Generation of maternal-zygotic KO embryos.

A. Schemes of mKO2-labeled Oct4 KO (Oct4mKO2) and Oct4 flox alleles. B. Schemes of EGFP-labeled Sox2 KO (Sox2EGFP) and Sox2 flox alleles. C. Mating strategy for Oct4 Ctrl and KO embryos. D. Mating strategy for Sox2 Ctrl and KO embryos. E. Immunostaining with embryos separated based on the mKO2 fluorescence. F. Immunostaining with embryos separated based on the EGFP fluorescence. N, the number of embryos. Scalebar, 20 μm.

Oct4 and Sox2 regulate chromatin landscape and transcriptome in the ICM.

A. PCA plot of all the identified ATAC-seq peaks. B. Number of differentially accessible ATAC- seq peaks in KO vs Ctrl samples. Cutoff, adjusted p-value <0.05. C. k-means clustering of all the significantly differential ATAC-seq peaks in KO vs Ctrl in Figure 2B. The heatmap is sorted by clusters, motifs and TSS. Peaks located within 100 bp from TSS were considered as TSS peaks. OCT-SOX, OCT or SOX motifs indicate that the peak contains the canonical OCT- SOX, OCT or SOX motif, respectively, while OCT&SOX motif indicates that separate OCT and SOX motifs were discovered in one peak. Cutoff, adjusted p-value <0.05. D. Number of differentially expressed genes in KO vs Ctrl samples. Cutoff, adjusted p-value <0.05 and log2 fold change ≥1. E. GSEA shows the correlation between significantly changed ATAC-seq peaks and the transcription of genes located within 10 kb of the peaks in Oct4- or Sox2-KO late ICMs. NES, normalized enrichment score. F. Examples of down- and up-regulated genes. The underlined numbers represent the adjusted p values. G. The ATAC-seq profiles surrounding the genes in E. Boxes mark the differentially accessible peaks, and red boxes specifically mark those with the OCT-SOX motif.

Oct4 and Sox2 activate EPI-specific genes and suppress TE-specific genes in ICM.

A. A scatter plot shows the log2 fold change of ATAC-seq signals in the Oct4-KO and Sox2-KO late ICM. Colored dots represent the significantly changed peaks. Cutoff, adjusted p-value <0.05. B. GREAT ontology enrichment analysis of the significantly changed peaks shared in the Oct4-KO and Sox2-KO late ICM. Red terms are related to the pluripotency and preimplantation embryonic development, and green ones are related to the development of embryonic and extraembryonic lineages in the post-implantation embryos. C. A scatter plot shows the log2 fold changes of RNA-seq signals in the Oct4-KO and Sox2-KO late ICM. Cutoff, adjusted p-value <0.05 and log2 fold change ≥1. D. Violin plots of RNA-seq rlog values for Nanog, Esrrb and Klf4 in the embryos. The underlined numbers represent the adjusted p values. E. Immunostaining of Pecam1 in the late blastocysts (E2.5+2 days). Scalebar, 50 μm. F. The accessibility of putative enhancers around Pecam1 in ICMs. Boxes mark the decreased peaks and red box marks the peak with the OCT-SOX motif.

Oct4 and Sox2 activate OCT-SOX enhancers cooperatively and independently in ICM.

A. Motif enrichment analysis of significantly changed ATAC-seq peaks in the Oct4- KO and Sox2-KO ICMs. B. Occurrence of the motifs in the ranked peaks. The decreased enhancers were ranked by the fold reduction. The cumulative percentages of peaks containing at least one sequence for a given motif are plotted against ranks of peaks. C. Average profiles of the 8,993 OCT-SOX peaks across all the Ctrl and KO ICM samples. D. Scatter plots show the log2 fold change of 8,993 OCT-SOX peaks in the Oct4- and Sox2-KO late ICMs. E. The profiles of ATAC-seq in early embryos, Sox2 CUT&RUN in E4.5 EPI (Li et al, 2023) and Oct4 and Sox2 ChIP-seq in ESCs (Marson et al, 2008) around the known OCT-SOX enhancers of Klf4 and Dppa3. The red boxes mark the OCT-SOX enhancers.

Oct4 and Sox2 promote the developmental trajectory from the morula to the ICM.

A. Bar graphs show the dynamics of the decreased peaks (left) and genes (right) in Oct4- and Sox2-KO early ICMs from the morula to the early ICM. B&D. Alluvial plots show the dynamics of chromatin accessibility (B) and transcriptome (D) from morula to late ICM in the Ctrl embryos. Green, grey and red lines represent the decreased, unchanged and increased peaks/genes, respectively. In D, only genes significantly up- or down-regulated in both Oct4 Ctrl and Sox2 Ctrl embryos were considered as up- or down-regulated genes, while all the rest were considered as unchanged genes. C&E. GSEA plots show the enrichment of the 21,731 ATAC-seq peaks (C) and 1,115 genes (E) in Oct4-KO and Sox2-KO early ICMs. NES, normalized enrichment score. F. The bar chart illustrates the GSEA Wikipathway enrichment in WebGestalt. The log2 foldchange values of the 1,115 upregulated genes (Figure 5D) in Sox2 KO vs Ctrl early ICM were used in this analysis. FDR≤0.05. FC, foldchange. G. GSEA enrichment plot of the term PluriNetWork in Figure 5F. NES, normalized enrichment score. H. Upper panel: IGV tracks displaying ATAC-seq and Sox2 CUT&RUN profiles (Li et al, 2023), along with ChIP-seq profiles of Oct4 and Sox2 in ESCs (Marson et al, 2008), centered around the genomic locus of Il6st. Red box mark the OCT-SOX enhancer. Lower panel: violin plot showing the rlog values of Il6st. The underlined numbers represent the adjusted p values. I. Model of the activation of pluripotency-related genes in the early embryos.

