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

Nucleic Acids Res. 2012 Apr;40(8):3364-77. doi: 10.1093/nar/gkr1253. Epub 2011 Dec 30.

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.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • 5-Methylcytosine / analogs & derivatives
  • Animals
  • Cells, Cultured
  • Cytosine / analogs & derivatives*
  • Cytosine / metabolism
  • DNA (Cytosine-5-)-Methyltransferases / metabolism
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / physiology*
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / enzymology*
  • Embryonic Stem Cells / metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Homeodomain Proteins / metabolism
  • Leukemia Inhibitory Factor / metabolism*
  • MAP Kinase Signaling System
  • Mice
  • Nanog Homeobox Protein
  • Proto-Oncogene Proteins / antagonists & inhibitors
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / physiology*
  • RNA Interference
  • STAT3 Transcription Factor / metabolism*
  • Signal Transduction

Substances

  • DNA-Binding Proteins
  • Homeodomain Proteins
  • Leukemia Inhibitory Factor
  • Lif protein, mouse
  • Nanog Homeobox Protein
  • Nanog protein, mouse
  • Proto-Oncogene Proteins
  • STAT3 Transcription Factor
  • Stat3 protein, mouse
  • TET1 protein, mouse
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA methyltransferase 3B