Fig 1: TET1 and TET2 Positively Correlate with the TSC State(A) qRT-PCR analysis of Tet1,2,3 mRNA expression in TSCs cultured in stem cell media (SCM) or differentiation media (DM) over 3 days. Data are normalized against housekeeping genes Sdha and Dynein, and are displayed as mean ± SEM; *p < 0.05, **p < 0.01 (ANOVA with Holm-Bonferroni post-hoc test); n = 3 independent replicates each.(B) Immunofluorescence (IF) staining for CDX2, TET1, and TET2 in TSCs cultured in SCM and DM. Scale bar, 100 µm.(C) Quantification of total mean cell fluorescence of (B). *p < 0.05, **p < 0.01; ***p < 0.001 (unpaired, one-sided t test). Measurements are of =100 cells each.(D) IF staining for TET1 in independent vector (v) control and Tet1 KO and Tet1/2 DKO clones. Scale bar, 100 µm.(E) Western blot for TET2 on vector control, Tet1 KO and Tet1/2 DKO TSCs. Tubulin was used as loading control.(F) Phase contrast images of vector control, Tet1 KO and Tet1/2 DKO clones grown in SCM. Yellow dotted lines indicate enlarged giant cell-like cells. Scale bar, 100 µm.(G) qRT-PCR analysis of TSCs and differentiation markers in vector control (set to 1) and mutant clones. SpTr, spongiotrophoblast; TGC, trophoblast giant cells. Data are mean ± SEM; *p < 0.05 (ANOVA with Holm-Bonferroni post-hoc test); n = 4 clones as independent replicates each.(H) Analysis of proliferation rates over a 4-day period. ****p < 0.0001 (two-way ANOVA with Holm-Sidak's multiple comparisons test); n = 5 independent replicates each.See also Figures S1–S3.
Fig 2: Oocytes impact the expression of Dnmt1 and Tet2 in granulosa cells and cumulus cells.a The effect of coculture with denuded oocytes on the expression of Dnmt1 and Tet2 in granulosa cells. Granulosa cells were collected from ovaries of 3-week-old mice after treatment with eCG for 6 h and were cultured in medium supplemented with FSH plus T in the presence of 1% FBS. GV-stage oocytes were collected from COCs. None or 90 GV-stage oocytes/150 µl were cocultured with granulosa cells. Values are represented as the mean ± SEM (n = 3 biological replicates). Levels of mRNA were normalized to that of L19. FSH + T. Granulosa cells were cultured with FSH (100 ng/ml) and testosterone (10 ng/ml) for 48 h. RA, RA (1 µM) was added to medium in which granulosa cells were cultured with 90 GV-stage oocytes. Significant differences were observed compared with cells treated with no oocytes or cells treated with 90 oocytes per 150 µl of medium (p < 0.05). b The effects of GDF9 on the expression of Dnmt1 and Tet2 in granulosa cells. Granulosa cells were collected from ovaries of 3-week-old mice after treatment with eCG for 6 h and were cultured in each treatment group in the presence of 1% (v/v) FBS. control (C); granulosa cells were cultured without any hormones for 48 h. FSH + T, granulosa cells were cultured with FSH (100 ng/ml) and testosterone (10 ng/ml) for 48 h. FSH + T + GDF9, granulosa cells were cultured with FSH + T and mouse recombinant GDF9 (100 ng/ml). FSH + T + GDF9 + RA, Granulosa cells were cultured with FSH + T + GDF9 and RA (1 µM) for 48 h. Levels of mRNA were normalized to that of L19. Values are represented as the mean ± SEM (n = 6 biological replicates). Significant differences were observed among the treatment groups (p < 0.05).
