Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse early embryos

Reviewer #1 (Public review):

Summary:

Numerous mechanism and structural studies reported the cooperative role of Oct4 and Sox2 during the establishment of pluripotency during reprogramming. Due to the difficulty in sample collection and RNA-seq with low-number cells, the precise mechanisms remain in early embryos. This manuscript reported the role of OCT4 and SOX2 in mouse early embryos using knockout models with low-input ATAC-seq and RNA-seq. Compared to the control, chromatin accessibility and transcriptome were affected when Oct4 and Sox2 were deleted in early ICM. Specifically, decreased ATAC-seq peaks showed enrichment of Motifs of TF such as OCT, SOX, and OCT-SOX, indicating their importance during early development. Moreover, by deep analysis of ATAC-seq and RNA-seq data, they found Oct4 and Sox2 target enhancer to activate their downstream genes. In addition, they also uncovered the role of OS during development from the morula to ICM, which provided the scientific community with a more comprehensive understanding.

Strengths:

On the whole, the manuscript is innovative, and the conclusions of this paper are mostly well supported by data, however, there are some issues that need to be addressed.

Weaknesses:

Major Points:

(1) In Figure 1, a more detailed description of the knockout strategy should be provided to clarify itself. The knockout strategy in Fig1 is somewhat obscure, such as how is OCT4 inactivated in Oct4mKO2 heterozygotes. As shown in Figure 1, the exon of OCT4 is not deleted, and its promoter is not destroyed. Therefore, how does OCT4 inactivate to form heterozygotes?

(2) Is ZP 3-Cre expressed in the zygotes? Is there any residual protein?

(3) What motifs are enriched in the rising ATAC-seq peaks after knocking out of OCT4 and SOX2?

(4) The ordinate of Fig4c is lost.

(5) Signals of H3K4me1, H3K27ac, and so on are usually used to define enhancers, and the loci of enhancers vary greatly in different cells. In the manuscript, the authors defined ATAC-seq peaks far from the TSS as enhancers. The definition in this manuscript is not strictly an enhancer.

(6) If Oct4 and Sox2 truly activate sap 30 and Uhrf 1, what effect does interfering with both genes have on gene expression and chromatin accessibility？

Reviewer #2 (Public review):

In this manuscript, Hou et al. investigate the interplay between OCT4 and SOX2 in driving the pluripotent state during early embryonic lineage development. Using knockout (KO) embryos, the authors specifically analyze the transcriptome and chromatin state within the ICM-to-EPI developmental trajectory. They emphasize the critical role of OCT4 and the supportive function of SOX2, along with other factors, in promoting embryonic fate. Although the paper presents high-quality data, several key claims are not well-supported, and direct evidence is generally lacking.

Major Points:

(1) Although the authors claim that both maternal KO and maternal KO/zygotic hetero KO mice develop normally, the molecular changes in these groups appear overestimated. A wildtype control is recommended for a more robust comparison.

(2) The authors assert that OCT4 and SOX2 activate the pluripotent network via the OCT-SOX enhancer. However, the definition of this enhancer is based solely on proximity to TSSs, which is a rough approximation. Canonical enhancers are typically located in intronic and intergenic regions and marked by H3K4me1 or H3K27ac. Re-analyzing enhancer regions with these standards could be beneficial. Additionally, the definitions of "close to" or "near" in lines 183-184 are unclear and not defined in the legends or methods.

(3) There is no evidence that the decreased peaks/enhancers could be the direct targets of Oct4 and Sox2 throughout this manuscript. Figures 2 and 4 show only minimal peak annotations related to OCT and SOX motifs, and there is a lack of chromatin IP data. Therefore, claims about direct targets are not substantiated and should be appropriately revised.

(4) Lines 143-146 lack direct data to support the claim. Actually, the main difference in cluster I, 11 and 3, 8, 14 is whether the peak contains OCT-SOX motif. However, the reviewer cannot get any information of peaks activated by OCT4 rather than SOX2 in cluster I, 11.

Minor Points:

(1) Lines 153-159: The figure panel does not show obvious enrichment of SOX2 signals or significant differences in H3K27ac signals across clusters, thus not supporting the claim.

(2) Lines 189-190: The term "identify" is overstated for the integrative analysis of RNA-seq and ATAC-seq, which typically helps infer TF targets rather than definitively identifying them.

(3) The Discussion is lengthy and should be condensed.

