Molecular interactions of TET proteins in pluripotent cells
Pantier, Raphaël Pierre
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Ten-Eleven-Translocation (TET) proteins form a family of enzymes responsible for active DNA demethylation by oxidation of 5-methylcytosine. TET proteins play a key role in genomic reprogramming in vitro and in vivo. Although TET proteins are expressed in embryonic stem cells (ESCs), their role in regulating pluripotency remains unclear. In addition, the mechanisms by which TET proteins are recruited to chromatin are largely unknown. To visualise TET protein dynamics during pluripotency and differentiation, the endogenous Tet1/2/3 alleles were fused to epitope tags in ESCs using CRISPR/Cas9. Characterisation of these cell lines showed that TET1 is the highest expressed TET protein in both naïve and primed pluripotent cells. In contrast, TET2 is expressed heterogeneously in ESCs and marks cells with a high self-renewal capacity. To assess the function of Tet genes in pluripotent stem cells, the endogenous Tet1/2/3 ORFs were removed using CRISPR/Cas9. Comparative analysis of single and combined Tet gene knockout ESC lines indicated that Tet1 and Tet2, but not Tet3, play redundant roles to promote loss of pluripotency. Furthermore, Tet-deficient cells retained a naïve morphology in differentiating conditions, suggestive of a LIF-independent self-renewal phenotype. To characterise physiological TET1 protein-protein interactions, TET1 protein partners were identified in ESCs by mass spectrometry and co-immuno-precipitations. This revealed that TET1 interacts with multiple epigenetic and pluripotency-related factors in ESCs. Moreover, detailed characterisation of the interaction between TET1 and NANOG identified three regions of TET1 involved in protein-protein interactions that are conserved in evolution. To investigate TET1 chromatin binding in ESCs, both at the molecular and cellular levels, TET1 was characterised by ChIP-seq analysis and live imaging experiments. Interestingly, TET1 is targeted to chromatin by two different mechanisms, involving distinct protein regions. The interaction with multiple protein partners, including NANOG, might enable TET1 to be targeted to specific chromosomal locations. Additionally, TET1 has the unusual ability to bind mitotic chromatin through its N-terminus, independently of its interaction with NANOG. Together these analyses provide a new understanding of the role of TET proteins in pluripotent cells, as well as a detailed map of TET1 residues involved in protein-protein interactions and mitotic chromatin binding.