Abstract
As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet1 has been implicated in regulating global DNA demethylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears primarily to repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, whereas ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.
Highlights
O-GlcNAc transferase (Ogt) N-acetylglucosylates proteins and plays an important role in mouse embryonic stem (ES) cells
Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt
Endogenous Tet1 Interacts with Repression-associated Chromatin Factors—To better understand how Tet1 carries out its function in regulating gene expression in ES cells, we performed large scale IP followed by mass spectrometry analysis using mouse ES cells and an antibody against endogenous Tet1 [18]
Summary
Results: The DNA demethylation enzyme Tet interacts with Ogt and is O-GlcNAcylated. Conclusion: Tet protein stability is positively regulated by O-GlcNAcylation, and its repression function on targeting genes is dependent on Ogt. Significance: Ogt-Tet interaction should further our understanding of how O-GlcNAcylation is integrated into ES cell regulatory networks. As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet has been implicated in regulating global DNA demethylation and gene expression. To understand how Tet may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet in mouse ES cells. Our results suggest that O-GlcNAcylation can positively regulate Tet protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt
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