Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. Here we report the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. We demonstrate that the complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, we show that H3-K27 methylation colocalizes with, and is dependent on, E(Z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (PC), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing.

5-methylcytosine (5mC) in DNA plays an important role in gene expression, genomic imprinting, and suppression of transposable elements. 5mC can be converted to 5-hydroxymethylcytosine (5hmC) by the Tet proteins. Here we show that, in addition to 5hmC, the Tet proteins can generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC in an enzymatic activity-dependent manner. Furthermore, we reveal the presence of 5fC and 5caC in genomic DNA of mouse ES cells and mouse organs. The genomic content of 5hmC, 5fC, and 5caC can be increased or reduced through overexpression or depletion of Tet proteins. Thus, we identify two new cytosine derivatives in genomic DNA as the products of Tet proteins. Our study raises the possibility that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation.

DNA methylation is one of the best-characterized epigenetic modifications 1–4. While the enzymes that catalyze DNA methylation have been characterized, enzymes responsible for the reversal process have been elusive 5. A recent study indicates that the human Tet1 protein could catalyze the conversion of 5-methyl-C (5mC) of DNA to 5-hydroxyl-methyl-C (5hmC), raising the possibility that DNA demethylation may be a Tet1-mediated process 6. Here we extended this study by demonstrating that all three mouse Tet proteins can also catalyze a similar reaction. Interestingly, Tet1 plays an important role in mouse ES cell maintenance through maintaining the expression of Nanog in ES cells. Importantly, Tet1 knockdown-mediated down-regulation of Nanog correlated with its promoter methylation, supporting a role for Tet1 in regulating DNA methylation status. Furthermore, knockdown of Tet1 in preimplantation embryos resulted in a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and ICM cell specification.