Validation of the transgenic embryos (related to Figure 1).

A. The mKO2-labeled Oct4-KO embryos were identified under fluorescent microscopy at E2.5+1 day. B. The EGFP-labeled Sox2-KO embryos were identified under florescent microscopy at E2.5+2 days.

Validation of the effect of MEK inhibitor (related to Figure 1).

A. The scheme of MEKi (PD0325901, 1 μM) treatment. B. Immunostaining of Sox17 and Nanog in the late blastocysts treated with or without MEKi. C. The number of Sox17+ or Nanog+ cells in each blastocyst.

Quality check of the low-input ATAC-seq (related to Figure 2).

A. The location of ATAC-seq peaks relative to TSSs. The number in each bar represents the number of peaks in each group. B&C. ATAC-seq (B) and Sox2 CUT&RUN (C) profiles (Li et al, 2023) over the decreased, unchanged and increased ATAC-seq peaks identified in our Sox2-KO late ICMs. D. ChIP-seq profiles of Oct4, Sox2, Nanog and H3K27ac in ESCs (Marson et al, 2008) over the decreased, unchanged and increased ATAC-seq peaks in our Sox2-KO late ICMs. To exclude spurious peaks, only strong unchanged peaks were used in the analysis from C to E. E. IGV tracks displaying ATAC-seq and Sox2 CUT&RUN profiles (Li et al, 2023) in late ICMs, along with ChIP-seq profiles of Oct4, Sox2, Nanog and H3K27ac in ESCs (Marson et al, 2008; Whyte et al, 2012), centered around the genomic locus of Sap30. Arrows mark the decreased ATAC-seq peaks.

Quality check of the low-input RNA-seq (related to Figure 2).

A. PCA plot illustrating the distribution of RNA-seq samples with the indicated stages and genotypes. B-D. GSEA plots of down- and up-regulated genes identified in our Oct4-KO early ICM (B), Oct4-KO late ICM (C) and Sox2-KO early ICM (D) in the scRNA-seq dataset of ICM samples without MEKi (Stirparo et al., 2021; Li et al., 2023). NES, normalized enrichment score.

The correlation between chromatin accessibility and the transcription activity of surrounding genes (related to Figure 2).

Heatmap of GSEA enrichment Oct4-KO (A) and Sox2-KO (B) samples. The analysis utilized genes with TSS falling within a 10 kb proximity to the decreased or increased ATAC-seq peaks. The number in parentheses denotes the count of genes with TSS located within 10 kb of the significantly differential peaks in each module. NES, normalized enrichment score.

GREAT Biological Process enrichment analysis of ATAC-seq peaks specifically decreased in the Oct4-KO or Sox2-KO late ICMs (related to Figure 3).

Terms highlighted in red are related to the pluripotency and preimplantation embryonic development. Example genes of associated peaks are listed.

Motif enrichment analysis of significantly changed ATAC-seq peaks that are distal to TSSs (related to Figure 4).

The values are –log10(p-value).

MA plots show the log2 fold change of the 8,993 OCT-SOX peaks in Oct4- and Sox2-KO ICMs (related to Figure 4).

Black and red dots represent the unchanged and significantly changed peaks, respectively. Grey lines are the regression curves of the 8,993 OCT-SOX peaks.

The dynamics of chromatin accessibility and transcriptome from morula to ICM (related to Figure 5).

A GREAT analysis was conducted on the most prominently elevated ATAC-seq peaks in Ctrl early ICM vs Ctrl morula. A log2 fold change cutoff of >3 was applied due to the limits of GREAT analysis with fewer than 10,000 peaks. B. Motif enrichment analysis with PScan of the increased ATAC-seq peaks in Ctrl early ICM vs Ctrl morula. Cutoff, log2 fold change >3. C. Expression of Sox2, Oct4, Nanog, Esrrb and Klf4 in morulae and ICMs (related to Figure 5). D. Bar chart illustrating the GSEA Wikipathway enrichment in WebGestalt. The log2 foldchange values of the 1,115 upregulated genes (Figure 5D) in Oct4 KO vs Ctrl early ICM were used in this analysis. FDR >0.05. E. Venn diagram showing the overlap between genes upregulated in early ICMs and those downregulated in Oct4-KO or Sox2-KO early ICMs. Pathway over-representation analysis was conducted on the genes upregulated in Ctrl early ICMs vs Ctrl Morula and downregulated in Oct4 KO vs Ctrl early ICM.