Abstract
Ten-eleven translocation (Tet) family proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. We show that mouse embryonic stem cells (ESCs) depleted of Tet1 and/or Tet2 by RNAi exhibit short telomeres and chromosomal instability, concomitant with reduced telomere recombination. Tet1 and Tet2double-knockout ESCs also display short telomeres but to a lesser extent. Notably, Tet1/2/3 triple-knockout ESCs show heterogeneous telomerelengths and increased frequency of telomere loss and chromosomal fusion. Mechanistically, Tets depletion or deficiency increases Dnmt3b and decreases 5hmC levels, resulting in elevated methylation levels at sub-telomeres. Consistently, knockdown of Dnmt3b or addition of 2i (MAPK and GSK3β inhibitors), which also inhibits Dnmt3b, reduces telomere shortening, partially rescuing Tet1/2 deficiency.Interestingly, Tet1/2 double or Tet1/2/3 triple knockout in ESCs consistently upregulates Zscan4, which may counteract telomere shortening. Together, Tet enzymes play important roles in telomere maintenance and chromosomal stability of ESCs by modulating sub-telomeric methylation levels.
Highlights
Ten-eleven translocation (Tet) family proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate that can lead to DNA demethylation (Kohli and Zhang, 2013; Tahiliani et al, 2009)
Tet3 is highly expressed in mouse embryonic fibroblasts (MEFs) and various somatic tissues but low in embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs)
To test whether Tet1 and Tet2 play a role in telomere maintenance, we depleted Tet1 and/or Tet2 in mouse ESCs by RNAi using at least two sequences targeting Tet1, Tet2, or both genes (Ficz et al, 2011; Ito et al, 2010; Experimental Procedures)
Summary
Ten-eleven translocation (Tet) family proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate that can lead to DNA demethylation (Kohli and Zhang, 2013; Tahiliani et al, 2009). Some Tet mutant mice have a slightly smaller body size at birth (Dawlaty et al, 2011), which might reflect a developmental delay, and are subfertile (Yamaguchi et al, 2012). These enzymes may have overlapping roles in development. This seems to be difficult to reconcile with the roles of Tets in selfrenewal and pluripotency of ESCs/induced pluripotent stem cells (iPSCs) (Costa et al, 2013; Ficz et al, 2011; Ito et al, 2010)
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