TET1 interacts directly with NANOG via independent regions containing hydrophobic and aromatic residues

Abstract

<jats:title>Abstract</jats:title><jats:p>The DNA demethylase TET1 is highly expressed in embryonic stem cells. Knockout experiments indicate that TET1 is important for lineage commitment, and paradoxically, also for reprogramming to naïve pluripotency. TET1 binds to promoters through a CXXC domain which recognises unmethylated CpG dinucleotides. TET1 also binds to enhancers, presumably via interactions with partner proteins. The transcription factor NANOG interacts with TET1 and is predominantly localised at enhancers in ESCs. Therefore, NANOG may contribute to TET1 biological activity in pluripotent cells. However, the regions of TET1 involved in protein-protein interactions are mostly 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. 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 is targeted to specific sites of action at enhancers by direct interaction with a transcription factor.</jats:p><jats:sec><jats:title>Highlights</jats:title><jats:list list-type="bullet"><jats:list-item><jats:p>NANOG and TET1 have regulatory roles in maintaining and reprogramming pluripotency</jats:p></jats:list-item><jats:list-item><jats:p>TET1 and NANOG interact via multiple independent binding regions</jats:p></jats:list-item><jats:list-item><jats:p>TET1 and NANOG interactions are mediated by aromatic and hydrophobic residues</jats:p></jats:list-item><jats:list-item><jats:p>TET1 residues that bind NANOG are highly conserved in mammals</jats:p></jats:list-item><jats:list-item><jats:p>Co-localisation of TET1 and NANOG on chromatin is enriched at NANOG target genes</jats:p></jats:list-item></jats:list></jats:sec>