Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

JM Freudenberg, S Ghosh, BL Lackford… - Nucleic acids …, 2012 - academic.oup.com
JM Freudenberg, S Ghosh, BL Lackford, S Yellaboina, X Zheng, R Li, S Cuddapah
Nucleic acids research, 2012academic.oup.com
The TET family of FE (II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote DNA
demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which
they further oxidize into 5-formylcytosine and 5-carboxylcytosine. Tet1 is robustly expressed
in mouse embryonic stem cells (mESCs) and has been implicated in mESC maintenance.
Here we demonstrate that, unlike genetic deletion, RNAi-mediated depletion of Tet1 in
mESCs led to a significant reduction in 5hmC and loss of mESC identity. The differentiation …
Abstract
The TET family of FE(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which they further oxidize into 5-formylcytosine and 5-carboxylcytosine. Tet1 is robustly expressed in mouse embryonic stem cells (mESCs) and has been implicated in mESC maintenance. Here we demonstrate that, unlike genetic deletion, RNAi-mediated depletion of Tet1 in mESCs led to a significant reduction in 5hmC and loss of mESC identity. The differentiation phenotype due to Tet1 depletion positively correlated with the extent of 5hmC loss. Meta-analyses of genomic data sets suggested interaction between Tet1 and leukemia inhibitory factor (LIF) signaling. LIF signaling is known to promote self-renewal and pluripotency in mESCs partly by opposing MAPK/ERK-mediated differentiation. Withdrawal of LIF leads to differentiation of mESCs. We discovered that Tet1 depletion impaired LIF-dependent Stat3-mediated gene activation by affecting Stat3's ability to bind to its target sites on chromatin. Nanog overexpression or inhibition of MAPK/ERK signaling, both known to maintain mESCs in the absence of LIF, rescued Tet1 depletion, further supporting the dependence of LIF/Stat3 signaling on Tet1 . These data support the conclusion that analysis of mESCs in the hours/days immediately following efficient Tet1 depletion reveals Tet1 's normal physiological role in maintaining the pluripotent state that may be subject to homeostatic compensation in genetic models.
Oxford University Press
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