TET1 Interacts Directly with NANOG via Independent Domains Containing Hydrophobic and Aromatic Residues

Raphaël Pantier,Nicholas Mullin,Elisa Hall-Ponsele,Ian Chambers

doi:10.1016/j.jmb.2020.10.008

Raphaël Pantier, Nicholas Mullin + Show 2 more

Open Access

https://doi.org/10.1016/j.jmb.2020.10.008

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Journal: Journal of molecular biology	Publication Date: Oct 13, 2020
Citations: 3	License type: cc-by

Affiliation: University of Edinburgh

Abstract

The DNA demethylase TET1 is highly expressed in embryonic stem cells and is important both for lineage commitment, and reprogramming to naïve pluripotency. TET1 interacts with the pluripotency transcription factor NANOG which may contribute to its biological activity in pluripotent cells. However, how TET1 interacts with other proteins is largely unknown. Here, we characterise the physical interaction between TET1 and NANOG using embryonic stem cells and bacterial expression systems. TET1 and NANOG interact through multiple binding sites that act independently. Critically, mutating conserved hydrophobic and aromatic residues within TET1 and NANOG abolishes the interaction. On chromatin, NANOG is predominantly localised at ESC enhancers. While TET1 binds to CpG dinucleotides in promoters using its CXXC domain, TET1 also binds to enhancers, though the mechanism involved is unknown. Comparative ChIP-seq analysis identifies genomic loci bound by both TET1 and NANOG, that correspond predominantly to pluripotency enhancers. Importantly, around half of NANOG transcriptional target genes are associated with TET1-NANOG co-bound sites. These results indicate a mechanism by which TET1 protein may be targeted to specific sites of action at enhancers by direct interaction with a transcription factor.

Highlights

Ten-eleven-translocation (TET) family proteins are responsible for active DNA demethylation by oxidation of 5-methylcytosine[1,2] and play important roles during embryonic development and various physiological processes.[3]
We analysed the interaction between endogenous TET1 and NANOG in pluripotent cells using nuclear protein extracts from Tet1-(Flag)[3] ESCs21 immunoprecipitated with an anti-Flag antibody
Relative to controls from E14Tg2a embryonic stem cell (ESC) which showed only background binding, FLAG immunoprecipitates from Tet1(Flag)[3] ESCs were strongly enriched for NANOG (Figure S1(b))

Summary

Introduction

Ten-eleven-translocation (TET) family proteins are responsible for active DNA demethylation by oxidation of 5-methylcytosine[1,2] and play important roles during embryonic development and various physiological processes.[3] TET proteins contribute to DNA demethylation in naïve embryonic stem cells (ESCs),[4,5,6,7] in particular at enhancers.[8,9,10,11,12,13] TET protein activity is required both for proper differentiation[14,15] and reprogramming to pluripotency.[16,17,18] TET1 is the most highly expressed TET family protein both in pluripotent cells and during early development.[19,20,21] TET1 predominantly binds to promoters via its N-terminal CXXC domain which recognises unmethylated CpG dinucleotides.[22,23,24,25] TET1 binding at enhancers in ESCs26–28 could be mediated by interactions with the pluripotency factors NANOG, PRDM14, OCT4 and SOX2.29–32 Interestingly, co-expression of TET1 and NANOG in pre-iPS cells synergistically enhances reprogramming to pluripotency.[29] how TET1 might be recruited to chromatin via protein–protein interactions remains poorly understood with little known about the residues involved in protein binding. Comparison of TET1 and NANOG ChIP-seq datasets identified genomic loci that are putatively regulated by the TET1-NANOG complex

Results

Discussion

Materials and Methods