Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity.
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.
Systems Biology Section, Biostatistics Branch, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA.
Johannes M
JM
Freudenberg
Swati
S
Ghosh
Brad L
BL
Lackford
Sailu
S
Yellaboina
Xiaofeng
X
Zheng
Ruifang
R
Li
Suresh
S
Cuddapah
Paul A
PA
Wade
Guang
G
Hu
Raja
R
Jothi
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D002478:Cells, Cultured; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000378:metabolism; D053605:Leukemia Inhibitory Factor / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D051379:Mice; D000071317:Nanog Homeobox Protein; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D000097584:DNA Methyltransferase 3B
D044503:5-Methylcytosine; D003596:Cytosine; D004248:DNA (Cytosine-5-)-Methyltransferases; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D053605:Leukemia Inhibitory Factor; D000071317:Nanog Homeobox Protein; D011518:Proto-Oncogene Proteins; D050796:STAT3 Transcription Factor; D000097584:DNA Methyltransferase 3B; C507838:Lif protein, mouse; C475540:Nanog protein, mouse; C494087:Stat3 protein, mouse; C540839:TET1 protein, mouse; C011865:5-hydroxymethylcytosine