABSTRACT: In defense of deleterious retrotransposition of intracisternal A particle (IAP) elements, IAP loci are heavily methylated and silenced in mouse somatic cells. To determine whether IAP is also repressed in pluripotent stem cells by DNA methylation, we examined IAP expression in demethylated mouse embryonic stem cells (mESCs) and epiblast-derived stem cells. Surprisingly, in demethylated ESC cultures carrying mutations of DNA methyltransferase I (Dnmt1), no IAP transcripts and proteins are detectable in undifferentiated Oct4(+) ESCs. In contrast, approximately 3.6% of IAP-positive cells are detected in Oct4(-) Dnmt1(-/-) cells, suggesting that the previously observed increase in IAP transcripts in the population of Dnmt1(-/-) ESCs could be accounted for by this subset of Oct4(-) Dnmt1(-/-) ESCs undergoing spontaneous differentiation. Consistent with this possibility, a dramatic increase of IAP mRNA (>100-fold) and protein expression was observed in Dnmt1(-/-) ESC cultures upon induction of differentiation through the withdrawal of leukemia-inhibitory factor for 6 or more days. Interestingly, both mRNAs and proteins of IAP can be readily detected in demethylated Oct4(+) epiblast-derived stem cells as well as differentiated mouse embryo fibroblasts, neurons, and glia upon conditional Dnmt1 gene deletion. These data suggest that mESCs are a unique stem cell type possessing a DNA methylation-independent IAP repression mechanism. This methylation-independent mechanism does not involve Dicer-mediated action of microRNAs or RNA interference because IAP expression remains repressed in Dnmt1(-/-); Dicer(-/-) double mutant ESCs. We suggest that mESCs possess a unique DNA methylation-independent mechanism to silence retrotransposons to safeguard genome stability while undergoing rapid cell proliferation for self-renewal.

SUBMITTER: Hutnick LK

PROVIDER: S-EPMC2898347 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Repression of retrotransposal elements in mouse embryonic stem cells is primarily mediated by a DNA methylation-independent mechanism.

Hutnick Leah K LK Huang Xinhua X Loo Tao-Chuan TC Ma Zhicheng Z Fan Guoping G

The Journal of biological chemistry 20100419 27

In defense of deleterious retrotransposition of intracisternal A particle (IAP) elements, IAP loci are heavily methylated and silenced in mouse somatic cells. To determine whether IAP is also repressed in pluripotent stem cells by DNA methylation, we examined IAP expression in demethylated mouse embryonic stem cells (mESCs) and epiblast-derived stem cells. Surprisingly, in demethylated ESC cultures carrying mutations of DNA methyltransferase I (Dnmt1), no IAP transcripts and proteins are detecta ...[more]

PMID: 20404320

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Project description:Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have previously demonstrated that double-strand breaks near telomeres in mammalian cells result in either the addition of a new telomere at the site of the break, termed chromosome healing, or sister chromatid fusion that initiates chromosome instability. In the present study, we have investigated the role of telomerase in chromosome healing and the importance of chromosome healing in preventing chromosome instability. In embryonic stem cell lines that are wild type for the catalytic subunit of telomerase (TERT), chromosome healing at I-SceI-induced double-strand breaks near telomeres accounted for 22 of 35 rearrangements, with the new telomeres added directly at the site of the break in all but one instance. In contrast, in two TERT-knockout embryonic stem cell lines, chromosome healing accounted for only 1 of 62 rearrangements, with a 23 bp insertion at the site of the sole chromosome-healing event. However, in a third TERT-knockout embryonic stem cell line, 10PTKO-A, chromosome healing was a common event that accounted for 20 of 34 rearrangements. Although this chromosome healing also occurred at the I-SceI site, differences in the microhomology at the site of telomere addition demonstrated that the mechanism was distinct from that in wild-type embryonic stem cell lines. In addition, the newly added telomeres in 10PTKO-A shortened with time in culture, eventually resulting in either telomere elongation through a telomerase-independent mechanism or loss of the subtelomeric plasmid sequences entirely. The combined results demonstrate that chromosome healing can occur through both telomerase-dependent and -independent mechanisms, and that although both mechanisms can prevent degradation and sister chromatid fusion, neither mechanism is efficient enough to prevent sister chromatid fusion from occurring in many cells experiencing double-strand breaks near telomeres.

Dataset Information

Repression of retrotransposal elements in mouse embryonic stem cells is primarily mediated by a DNA methylation-independent mechanism.

Publications

Repression of retrotransposal elements in mouse embryonic stem cells is primarily mediated by a DNA methylation-independent mechanism.

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