Author response:

Public Reviews:

Reviewer #1 (Public review)

Summary:

Numerous mechanism and structural studies reported the cooperative role of Oct4 and Sox2 during the establishment of pluripotency during reprogramming. Due to the difficulty in sample collection and RNA-seq with low-number cells, the precise mechanisms remain in early embryos. This manuscript reported the role of OCT4 and SOX2 in mouse early embryos using knockout models with low-input ATAC-seq and RNA-seq. Compared to the control, chromatin accessibility and transcriptome were affected when Oct4 and Sox2 were deleted in early ICM. Specifically, decreased ATAC-seq peaks showed enrichment of Motifs of TF such as OCT, SOX, and OCT-SOX, indicating their importance during early development. Moreover, by deep analysis of ATAC-seq and RNA-seq data, they found Oct4 and Sox2 target enhancer to activate their downstream genes. In addition, they also uncovered the role of OS during development from the morula to ICM, which provided the scientific community with a more comprehensive understanding.

Strengths:

On the whole, the manuscript is innovative, and the conclusions of this paper are mostly well supported by data, however, there are some issues that need to be addressed.

Weaknesses:

Major Points:

(1) In Figure 1, a more detailed description of the knockout strategy should be provided to clarify itself. The knockout strategy in Fig1 is somewhat obscure, such as how is OCT4 inactivated in Oct4mKO2 heterozygotes. As shown in Figure 1, the exon of OCT4 is not deleted, and its promoter is not destroyed. Therefore, how does OCT4 inactivate to form heterozygotes?

Thank you for your kind suggestions. We will add a detailed description of the knockout strategy in the legends for Figure 1A and 1B, as shown below:

Figure 1A. Schemes of mKO2-labeled Oct4 KO (Oct4mKO2) and Oct4 flox alleles. In the Oct4mKO2 allele, a PGK-pac∆tk-P2A-mKO2-pA cassette was inserted 3.6 kb upstream of the Oct4 transcription start site (TSS) and a promoter-less FRT-SA-IRES-hph-P2A-Venus-pA cassette was inserted into Oct4 intron 1. The inclusion of a stop codon followed by three sets of polyadenylation signal sequences (pA) after the Venus cassette ensures both transcriptional and translational termination, effectively blocking the expression of Oct4 exons 2–5.

Figure 1B. Schemes of EGFP-labeled Sox2 KO (Sox2EGFP) and Sox2 flox alleles. In the Sox2EGFP allele, the 5’ untranslated region (UTR), coding sequence and a portion of the 3’ UTR of Sox2 were deleted and replaced with a PGK-EGFP-pA cassette. Notably, 1,023 bp of the Sox2 3’UTR remaine intact.

(2) Is ZP 3-Cre expressed in the zygotes? Is there any residual protein?

Thank you for the question. While we have not directly tested for ZP3-Cre expression in zygotes, the published transcriptome and proteomics data shows that ZP3 is present at both the transcriptional and protein levels in wild-type zygotes (Deng et al., Science, 2014; Gao et al., Cell Reports, 2017). This suggests that ZP3-Cre could potentially be expressed in zygotes as well.

(3) What motifs are enriched in the rising ATAC-seq peaks after knocking out of OCT4 and SOX2?

Thank you for the question. The enriched motifs in the rising ATAC-seq peaking in Oct4 KO and Sox2 KO ICMs are the GATA, TEAD, EOMES and KLF motifs, as shown in Figure 4A and Figure supplement 7.

(4) The ordinate of Fig4c is lost.

Thank you for the question. The y-axis is average normalized signals (reads per million-normalized pileup signals). We will add it in the revised version.

(5) Signals of H3K4me1, H3K27ac, and so on are usually used to define enhancers, and the loci of enhancers vary greatly in different cells. In the manuscript, the authors defined ATAC-seq peaks far from the TSS as enhancers. The definition in this manuscript is not strictly an enhancer.

Thank you for this insightful comment. We will search for and analyze published omics data on H3K4me1 and H3K27ac in early embryos or mouse embryonic stem cells to conduct this analysis.

(6) If Oct4 and Sox2 truly activate sap 30 and Uhrf 1, what effect does interfering with both genes have on gene expression and chromatin accessibility?

Thank you for the interesting question. Unfortunately, we have not conducted this specific experiment, so we do not have direct results. However, Sap30 is a key component of the mSin3A corepressor complex, while Uhrf1 regulates the establishment and maintenance of DNA methylation. Both proteins are known to function as repressors. Therefore, we hypothesize that interfering with these two genes could alleviate repression of some genes, such as trophectoderm markers, similar to what we have observed in Oct4 KO and Sox2 KO ICMs.

Reviewer #2 (Public review):

In this manuscript, Hou et al. investigate the interplay between OCT4 and SOX2 in driving the pluripotent state during early embryonic lineage development. Using knockout (KO) embryos, the authors specifically analyze the transcriptome and chromatin state within the ICM-to-EPI developmental trajectory. They emphasize the critical role of OCT4 and the supportive function of SOX2, along with other factors, in promoting embryonic fate. Although the paper presents high-quality data, several key claims are not well-supported, and direct evidence is generally lacking.

