Fig 2: Dnmt3b interacts with Smad2/3.A Venn diagram of Smad2/3 co-IP and Mass spectrometry (IP-MS) analysis. Smad2/3 IP-MS were performed in D3 EBs from WT and S4KO, including non-treated, treated with SB or AC. 16 proteins were identified in all three comparisons (S4KO vs. WT, S4KO + AC vs. S4KO + SB, S4KO vs. S4KO + SB) including Dnmt3b. B Smad2/3 immunoprecipitation in WT and S4KO D3 EBs under SB or AC addition were analyzed by western blot using Dnmt3b, pSmad2/3, Smad2/3 and Smad4 antibodies. This experiment was repeated 3 times. C Western blot analysis of Dnmt3b, pSmad2/3, Smad2/3 and Smad4 for the anti-HA immunoprecipitation in Smad2/3/4TKO cells in which HA tagged Smad2/3 (HA-S2/3) or/and Smad4 (HA-S4) were overexpressed as indicated in the figure. Cells were collected after SB or AC treatment. This experiment was repeated 3 times. D Smad2/3 immunoprecipitation of chromatin-bound proteins in D3 EBs of WT and S4KO with AC treatment were analyzed by western blot using Dnmt3b, Smad2/3, Smad4 and Otx2 antibodies. This experiment was repeated 3 times. E Representative confocal images of PLA experiments on WT, S4KO and S2/3DKO D3 EBs. This experiment was repeated 3 times. Green or gray punctate, positive PLA signal; blue, DAPI. Scale bars, 20 μm. F Overexpression of HA tagged Smad2 (HA-S2) or Smad3 (HA-S3), or/and Flag tagged Dnmt3b (Flag-3b) in 293 T cells were shown in the figure. Western blot of Flag and HA were detected after anti-Flag immunoprecipitation. This experiment was repeated 3 times. G GST pull-down assays using bacterially expressed GST-Dnmt3b or GST-GFP, and 6xHistidine-tagged Smad2 (His-S2, left) or Smad3 (His-S3, right). Elution proteins were analyzed using anti-GST and anti-His antibodies. This experiment was repeated 3 times. H His pull-down assays using bacterially expressed GST-Dnmt3b or GST, and 6xHistidine-tagged Smad2 (His-S2, left) or Smad3 (His-S3, right). Elution proteins were analyzed using anti-GST and anti-His antibodies. This experiment was repeated 3 times.

Fig 3: Epiblast occurs in the absence of Smad4.A The scheme showed the procedure of EpiLC transition from mESC (top). Briefly, mESCs turned into EpiLCs after mESCs were shifted from 2i+LIF medium to N2B27 medium with Activin A and bFGF for 6 days. This experiment was repeated 5 times. Representative images showing cell morphology changes during the transition in WT, S4KO, S2/3DKO, and S2/3DKO rescued with Smad2/3 (bottom) are presented here. Scale bar: 100 μm. B mRNA levels of pluripotent genes (Nanog, Oct4) and post-implantation epiblast genes (Fgf5, Dnmt3a/3b, T) during the induction of EpiLCs were analyzed by qRT-PCR. Data are presented as mean values +/- SD. n = 3 independent experiments. P values were calculated by two-way ANOVA test with Geisser-Greenhouse correction. C Venn diagram indicated the overlapping peaks among Smad4 ChIP-seq in WT, Smad2/3 ChIP-seq in WT and Smad2/3 ChIP-seq in S4KO. Genes adjacent to 7926 peaks were annotated, in which primed epiblast genes were marked below. D Heatmap of ChIP-seq tag densities for Smad2/3 and Smad4 were within −1.5/ + 1.5 kb genomic regions surrounding the centers of 11,080 high-confidence Smad2/3 binding sites in S4KO D3 EBs with AC treatment. SB, SB431542, a selective inhibitor of TGFβR; AC, Activin A, an available ligand for Nodal/Activin receptors. E Gene track view of the Fgf5, Dnmt3a, Dnmt3b and T loci. Smad2/3 and Smad4 ChIP-seq were performed in SB- or AC-treated D3 EBs and in D0 non-treated mESCs. Tag densities were normalized to reads per kilobase per million mapped reads (RPKM). The gene structures from RefSeq are schematically represented at the bottom.

Fig 4: Upregulation of Dnmt3b by Smad2/3 is important for epiblast formation.A Western blot analysis of Dnmt3a, Dnmt3b, Eomes, Smad2/3 and Smad4 protein levels during EB differentiation (D0, D3 and D4) in WT, S4KO, S2/3DKO, and S2/3/4TKO cells. Tubulin serves as a loading control. This experiment was repeated 3 times. B The two-step induction scheme of mESC-EpiLC-ME was presented (top). In brief, after changing into N2B27 medium with Activin A and bFGF for 6 days, EpiLCs were further cultured in N2B27 medium plus CHIR99021 for 24 h to form ME. Western blot showed the expression of Dnmt3a, Dnmt3b, Eomes, Smad4 and Smad2/3 in WT, S4KO and S2/3DKO. Tubulin serves as a loading control. This experiment was repeated 3 times. C qRT-PCR analysis of primed epiblast genes (Dnmt3b, Fgf5) and pluripotent gene Oct4 during EB differentiation (D0, D3) and EpiLC induction (D0, D4, D6) derived from WT, S2/3DKO, S4KO, and S4KO with Dnmt3b shRNA 1 or 2 cells. Data are presented as mean values +/- SD. n = 3 independent experiments. P values were calculated by two-way paired t-test. D Representative confocal microscope images of 5-mC (Red) and DAPI (blue) staining in WT, S4KO and S2/3DKO EBs. Scale bars, 20 μM. This experiment was repeated 3 times. E IGV view showed the WGBS signal profiles of Sox2 locus at ES and EpiLC stages from WT and Dnmt3b knockout (3bKO). The CpG island (purple) and differentially methylated regions (DMRs) (red) were labeled as well. MeDIP-qPCR analysis of DNA methylation levels on Sox2 DMR1 and DMR3 were carried out in D3 EBs derived from WT, S2/3DKO and S4KO. Data are presented as mean values +/- SD. n = 4 independent experiments. P values were calculated by two-sided Mann-Whitney test. F The mRNA level of Sox2 in WT, S2/3DKO, and S4KO cells before and after EB differentiation (D0, D3, D4) were measured by qRT-PCR. Data are presented as mean values +/- SD. n = 4 independent experiments. P values were calculated by two-way ANOVA test.

Fig 5: A two-step model describes the transition from naïve to primed epiblast and subsequent differentiation.In the canonical TGFβ signaling pathway, TGFβ ligands bind to TGFβ receptors. Type II receptors phosphorylate and activate Type I receptors, which then phosphorylate Smad2/3. Phosphorylated Smad2/3 form a trimer either with or without Smad4, and transfer into nucleus. First, Smad2/3 play a dominant role in orchestrating the transition from the naïve mESC to the primed epiblast stage by inducing Dnmt3b. On one hand, Dnmt3b establishes DNA methylation patterns and ensures the timely downregulation of pluripotent factors like Sox2 by methylating its DNA at DMRs. On the other hand, Dnmt3b interacts with Smad2/3 to facilitate their binding to the hypomethylated centers of promoters and enhancers of epiblast genes, likely relying on H3K36me2/3. Second, Smad2/3-Smad4 complex plays a key role during the mesendoderm differentiation from the primed EpiLCs, in which Dnmt3b is downregulated and becomes less actively involved. The Smad2/3-Smad4 complex binds to the promoters and enhancers of mesendoderm genes, with the prebound pioneer factor Foxh162.