Fig 2: Dppa3 facilitates the chromatin conversion to resembling that of two-cell embryos. (A) Average density of ATAC-Seq reads around TSSs (±5 kb) for the 2C related gene set. The red line represents the signal distribution of ATAC in 2C-specific genes set in Con group, the blue line represents the signal distribution of ATAC in 2C-specific genes set in the Dppa3 OE group. (B) Expression of Zscan4b, Zscan4c, Zscan4d, and Zscan4f in Dppa3 OE ESCs compared to those in Con ESCs. (C,D) Genome browser view of the distribution of ATAC-Seq signals at the 2C-related genes in Dppa3 OE and Con ESCs. Shown are Zscan4 (at Chr7) (C) and MERVL (at Chr3 and Chr9) (D). (E) Immunofluorescence of H3K4me3 (red) and Dppa3 (green) in Dppa3 OE, Dppa3 KD, and control ESCs. Scale bar = 10 µm. (F) Quantification of numbers of H3K4me3 foci in Dppa3 OE and Con ES cells (n = 4; *p < 0.05; each dot represents the fluorescence intensity in each cell in one replicate experiment). (G) Western blot analysis of histone modifications in Dppa3 OE, Dppa3 KD, and control ESCs.

Fig 3: Dppa3 overexpression leads to DNA demethylation in ESCs. (A) Immunofluorescence staining for 5mC (red) and Dppa3 (green) in Dppa3 OE and Con ESCs. Right panel: quantification of 5mC foci in Dppa3 OE and Con ESCs. Scale bar represents 10 µm. Data are presented as mean ± SEM (n = 3, ** p < 0.01). (B) Hierarchical clustering of differentially methylated sites between Dppa3 OE and control ESCs. Heat map displays methylation levels for whole genomes as measured by Reduced Representation Bisulfite Sequencing separated into groups by genomic features. Each row indicates a CpG site and the color scale represents the methylation level. CGI, CpG island; TSS, transcription start site; TES, terminate sequence. (C) DMRs methylation levels as measured by RRBS in Dppa3 OE and control ESCs. Horizontal bars represent the median values. Inside is the boxplot. Violin flanking represents the number of genes at this methylation level. (D) The genomic distribution of hypomethylated DMRs in Dppa3 OE ESCs. (E) Overlap between hypo-DMR-associated promoter genes (DMPs) and 2C-like genes.

Fig 4: Knockdown of Dppa3 impair the 2CLCs conversion in ESCs. (A) Confirmation of the expression level of Dppa3 in transient Dppa3 KD and control ESCs by quantitative PCR (n = 2, *p < 0.05). (B,C) The transient knockdown of Dppa3 decreases the Zscan4 protein level analyzed by western blot in D3 ESCs (B) and J1 ESCs (C). (D) The transient Dppa3 KD reduces the Zscan4 positive cells by immunofluorescence staining of Zscan4 (green) and Dppa3 (red) in ESCs. Scale bar represents 100 μm. Right: The number of Zscan4 positive cells was counted (n = 4; *p < 0.05; each dot corresponds to one replicate experiment, in which 200 cells were counted). (E) FACS profiles of Zscan4+ ESCs in transient Dppa3 KD ESCs. (F) qRT-PCR analysis of selected 2C-specific genes and retroviral elements’ expression in transient Dppa3 KD ESCs. Data are presented as mean ± SEM (n = 3, *p < 0.05; **p < 0.01).

Fig 5: Overexpression of Dppa3 activates a 2C-like gene expression. (A) Morphology of ESCs with an ectopic expression of Dppa3. Scale bars represent 100 μm. (B) Relative expression level of Dppa3 in Dppa3 OE and control (Con) ESCs. (C) Western blot analysis of Dppa3 protein level in Dppa3 OE and Con ESCs. Right: Quantification of relative Dppa3 protein levels normalized to Tubulin by ImageJ software (n = 3; *p < 0.05). (D) Scatter plot showing differentially expressed genes following the ectopic expression of Dppa3 in ESCs. Representative 2C genes that are upregulated in Dppa3 OE ESCs are marked in red. Two-tailed Student’s t-tests were used to derive the p values. (E) GSEA indicating that upregulated genes in Dppa3 OE ESCs were highly enriched in the two-cell embryo gene set. (F) qRT-PCR analysis of selected 2C-specific genes and retroviral elements expression in transient Dppa3 OE ESCs. Data are presented as mean ± SEM (n = 3, *p < 0.05; **p < 0.01). (G,H) Western blot analysis of Zscan4 protein level in stable Dppa3 overexpressed D3 (G) and J1 (H) ES cell lines. Right: Quantification of relative Zscan4 protein levels normalized to H3 by ImageJ software (n = 3; *p < 0.05). (I) Dppa3 overexpression increases Zscan4 positive cells by immunofluorescence staining of Zscan4 (red) and Dppa3 (green) in stable Dppa3 overexpressed D3 ESCs. Scale bar represents 10 μm. Below: Quantification of Zscan4 positive cells in Dppa3 OE and control (Con) ESCs (n = 4; *p < 0.05; each dot corresponds to one replicate experiment, in which 200 cells were counted). (J) FACS profiles of Zscan4+ ESCs population in stable Dppa3 OE ESCs.