Fig 3: Tet2CD rescues Ig? expression, promotes demethylation and accessibility of 3’E? and distal E? enhancers.(A–B) The enzymatic activity of Tet2 is required to promote Irf4 expression and Ig? germline transcription. BCR-Abl-transformed Tet2/3 DKO pre-B cells were transduced with retrovirus expressing Tet2CD, the corresponding Tet2CD HxD catalytic inactive mutant or empty vector (mock) together with a blasticidin resistance gene. Seven days post blasticidin selection, expression of Tet2 (A), C?, Ig? germline transcripts and Irf4 was determined by real time PCR (B). Error bars represent the standard deviation of three independent experiments. *, p<0.01 in Student’s t test. (C) The enzymatic activity of Tet2 is required to promote ‘demethylation’ (loss of 5mC + 5 hmC) at Ig? enhancers. BCR-Abl-transformed Tet2/3 DKO pre-B cells were transduced as in (B), and the DNA modification status of the 3’ and distal E? enhancers was analyzed by bisulfite sequencing. Error bars indicate the range of values obtained in two independent experiments. (D) Tet2 associates with the Igk enhancers. WT, Tet2/3 DKO, and DKO transduced with Tet2CD or Tet2CD HxD Abl-transformed pre-B cells were used as input and Tet2 binding to indicated regions was detected by ChIP-qPCR. The signal was normalized to corresponding samples immunoprecipitated with Ig. (E) Tet2CD restores chromatin accessibility at the Ig? enhancers in Tet2/3 DKO cells. Tet2/3 DKO pro-B cells were transduced with retrovirus containing Tet2CD-IRES-Thy1.1 (Tet2CD), Tet2CD HxD mutant-IRES-Thy1.1 (Tet2CD HxD) or empty vector (mock). Thy1.1+ cells were sorted and chromatin accessibility was analyzed through ATAC-seq. (F) Tet2 requires its enzymatic activity in promoting chromatin accessibility. TET-regulated accessible regions (WT>DKO DARs) were identified as in Figure 4A, and chromatin accessibility of these regions were plotted in WT pro-B cells, or Tet2/3 DKO pro-B cells reconstituted with Tet2CD, Tet2CD HxD or empty vector (mock). Histogram shown is distribution of ATAC-seq reads (normalized to 10 million reads depth) over 750 bp downstream or upstream of the peak center. Verification of Tet2 expression by immunoloblotting is shown in Figure 8A. (G) Tet2CD restores V?J recombination in Tet2/3 DKO pre-B cells. Abl-transformed pre-B cells transduced with Tet2CD or Tet2CD HxD were treated with Gleevec to induce V?J rearrangement and analyzed as in Figure 2C by ligation-mediated-PCR.DOI: http://dx.doi.org/10.7554/eLife.18290.017
Fig 4: Lineage-specific transcription factors cooperate with TET protein in inducing Ig? enhancer demethylation.(A) Depletion of E2A increases DNA modification (5mC + 5 hmC) at the distal E? enhancer. WT BCR-Abl-transformed pre-B cells were transduced with two independent shRNAs against E2A or scrambled shRNA as a control. Methylation of 3’E? and dE? was determined by bisulfite sequencing in the control or E2A-depleted cells. E2A knockdown is verified by immunoblotting (Figure 6—figure supplement 1A). Data are the summary of two independent experiments. Error bars show the range of duplicates. (B) Depletion of PU.1 increases DNA modification (5mC + 5 hmC) at the distal E? enhancer. As in (A), methylation of 3’E? and dE? enhancers was analyzed by bisulfite sequencing after PU.1 knockdown. PU.1 knockdown is verified by immunoblotting (Figure 6—figure supplement 1B). Error bars show the range of duplicates. (C) Tet2 directly interacts with PU.1 and E2A. E2A and PU.1 were immunoprecipitated from WT or Tet2/3 DKO BCR-Abl pre-B cell nuclear extract in the presence of ethidium bromide and benzonase to prevent indirect 'interaction' via DNA. Co-immunoprecipitated proteins were probed with anti-Tet2. Input loaded was 2.5%. Note that a different secondary antibody was used for PU.1 and E2A to avoid the interference of IgH and IgL and thus weaker signal. (D–E) E2A and PU.1 facilitate the binding of Tet2 to dEk. E2A (D) and PU.1 (E) were depleted by shRNAs as in (A) and (B) and the association of Tet2 to Ig? enhancers or control regions (Gadph and Ig? introns) were assessed by ChIP-qPCR. Data are representative for at least two experiments.DOI: http://dx.doi.org/10.7554/eLife.18290.014
Fig 5: Loss-of-function effect of Tet2 and Tet3 on cell proliferation, survival, and differentiation AmRNA expression of Tet1, Tet2, and Tet3 in BMMs stimulated with RANKL for 0 and 2 days (RNA-seq analysis; n = 3 biological replicates). Data denote mean ± s.e.m.BmRNA expression of Tet2 and Tet3 in control- and Tet2Rank –/–; Tet3aRank +/– -derived BMMs cultured in the absence and presence of RANKL for 2 days (quantitative RT–PCR analysis; n = 3 biological replicates). Data denote mean ± s.e.m.CProtein expression in control- and Tet2Rank –/–; Tet3aRank +/– -derived BMMs cultured in the absence and presence of RANKL for 2 days. Arrows and arrowhead indicate Tet2 and Tet3 proteins, respectively.D, EPercentage of BrdU-labeled CD11b+ BMMs (D) and Annexin V+ CD11b+ BMMs (E) derived from control- and Tet2Rank –/–; Tet3aRank +/– mice. Data denote mean ± s.e.m. NS, not significant (n = 5 (D) and n = 3 (E) biological replicates; t-test).
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