NCAPG Dynamically Coordinates the Myogenesis of Fetal Bovine Tissue by Adjusting Chromatin Accessibility

Round 1

Reviewer 1 Report

The manuscript by XIN Hu et al.NCAPG coordinates myogenesis  in bovine fetal tissue. The paper have good a mechanistic approach, but it provides the data that can be the basis for further research regarding mechanisms, in which in vitro model of studying biological relevance of NCAPG react to the altered myogenesis state of bovine fatal tissue. Therefore, it can be of interest to reproductive biologists and clinicians. My detailed comments and suggestions are as follows:

1) l. 19, Abstract - please rearrange the sentence in the text because it's not clear.

2) in all results the author is missing scrambled siRNA for negative control in experimental procedure?

3). In PCR experiments the author used 18S and did you check other housekeeping gens?

4) In Fig 1. explain relative expression of which genes? 

Author Response

Response to Reviewer 1 Comments

Point 1: 19, Abstract - please rearrange the sentence in the text because it's not clear.

Response 1: Thanks for your comments. We revised the sentence in line 19-21.

Point 2: in all results the author is missing scrambled siRNA for negative control in experimental procedure?

Response 2: Thanks for your suggestions. In all results, the NC refers to scrambled siRNA control. Also, we revised the description in line 342.

Point 3: In PCR experiments the author used 18S and did you check other housekeeping gens?

Response 3: No, I haven’t check other housekeeping genes. Some studies indicated that 28S rRNA and 18S rRNA is recommended as internal standards of mRNA quantification [1]. Meanwhile, the use of only one internal standard is nevertheless acceptable [2,3]. Also, some studies showed that bovine 18S ribosomal RNA (18S) was used as the reference gene [4,5].

[1] Thellin O, Zorzi W, Lakaye B, et al. Housekeeping genes as internal standards: use and limits. J Biotechnol. 1999;75(2-3):291–295. doi:10.1016/s0168-1656(99)00163-7

[2] Lemay S, Mao C, Singh AK. Cytokine gene expression in the MRL/lpr model of lupus nephritis. Kidney Int. 1996;50(1):85–93. doi:10.1038/ki.1996.290

[3] Tang WW, Qi M, Van GY, Wariner GP, Samal B. Leukemia inhibitory factor ameliorates experimental anti-GBM Ab glomerulonephritis. Kidney Int. 1996;50(6):1922–1927. doi:10.1038/ki.1996.514

[4] Zhang, W. W., Sun, X. F., Tong, H. L., Wang, Y. H., Li, S. F., Yan, Y. Q., & Li, G. P. (2016). Effect of differentiation on microRNA expression in bovine skeletal muscle satellite cells by deep sequencing. Cellular & molecular biology letters, 21, 8. doi:10.1186/s11658-016-0009-x

[5] Vaughn, M. A., Lancaster, P. A., Roden, K. C., Sharman, E. D., Krehbiel, C. R., Horn, G. W., & Starkey, J. D. (2019). Effect of stocker management program on beef cattle skeletal muscle growth characteristics, satellite cell activity, and paracrine signaling impact on preadipocyte differentiation. Journal of animal science and technology, 61(5), 260–271. doi:10.5187/jast.2019.61.5.260

Point 4: In Fig 1. explain relative expression of which genes?

Response 4: Thanks for your suggestions. We modified the legend of Fig 1.

 

Reviewer 2 Report

This paper focuses on the impact of condensins, and more specifically NCAPG on the myogenesis in cattle muscle. The studies are carried out using primary cultures of isolated myoblasts from bovine 90-120 day fetuses. The authors show during the culture in this specific model, a deregulation of myogenic differentiation and of markers specific of this phenomena, such as MYF5 and MyoD which are downregulated and MYH proteins that are up-regulated, similarly to the differentiation process known essentially in mice (as reviewed for instance in Development 2017 144: 2104-2122, that could be quoted as useful for the reader). Blocking NCAPG using siRNA transfection triggers a longer mitosis phase in the myoblasts, alter the expression of specific genes of myoblast differentiation oppositely at 1.5 days post knock-down versus 4 days (induction and repression of MYF5, MYOD, MYOG, and MYH, respectively) , induces an increased apoptosis at day 4. The authors then analyzed the open chromatin state with ATAC seq in the KD condition, in particular along with the marks H4K20me1 (chromatin compaction) and H4K16ac (chromatin open). Silencing of NCAPG induced an increased binding around the TSS of genes, but with decreased accessibility, especially nearby TF binding sites for AP1 subunits.

