Fig 2: TET2 catalytic activity is required to fine tune Th-POK expression in cooperation with TET3 in CD4 SP cells and iNKT cells. (A) Representative histogram depicting Th-POK expression, assessed by flow cytometry, in control (black), Tet2/3 DKO (purple), Tet2 CD Tet3 KO (cyan) in CD4 SP cells. (B) Median fluorescence intensity (MFI) of Th-POK expression in control (in black), Tet2/3 DKO (in purple) and Tet2 CD Tet3 KO (in cyan) CD4 SP cells. Each dot represents a mouse. 5 independent experiments were performed. Male and female mice for each genotype were analyzed. Control n=10, Tet2/3 DKO n=6, Tet2 CD Tet3 KO n=7 **** (p < 0.0001), ns: non-significant, unpaired t test. Horizontal lines indicate the mean (s.e.m.). (C) Representative histogram depicting Th-POK expression, assessed by flow cytometry, in control (black), Tet2/3 DKO (purple), Tet2 CD Tet3 KO (cyan) in iNKT cells. (D) Median fluorescence intensity (MFI) of Th-POK expression in control (in black), Tet2/3 DKO (in purple) and Tet2 CD Tet3 KO (in cyan) iNKT cells. Each dot represents a mouse. 5 independent experiments were performed. Male and female mice for each genotype were analyzed. Control n=7, Tet2/3 DKO n = 7, Tet2 CD Tet3 KO n = 7 **** (p < 0.0001), ns, non-significant, unpaired t test. Horizontal lines indicate the mean (s.e.m.).

Fig 3: Kinetic changes in the expression levels of genes involved in epigenetic regulatory mechanisms in mouse granulosa cells during follicular development.a The expression levels of epigenetic-related genes in granulosa cells. Levels of mRNA were normalized to that of L19. Data represents the mean ± SEM (n = 3 biological replicates). Significant differences were observed between SF and eCG at 0 h (p < 0.05). *Significant differences were induced by eCG injection compared with before eCG injection (0 h) (p < 0.05). SF, secondary follicles. b, c Temporal changes of DNMT1 (b) and TET2 (c) in granulosa cells of secondary follicles (SF) with multilayered granulosa cells and antral follicles before or after eCG stimulation. β-Actin was used as a loading control. The results are representative of three independent experiments. Immunoblots shown are representative of three independent experiments. In each experiment, the different protein samples were used (n = 3 biological replicates). Quantitative expression of DNMT1 and TET2 relative to the expression of β-actin (control), as determined by western blotting. The SF value was set as 1 and the data are expressed as fold induction. The values are the mean ± SEM (n = 3 biological replicates). Significant differences were observed between SF and eCG at 0 h (p < 0.05). *Significant differences were induced by eCG injection compared with before eCG injection (0 h) (p < 0.05). Full-length blotting images of DNMT1, TET2, and β-actin are shown in Supplementary Fig. 8. d The localization of DNMT1, TET2, and PCNA in secondary follicles (SFs) and antral follicles (AFs) of 3-week-old immature mice or preovulatory follicles (PFs) of ovaries collected 48 h after eCG injection. DNMT1 and TET2 were visualized with Cy3, PCNA was visualized with FITC, and nuclei were stained with DAPI in the ovaries of 3-week-old mice treated with or without eCG. All of different length scale bars show 50 μm.

Fig 4: Loss of TET2 leads to dysregulated lobuloalveolar development and impaired luminal lineage commitment.a Immunoblot showing TET2 protein expression in WT, HET, and KO mammary tissues. b, c Whole mount, H&E, and Masson’s Trichrome staining of mammary tissues from 7-week-old WT, HET, and KO virgin female mice. Arrow indicating light blue staining of collagen enriched fibrosis regions (scale bar: 50 µm). d, e H&E staining of mammary tissues from WT, HET, and KO pregnant mice (day 18.5, scale bar: 50 µm) and lactating mice (day 10, scale bar: 200 µm). Arrows indicating lipid droplets in pregnant mouse tissues (d) and milk in lactating mouse tissues (e), respectively. f Immunoblots showing expression of ß-casein and estrogen receptor-a (ERa). g Flow cytometry analysis showing the percentage of the basal MaSC-enriched cell population (MaSCe, Lin-CD24+CD29hi, indicated by a red circle) and luminal cell population (Lum, Lin-CD24+CD29lo, indicated by a green circle), and h the percentage of basal MaSC-enriched cell population (MaSCe, Lin-CD24+CD29hiCD61hi, indicated by a red circle), luminal progenitor cell population (LP, Lin-CD24+CD29loCD61hi, indicated by a black circle), and mature luminal cell population (ML, Lin-CD24+CD29loCD61lo, indicated by a green circle) isolated from n = 3 biologically independent mammary epithelial cell samples of 7-week-old- WT, HET, and KO female mice (n = 3 animals/group). i Representative image and the number of primary mammospheres (scale bar: 50 µm) generated from WT, HET, and KO mammary epithelial cells per 1000 seeding cells (n = 3 independent experiments), and j the number of the serially passaged spheres generated from WT and KO cells (n = 3 independent experiments). Data were presented as mean ± SD. p-values were determined by two-sided Student’s t-test; asterisk indicates p < 0.05, double asterisks indicate p < 0.01 (compared to WT). k, l Representative confocal immunofluorescence images showing co-staining of CK8 with CK14 or MUC1 with SMA in mammary glands from 7-week-old- WT and KO female mice (scale bar: 50 µm). Source data are provided as a source data file.

Fig 5: Tet2 and Tet3 are upregulated during T cell lineage specification and are required for optimal Th-POK expression. (A) Gene expression of Tet2 (left) and Tet3 (right) in DP cells, CD4 SP cells and CD8 cells. (Source: Immunological Genome project, Immgen). (B) MA plot depicting log2 fold-change of gene expression in Tet2/3 DKO CD4 SP cells compared to control (wild type) CD4 SP cells. 561 were identified to be upregulated (padj < 0.01, in green) in Tet2/3 DKO SP cells, whereas 255 were downregulated (padj < 0.01, in red). Genes of interest are indicated. n=3 male control (wild type) and 2 male Tet2/3 DKO mice were used. (C) Venn diagram indicating the intersecting genes that are differentially expressed in Tet2/3 DKO iNKT cells compared to wild type iNKT cells (8) and Tet2/3 DKO CD4 SP cells compared to wild type CD4 SP cells. (D) Heatmap indicating the log2 fold-change of the overlapping genes identified in (C). The scale for evaluating differential gene expression is depicted. (E) Representative histogram comparing the protein expression levels of Th-POK in wild type (gray) and Tet2/3 DKO (purple) CD4 SP cells evaluated by flow cytometry. (F) Median Fluorescence Intensity (MFI) of Th-POK expression in CD4 SP cells as evaluated in (E). Each dot represents a mouse. WT n=10 (in black), Tet2/3 DKO n=6 (in purple). For each genotype male and female mice were analyzed with comparable findings. (G) Representative histogram comparing the protein expression levels of Th-POK in wild type (gray) and Tet2/3 DKO (purple) iNKT cells evaluated by flow cytometry. (H) Median Fluorescence Intensity (MFI) of Th-POK expression in iNKT cells as evaluated in (G). Each dot represents a mouse. WT n=7 (in black), Tet2/3 DKO n=7 (in purple). For each genotype male and female mice were analyzed with comparable findings. 5 independent experiments were performed. **** (p < 0.001), unpaired t test. Horizontal lines indicate the mean (s.e.m.).