Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells
, Nikolay D. Aksenov, Evgeny I. Bakhmet, Ekaterina V. Aleksandrova, Andrey A. Kuzmin and Alexey N. Tomilin *
Round 1
Reviewer 1 Report
The manuscript from Ermakova et al., is a very interesting and well written article, in which the authors explored the outcome of the traslocation of cis-regulatory elements within the Pou5f1 gene. The experiments are well designed and the results are opening doors to more inquisitive experiments aimed at finding new cis-regulatory elements that might be involved in the regulation of the Pou5f1 gene.
In my opinion it would be also very interesting to investigate more in detail the reason why and how the Pou5f1∆/∆;Rosa26Pou5f1/+ line is able to adapt to the Pou5f1 mis-regulation. The authors spculate 'epigenetic erasing', but the concept is not clear to me, especially why the epigenetic erasing does not apply to the other cell line Pou5f1flox/Δ.
Author Response
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Author Response.pdf
Reviewer 2 Report
Ermakova et al. used embryonic stem cells to investigate the effects of regulatory elements located upstream and downstream of Pou5f1 gene (Oct4-encoding gene) towards the self-renewal and differentiation process of pluripotent cells. They assess the proliferation and apoptosis for the self-renewal process and differentiation markers for fate into specific lineages. Through the above results, they found these genetic elements located outside of the previous Pou5f1 gene are essential for the regulation of transcription factor Oct4.
1. I would suggest it be proofread and edited by professional academic writers to further improve the overall quality of the manuscript.
2. Fig 1, for the allele diagrams, consider changing to a higher magnification one with a clear indication of previously reported Pou5f1 gene region, DE and PE regions, with the length of base pairs.
3. Fig 2a, is there any statistic insignificance you found by ANOVA? It seems like a ~25% decrease in proliferation rates to me.
4. Fig 2c, Eosin is under-stained in ectoderm and endoderm section of Pou5f1flox/Δ , while over-stained in all Pou5f1Δ/Δ;Rosa26Pou5f1/+. Consider finding new sections for staining. Also, it would be more convincing to perform immunostaining for markers of each germ layer.
5. Fig 3a-c, what’s your interpretation of these proliferation and apoptosis results? Also, what’s the boundary between early and late apoptosis?
6. Fig 3e, what’s your interpretation of the ectopic Oct4 plotting? Why would protein level increase dramatically in a knockout line?
7. Fig 3f, you need a higher magnification for readers to see individual cells.
8. Fig 4a, it would be more convincing that you perform a measurement than quantification for fluorescence intensity. Also, the images should be at a higher magnification to see individual cells. In order to show “a mosaic pattern” mentioned in line 138 and “a minor subset” in line 139, an even higher magnified image with high resolution with the target population pointed by arrows would be helpful.
9. Line 145-146, are there other EpiSC markers you could test? Oct4 is directly affected by allele knockout, only Oct6 wouldn’t be that convincing.
10. Generally, the research is properly designed and performed, however, I think more specific markers other than Oct4 should be included in the western blot or immunostaining experiment, Bulk-seq would be even ideal. Some data visualization and statistics issues are found but can be fixed with more careful interpretation.
11. You didn't explain statistics in Material & Method. For all the * in graphs, you also need to explain in the corresponding figure legend.
Proofreading and editing by professional academic writers would be beneficial for the overall quality of the manuscript.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript by Ermakova et al describes unnatural expression pattern of Pou5f1 gene transposed at Rosa26 locus even though the transposed gene included all elements for transcriptional regulation identified in previous studies. The aberrant expression pattern depended on culture condition and pluripotency status, and resulted in preferential differentiation into mesodermal lineage and altered expression pattern of Nanog and Oct6. Based on the results, the authors suggested that additional regulatory element(s) located outside of the transposed sequence should be required to recapitulate the natural expression pattern of the gene. Changes in expression and in accompanied gene expression and differentiation potential by the transposition of Pou5f1 gene were obviously demonstrated. Although position effect of transposed gene was considered by testing tdTomato expression under CAG promoter control, however, the long transposed Pou5f1 DNA segment could be much more susceptible to various modifications including methylation. Therefore, the presence of novel elements for transcriptional regulation of the Pou5f1 gene could be a possibility at this stage of study.
1. Graphs in figures should be prepared to show axes clearly.
2. Axis labels and legends of the supplementary figures are not clear to read. Change in character color is necessary.
3. How was the proliferation rate determined?
4. Quantitation of Western blot results in 1b is required. What would be the correlation of Oct4 expression with the proliferation rate?
5. What was the rationale to determine the level of Oct4 on day 11 in Figure 3? A Western blot might be required to show the time-dependent change of Oct4 expression.
6. What does it mean with ‘mosaic pattern’ of Oct4 expression in Figure 4a? It appears that all DAPI-positive spots are also Oct4-positive.
7. Methylation pattern of the Pou5f1 gene at Rosa26 locus should be compared with that in native locus in Pou5f1flox/D to further verify the necessity of additional elements for transcriptional regulation.
8. ‘Of note, recently described Pou5f1 elements may serve to this purpose [12,16]’ in lines 184~185 should be more specific and needs to be expanded.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
The authors addressed most of the issues raised in the previous review process. The manuscript is now considered publishable in the current form.
