Loss of Tet Enzymes Compromises Proper Differentiation of Embryonic Stem Cells

TLDR

Tet enzymes convert 5‑methylcytosine to 5‑hydroxymethylcytosine and are dynamically expressed during development, yet the consequences of complete loss of all three Tet proteins on embryogenesis and ESC differentiation are not yet understood. The authors generated Tet1/2/3 triple‑knockout mouse embryonic stem cells and evaluated their developmental potential. Loss of all three Tet enzymes depleted 5‑hmC, induced promoter hypermethylation and deregulation of developmental genes, and impaired ESC differentiation, producing poorly differentiated embryoid bodies, teratomas, and failure to contribute to chimeric embryos, highlighting the essential role of Tet‑mediated DNA demethylation in proper ESC differentiation and development.

Abstract

Highlights•Combined loss of all three Tet enzymes restricts normal differentiation of ESCs•Tet null ESCs contribute poorly to developing embryos and cannot support development•Tet loss causes promoter hypermethylation and deregulation of developmental genesSummaryTet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation, as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO ESCs contributed poorly to chimeric embryos, a defect rescued by Tet1 reexpression, and could not support embryonic development. Global gene-expression and methylome analyses of TKO EBs revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet- and 5hmC-mediated DNA demethylation in proper regulation of gene expression during ESC differentiation and development.