Major Points:

(1) Although the authors claim that both maternal KO and maternal KO/zygotic hetero KO mice develop normally, the molecular changes in these groups appear overestimated. A wildtype control is recommended for a more robust comparison.

Thank you for your valuable feedback. However, I’m unclear on what is meant by “the molecular changes in these groups appear overestimated.” Could the reviewer kindly provide more details or clarify which specific aspects of the molecular changes they are referring to? This would help us better address the concern.

(2) The authors assert that OCT4 and SOX2 activate the pluripotent network via the OCT-SOX enhancer. However, the definition of this enhancer is based solely on proximity to TSSs, which is a rough approximation. Canonical enhancers are typically located in intronic and intergenic regions and marked by H3K4me1 or H3K27ac. Re-analyzing enhancer regions with these standards could be beneficial. Additionally, the definitions of "close to" or "near" in lines 183-184 are unclear and not defined in the legends or methods.

Thank you for this insightful comment. We will search for and analyze published omics data on H3K4me1 and H3K27ac in early embryos or mouse embryonic stem cells to address the concern of “enhancer”.

The definition of "close to" or "near" in lines 183-184 is in the legend of Figure 2E and methods. In the GSEA analysis, Ensembl protein-coding genes with TSSs located within 10 kb of ATAC-seq peak centers were included.

(3) There is no evidence that the decreased peaks/enhancers could be the direct targets of Oct4 and Sox2 throughout this manuscript. Figures 2 and 4 show only minimal peak annotations related to OCT and SOX motifs, and there is a lack of chromatin IP data. Therefore, claims about direct targets are not substantiated and should be appropriately revised.

Thank you for the comment. In Figure Supplement 3C, we analyzed published Sox2 CUT&RUN data from E4.5 ICMs (Li et al., Science, 2023), which demonstrates that the reduced ATAC-seq peaks in our Sox2 KO ICMs are enriched with Sox2 CUT&RUN signals. This data suggests that decreased peaks/enhancers could be the direct targets of Sox2. Unfortunately, we did not to find similar published data for Oct4 in embryos.

(4) Lines 143-146 lack direct data to support the claim. Actually, the main difference in cluster 1, 11 and 3, 8, 14 is whether the peak contains OCT-SOX motif. However, the reviewer cannot get any information of peaks activated by OCT4 rather than SOX2 in cluster 1, 11.

Thank you for the comment. As the reviewer pointed out, we agree that clusters 3, 8, 14 is more enriched with OCT-SOX motifs than clusters 1/11. However, this is consistent with our observation that the accessibility of peaks in clusters 1 and 11 mainly relies on Oct4, while the accessibility of clusters 3, 8, 14 relies on both Oct4 and Sox2. Probably the word “activate” is not accurate. We will rearrange the texts as below:

“Notably, compared to the peaks dependent on Oct4 but not Sox2 (Figure 2B, clusters 1 and 11), those reliant on both Oct4 and Sox2 show greater enrichment of the OCT-SOX motif (Figure 2B, clusters 3, 8 and 14). The former group tended to be already open in the morula, while the latter group became open in the ICM. “

Minor Points:

(1) Lines 153-159: The figure panel does not show obvious enrichment of SOX2 signals or significant differences in H3K27ac signals across clusters, thus not supporting the claim.

Thank you for the comments.

Line 153-159 reference two datasets: Figure supplement 3C and 3D.

In Figure supplement 3C, the average plots above the heatmaps show that the decreased ATAC-seq peaks exhibited higher enrichment with Sox2 CUT&RUN signals compared to the increased or unchanged peaks.

Regarding Figure supplement 3D, we agree that the H3K27ac signal is only slightly more enriched on the decreased peaks than the unchanged peaks, However, it's important to note that only the top 57,512 strongest of the 142,096 unchanged peaks were included in the analysis. We excluded the weaker unchanged peaks because they are less informative. but if included, they could reduce the average H3K27ac signal for the unchanged peaks.

(2) Lines 189-190: The term "identify" is overstated for the integrative analysis of RNA-seq and ATAC-seq, which typically helps infer TF targets rather than definitively identifying them.

Thank you for the suggestion. We will replace “identify” with “infer”. The revised version is as below:

“In addition, integration of the ATAC-seq and RNA-seq data allowed us to infer previously unknown targets of Oct4 and Sox2, such as Sap30 and Uhrf1, which are essential for somatic cell reprogramming and embryonic development.”

(3) The Discussion is lengthy and should be condensed.

Thank you for the suggestion. We will shorten it.