The review article titled “RNA polymerase II pausing in development: orchestrating transcription” by Abuhashem et al. summarizes the studies and their findings regarding RNA polymerase II promoter-proximal pausing in metazoan development. I believe that this review is timely and useful for the field and contains interesting discussions and perspectives. However, I found that some information should be improved to be correct, unbiased, and sufficient. Some sentences and figures are unclear and difficult to digest as well. I suggest the authors to revise the manuscript according to the comments below:

In the abstract:

I suggest that RNA polymerase II promoter-proximal pausing be called “Pol II pausing” instead of pausing throughout the review.

RNA polymerase II promoter-proximal pausing is a common mechanism…  Please replace “common” or rewrite the sentence.

“call for” instead of calls for?

In the introduction:

In the beginning paragraph, it is written “The canonical control system includes an extracellular signal that binds a cell surface receptor…” In fact, an extracellular signal can bind either a cell surface receptor or nuclear receptor (e.g. estrogen/androgen receptors) or it can directly modulate a master transcription factor (hypoxia:HIF, heat:HSF1, or small chemicals that bind to transcription factors etc).

It is said “The phenomenon, referred to as… estimating that up to 40% of expressed genes experience pausing.” I think that “up to 40% of protein-coding genes” is more appropriate for the given references.

In the section of Pol II pausing and pause release:

The sentence “For example, inhibiting CDK9 does not necessarily inhibit the release of pausing because CDK9 is also required for efficient elongation” is suggested to be rewritten for clarity.

I strongly recommend that the findings about Pol II pausing in human cells be included in this section and wherever appropriate. Pol II pausing in human HSP70 or other stress-inducible genes has been studied by a few groups, which is entirely missing in the current version of manuscript.

Throughout the text and figures, “flavopiridole” should be corrected to flavopiridol.

In the section of “Effects of pausing on transcription:

The kinetics of Pol II pausing has been studied by at least a couple of groups (they provided somewhat different/similar durations of Pol II pausing). In the last sentence of the first paragraph, these studies can be briefly summarized and referred to.

In the section of Recent molecular insights into pausing:

SIRT6, a histone… FACT complex stabilize Pol II pausing  Note that the function of FACT complex is unlikely limited to stabilizing Pol II pausing but it is also involved in transcriptional productive elongation according to the literature. Refer to some of the papers below:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC262413/
https://pubmed.ncbi.nlm.nih.gov/15020050/
The findings of FACT in Pol II pausing and elongation need to be elaborated, avoiding biased descriptions.

There are more histone modifiers important for Pol II pausing regulation. I suggest more comprehensive descriptions including them rather than the current ones only with SIRT6 and FACT.

In the section of “Pausing in mammalian development:

Overall, this section should be reinforced and enhanced by including more recent studies and discussions regarding them.

“Pausing has been shown to be prevalent in mES cells, with…. A pausing index >4 …”  It says the gene body within 2 kilobases but doesn’t provide the length of TSS? Also, how significant is pausing index > 4?

Bivalent genes and developmental genes are not mutually exclusive. The sentence “at a comparable level at developmental and ‘bivalent genes’… “ should be revised for clarity.

“Whether pausing occurs at non-expressed genes in mES cells, as observed in heat shock genes…, is still unknown.  what are non-expressed genes? If they meant non-coding genes, some studies studied Pol II pausing at non-coding genes in mES cells, which can be referred to.

Some important papers have not been discussed in the review. For example, TIF1gamma regulating Pol II pausing for erythroid cell differentiation and fate control (PMID: 20603019), TIF1beta regulating Pol II pausing in mES cells (PMID: 25173174), Pol II pausing in the regulation of EMT (PMID: 25820424), etc.

In the section of Signaling and pausing: Mediating a transcriptional response to stimuli:

In the beginning of the second paragraph, the sentence “Notably, reports of a connection… in developmental processes” should be rewritten.

In the last paragraph, it says “Presumably, such factors directly or indirectly recruit P-TEFb and other… to mediate pause release. It has been reported by a few papers that MYC interacts with and recruits P-TEFb for transcriptional activation. I suggest this paragraph to be further elaborated.

In the section of Rate of induction and/or repression of active transcription:

Additionally and perhaps more importantly, absence of pERK… a hypothesis previously tested in heat shock recovery.  This can be confusing— Do the authors mean that unphosphorylated ERK establishes Pol II pausing at heat shock genes? Perhaps, the sentence can be rewritten to avoid the confusion. In addition, even during Pol II pause release in transcriptional activation, Pol II pausing occurs, yet for a much reduced duration. Could this support the function of the activation of major transcription factors to facilitate the turnover rate rather than preventing/repressing Pol II pausing (e.g. ERK- and HSF1-target genes)? I suggest the paragraph be revised to include this possibility and discussion.

In Figures:

In Fig. 1A, the description of activated transcription and Pol II pausing/pause release is inaccurate. When transcription is activated and Pol II pause is reversed, Pol II occupancy in the TSS doesn’t decrease but rather increase. Pausing index is decreased in transcriptional activation due to the increase of gene body Pol II, not for the decrease of TSS Pol II. There are many references showing this observations (e.g. PMID: 22252557; PMID: 31249142 and many more!).

In Fig. 2B, TPB  TBP? In addition, some more descriptions are required for readers to understand the figure. What are the flags in the histones?? Is this paused complex or elongating one?

In figures or text, please describe the three phosphorylation sites in P-TEFb.

In Fig. 4, again, the Pol II peak in the TSS appears dramatically smaller during the inactive state of transcription than in the active state. This is not observed in Pol II ChIP-seq in most studies and so is to be revised.

Fig. 5 is not self-explaining. The consequence of loss of pausing is unclear.

Fig. 6 is also not self-explaining and uncertain. Fate A and fate B could be defined.

The authors addressed all the concerns and suggestions. A couple of minor comments that may help the authors to improving the manuscript further are listed below.

In page 6: ~5-10 min to be changed to ~5–10 min; 1-2 min to 1–2 min.

In Fig. 2A, red flags are not defined whereas green flags are in Fig 2B?

In the Fig. 5 legend, please define what the different colors mean. For non-paused genes, overall it is confusing and unable to distinguish them with paused, un-induced genes. Please see and consider to revise the figure according to the paper, a Nat Rev Genet paper by Adelman and Lis (2012, PMCID: PMC3552498), in particular take a look at Fig. 2. In that figure, non-paused genes are depicted for when they are uninduced and induced.

In Fig. 6, the claim “pausing is dispensable (currently it is written dispensible)” for the genes with highly expressed, paused genes has not been established by research and so please revise it. Rapid turnover of pausing or lenient pausing? “Pausing is necessary” should also be revised accordantly.