                Technically, this is a superb work, extending the comprehension of myogenic mechanisms with enlargement to another model than mice in a species of economic interest, and pinpointing the impact of chromatin structure in these mechanisms. The techniques used are comprehensive.

My major concern is about the readability of the text that is replete with grammatical errors, verbs missing, words in the wrong order, etc. It has to be thoroughly read and edited by a native English speaker. The authors starts with a title that has no actual grammatical sense: it should be something like ‘through adjusting (orchestrating) chromatin accessibility’. ‘orchestrates’ is for the present tense.

Among minor questions:

Are the three siRNA used targeting different part of the mRNA? Wouldn’t it be more extinct using a combination of two or three of these siRNA? Apoptosis is marked in red not in green as wrongly claimed in the legend of figure 4 On the images when there is a quantification of the fluorescence signal how is it done to get objective data? It is written that it was done manually. Was it blinded? Did two investigators independently scrutinize the same experiment? How many fields were analyzed? In Table 4, MYH3 has twice the same primer for forward and reverse. How were validated the antibodies? Are they specific for cattle? The authors may add this reference: Liu Y, Duan X, Chen S, He H, Liu X. NCAPG is differentially expressed during longissimus muscle development and is associated with growth traits in Chinese Qinchuan beef cattle. Genet Mol Biol. 2015 Dec;38(4):450-6.

Author Response

Response to Reviewer 2 Comments

 

Point 1: My major concern is about the readability of the text that is replete with grammatical errors, verbs missing, words in the wrong order, etc. It has to be thoroughly read and edited by a native English speaker. The authors starts with a title that has no actual grammatical sense: it should be something like ‘through adjusting (orchestrating) chromatin accessibility’. ‘orchestrates’ is for the present tense.

 

Response 1: Thank you very much for your suggestions. We have revised our manuscript by native English speaker.

 

Point 2: Are the three siRNA used targeting different part of the mRNA? Wouldn’t it be more extinct using a combination of two or three of these siRNA?

 

Response 2: Thanks for your comments. Indeed, three designed siRNAs target different location of the NCAPG mRNA. Furthermore, the efficiency of siRNA003 reached 70% compared with NC group. So we did not try using two or more siRNAs together.

 

Point 3: Apoptosis is marked in red not in green as wrongly claimed in the legend of figure 4 On the images when there is a quantification of the fluorescence signal how is it done to get objective data? It is written that it was done manually. Was it blinded? Did two investigators independently scrutinize the same experiment? How many fields were analyzed?

 

Response 3: We corrected it in line 170. And we have revised method in line 397-398.

 

Point 4: In Table 4, MYH3 has twice the same primer for forward and reverse.

 

Response 4: We apologize for the error here. We have revised the primer sequence for forward primer of MYH3 in Table 4.

 

Point 5: How were validated the antibodies? Are they specific for cattle?

 

Response 5: Thanks for your suggestion. We selected the antibodies based on the species suggestion, supporting references, or high conservation between the amino acid sequence of antigen and bovine protein. We provide the product ID of antibodies in materials section.

 

Point 6: The authors may add this reference: Liu Y, Duan X, Chen S, He H, Liu X. NCAPG is differentially expressed during longissimus muscle development and is associated with growth traits in Chinese Qinchuan beef cattle. Genet Mol Biol. 2015 Dec;38(4):450-6.

 

Response 6: Thanks for your comments. We added this paper in line 37.

 

Reviewer 3 Report

Interesting article. Well designed and presented. The authors have succeeded to perform and exhibit an experimental study . It is an analytical study with clear results. I congratulate them !

Author Response

Response to Reviewer 3 Comments

 

Interesting article. Well designed and presented. The authors have succeeded to perform and exhibit an experimental study. It is an analytical study with clear results. I congratulate them!

 

Thank you very much for your review!