4d), indicating that Tet1 knockdown favors embryonic cell specification towards the TE lineage. a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but WHI-P97 also demonstrate a role for Tet1 in ES cell maintenance and ICM cell specification. The Tet family proteins include three members Tet1-3 (Fig. S1a). In addition to the dioxygenase motif involved in Fe(II) and alpha-ketoglutarate (-KG) binding, they also share a conserved cysteine-rich region (Fig. S1a, indicated by D and C, respectively). Recent demonstration that the human Tet1 WHI-P97 can convert 5mC to 5hmC in an Fe(II) and -KG-dependent manner6prompted us to evaluate whether mouse Tet1 and its homologs possess a comparable enzymatic activity. Results shown inFig. S2demonstrate that overexpression of both mouse Tet1 and Tet2 catalytic domains greatly reduced 5mC staining in both U2OS and HEK293T cells. In contrast, WHI-P97 overexpression of mouse Tet3 catalytic domain name in these cells has no apparent effect on 5mC staining. The reduced 5mC staining is not due to blocked access of the antibody by the overexpressed proteins as overexpression of a mutant Tet1 or Tet2 does not affect 5mC staining (Fig. S2a). These results suggest that the enzymatic activity of Tet1 is usually conserved from human to mouse and that both mouse Tet1 and Tet2 can reduce global 5mC levels when overexpressed in a manner that requires the presence of an intact Fe(II) binding site. Since human Tet1 is usually capable of converting 5mC to 5hmCin vitro6, we asked whether decreased 5mC staining in cells overexpressing Tet proteins is usually concomitant with generation of 5hmC. To this end, WHI-P97 we characterized a commercial 5hmC antibody by dot blot and exhibited that this antibody is usually 5hmC specific (Figs. S3ac). Competition assays further exhibited its specificity in immunostaining WHI-P97 (Fig. S3d). As expected, enforced expression of wild-type Tet1 and Tet2, but not their catalytic mutants, resulted in the generation of 5hmC (Fig. 1a), indicating that Tet proteins can convert 5mC to 5hmCin vivo(also seeFig. S3b). Interestingly, while enforced expression of Tet3 does not cause an obvious decrease in 5mC staining (Fig. S2), it does result in the generation of 5hmC (Fig. 1a), suggesting that Tet3 is indeed enzymatically activein vivo. == Physique 1. The Tet family proteins convert 5mC of DNA to 5hmC. == To evaluate the enzymatic activityin vitro, we purified Flag-tagged Tet catalytic domains as well as their corresponding catalytic mutants from baculovirus infected SF9 cells (Fig. S4a). Incubation of the purified proteins with methylated DNA substrates followed by restriction digestion, end labeling, and TLC assays (Fig. S4b, c)7demonstrated that wild-type recombinant Tet proteins, but not their corresponding catalytic mutants, were able to generate a radioactive product that co-migrated with 5hmC on TLC plates (compare lanes 4, 6, and 8 with lanes 5, 7, and 9). Given that enforced expression of Tet Mouse monoclonal to SMN1 proteins resulted in the generation of 5hmC (Fig. 1a), we conclude that all the mouse Tet proteins have the capacity to convert 5mC to 5hmC. To understand the biological function of the Tet proteins, we examined their expression patterns in various mouse tissues and cell types by RT-qPCR. Results shown inFig. S5revealed that both Tet1 and Tet2, but not Tet3, are expressed in ES cells. This unique expression pattern prompted us to inquire whether Tet1 or Tet2 posesses an important function in ES cells. Thus we generated two impartial lentiviral knockdown shRNAs for each of the Tet proteins and verified the knockdown efficiency by RT-qPCR (Fig. S6a). Interestingly, knockdown of Tet1, but not Tet2 or Tet3, resulted in morphological abnormality as well as decreased alkaline phosphatase (AP) activity (Fig. S6b). In addition to morphological changes, knockdown of Tet1 also resulted in a reduced ES cell growth rate (Fig. 2a), which is not due to a.