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Investigating the OGT-TET interaction in vitro and in mouse embryonic stem cells

Abstract

Proper spatial and temporal control of gene expression is necessary for cellular survival and proper function. Addition of a methyl group to the 5’ carbon of cytosine in DNA (5mC) is a major mechanism used to modulate gene expression. The Ten-Eleven Translocation (TET) family of enzymes iteratively oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC), and 5-carboxylcytosine (5caC). These modifications function both as stable epigenetic marks and transient intermediates in the demethylation of DNA. Improper placement of these epigenetic marks often causes death or disease. Among the proteins that interact with TET enzymes is O-linked N-acetylglucosamine (O-GlcNAc) Transferase (OGT). OGT is the sole enzyme responsible for attaching a GlcNAc sugar to serine, threonine, and cysteine residues of over 1,000 nuclear, cytoplasmic, and mitochondrial proteins. OGT has been termed a “nutrient sensor” because its activity requires the sugar donor UDP-GlcNAc, whose abundance is dependent upon the levels of various cellular metabolites. Thus the reversible O-GlcNAc modification dynamically regulates the functions of OGT’s targets in response to nutrient status. OGT stably interacts with and modifies TET proteins and its genome-wide distribution overlaps significantly with TETs. However, the significance of the OGT-TET interactions are poorly understood. In this work, we explore the consequences of the OGT-TET interactions in vitro and in mouse embryonic stem cells (mESCs). We show that OGT directly binds and modifies TET1 in vitro, and the O-GlcNAc modification enhances TET1 activity. We identify a point mutation in TET1 that disrupts its interaction with OGT and use this to interrogate the effects on TET activity, gene expression, and epigenetic patterning of disrupting the OGT-TET1 interaction in mESCs. To assess the importance of the OGT-TET interaction for OGT function, we use quantitative SILAC mass spectrometry to examine proteome-wide changes in O-GlcNAcylation in mESCs when Tets are deleted. We also identify sites of O-GlcNAcylation on TET1 and TET2, further analyze the interactions between OGT and all three TETs, and examine the effect of O-GlcNAcylation on TET2 and TET3 activity in vitro. Our results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

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