Figure 3. Nanog is a direct Tet1 target and Tet1 knockdown phenotypes can be partially rescued by expression of exogenous Nanog.

1. ChIP analysis demonstrates that Nanog is a direct target of Tet1. Top panel is a diagram of the Nanog gene with the four amplicons indicated. The proximal and distal T-DMR as well as regions I and II are defined as that in a previous publication ⁸. The numbers in the diagram refer to the gene coordinates. Bottom left panel is Tet1 enrichment in control and Tet1 knockdown cells relative to IgG controls. Bottom right panel is 5mC enrichment in Tet1 knockdown relative to control knockdown. Results presented are the average of three independent experiments with s.d.
2. Bisulfite sequencing results indicate that Tet1 knockdown in ES cells results in an increase in DNA methylation at the proximal T-DMR (Fig. 3a) of Nanog promoter. Open circles indicate unmethylated CpG dinucleotides, closed circles indicate methylated CpGs.
3. RT-qPCR analysis demonstrates that knockdown of Tet1 in wild-type J1 ES cells, but not in the DNMT TKO J1 ES cells, results in Nanog down-regulation. Error bars represent s.d. of two independent experiments.
4. AP staining of mouse ES cells after puromycin selection of cells infected with lentiviruses expressing control and Tet1 knockdown shRNAs with or without co-expression of Nanog.
5. The self-renewal defects associated with Tet1 knockdown can be partially rescued by expression of exogenous Nanog. Self-renewal assay was performed as that described in Fig. 2b.
6. RT-qPCR analysis demonstrates that expression of exogenous Nanog suppressed up-regulation of differentiation genes (Cdx2, GATA6) caused by Tet1 knockdown. Data shown is the average of three independent experiments with error bars (s.e.m).