Promoter-Adjacent DNA Hypermethylation Can Downmodulate Gene Expression: TBX15 in the Muscle Lineage

Round 1

Reviewer 1 Report

The manuscript titled “Promoter-adjacent DNA hypermethylation can downmodulate gene expression: TBX15 in the muscle lineage” by Dr. Ehrlich et.al proposed that DNA hypermethylation around the unmethylated promoter region of TBX15 correlated with the gene expression in myoblasts, highlighting the roles of DNA methylation on the regulation of TBX15 expression in different cells. The authors first identified the hypermethylation regions by NGS sequencing in myoblasts (EM-seq and WGBS) and skeletal muscle (WGBS), and then integrated the published WGBS data for systemic comparison among different cells. They also designed the cloned the differentially methylated sequences (DMRs) in CpG-free reporter vectors and tested them upon transient transfection in C2C12 and MCF-7. Based on these results, the authors proposed a model for the role of myoblast-hypermethylated DMRs adjacent to the TBX15 promoter in down-modulating expression of the transcriptionally active gene. These observations are quite interesting, and the evidence for support the claims are solid. The manuscript is well organized and is nearly ready to publish. My minor concerns are as following:

 

1.     In the model, the high level of methylation of promoter-adjacent DMRs is proposed to prevent their latent enhancer/promoter activity in vivo but to allow core promoter activity and downstream enhancer activity in myoblasts. However, in other cells, such as ESC, repressive histone modification (H3K27me3) is seen in 525 many cell and tissue types that have little or no DNA methylation at the DMRs. These results involved the relationship between DNA methylation and histone modification (H3K27me3 and H3K36me3), and how to explain these results? Does it mean, the histone modification (H3K27me3) signals are more important than DNA methylation in this case? It is better that the authors could discuss it with focusing on the crosstalk between histone modification and DNA methylation.

2.     Why the arrow for TSS was not indicated at transcript start site, but looks like at the CDS start (Figure 2A, 3B, 5A, and 6A)?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have well shown the effect of DNA hypermethylation in downmodulating TBX 15 gene expression in muscle lineage; however, the authors need to clarify some of the questions which deem important before publication.  

 

1.    The authors should discuss more about the functional significance of down regulation (but not silencing) of genes in virtue of DNA hypermethylation in context of biological relevance. Does downmodulation due to DNA hypermethylation is specific to certain cell types or for certain genes barring the others? If so, what could be the possible conditions/state where in a particular cell decides for a particular gene in question whether it should be down regulated or silenced? 

 

2.    Line 510 -511 As the authors argue against the fact that hypermethylation of DMRs turns off in cis an overlapping repressor, and further provide an explanation stating “A likely explanation for the lack of promoter-border DNA methylation in cells not transcribing TBX15 is that such cells have no need of fine-tuning TBX15 expression”. The explanation is however not straight and clear which can be argued with established facts. The author needs to strengthen the argument with a befitting explanation to counter the facts.  

 

3.    In the figure 3 panel C, D and E, the Y axis shows normalized activity. Since the activity refers to the luciferase activity, it should be better the authors report the Y axis as a function of Relative light units (RLU). However, if the authors wish to retain the term “normalized activity (or loss of activity)”, authors should define the activity in mathematical terms (emphasizing how RLU has been converted to activity units). 

 

4.    In the figure 3, Panel C & D as both the graphs show promoter activity assays for inserts, it may be combined. Further, a graph must show the statistical significance of comparing the activity brought about by different inserts (in the form of asterisks).  

 

5.    In the result section, the subheadings are like statements. These can be curtailed for better understanding and readability.  

Author Response

Please see attachment.

Author Response File: Author Response.pdf