Fragile nucleosomes are essential for RNA Polymerase II to transcribe in eukaryotes
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eLife Assessment
This study addresses an important problem in gene regulation, namely, which features of chromatin regulate potential RNA Polymerase 2 activity at a locus. The authors provided evidence that specific post-translational modifications of histones within the gene body are correlated with Pol II transcription, that these modifications are dynamic, and that they can be regulated by Pol II activity. The manuscript contributes to the concept of "fragile nucleosomes" as a unifying framework for key epigenetic drivers of transcription; however, the quality of the evidence provided is inadequate in support of the claims made, and further evidence teasing out the mechanistic aspects of the work would strengthen its impact. This work will be of interest to the fields of transcriptional regulation, chromatin structure, and epigenetics.
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Abstract
Nucleosomes are barriers to RNA Polymerase II (Pol II) transcribing along gene bodies in eukaryotes. We found that a fragile “tailless nucleosome” could be generated to resolve the Pol II pausing caused by the +1 nucleosome in metazoans. We hypothesized that “pan-acetylated nucleosomes” along gene bodies are fragile and are generated to resolve the resistance of nucleosomes to the elongation of Pol II. To confirm this hypothesis, we systematically analyzed pan-acetylation and pan-phosphorylation of the entire human genome in HEK293T cells. Herein, we show that both pan-acetylation and pan-phosphorylation of histones in nucleosomes along gene bodies are coupled with the activities of Pol II. Our results show that there are four major types of fragile nucleosomes required for Pol II to transcribe along gene bodies: nucleosomes without tails, nucleosomes with dominant acetylation, nucleosomes with dominant phosphorylation, and nucleosomes with both acetylation and phosphorylation.
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eLife Assessment
This study addresses an important problem in gene regulation, namely, which features of chromatin regulate potential RNA Polymerase 2 activity at a locus. The authors provided evidence that specific post-translational modifications of histones within the gene body are correlated with Pol II transcription, that these modifications are dynamic, and that they can be regulated by Pol II activity. The manuscript contributes to the concept of "fragile nucleosomes" as a unifying framework for key epigenetic drivers of transcription; however, the quality of the evidence provided is inadequate in support of the claims made, and further evidence teasing out the mechanistic aspects of the work would strengthen its impact. This work will be of interest to the fields of transcriptional regulation, chromatin structure, and epigenetics.
eLife Assessment
This study addresses an important problem in gene regulation, namely, which features of chromatin regulate potential RNA Polymerase 2 activity at a locus. The authors provided evidence that specific post-translational modifications of histones within the gene body are correlated with Pol II transcription, that these modifications are dynamic, and that they can be regulated by Pol II activity. The manuscript contributes to the concept of "fragile nucleosomes" as a unifying framework for key epigenetic drivers of transcription; however, the quality of the evidence provided is inadequate in support of the claims made, and further evidence teasing out the mechanistic aspects of the work would strengthen its impact. This work will be of interest to the fields of transcriptional regulation, chromatin structure, and epigenetics.
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Reviewer #1 (Public review):
Summary:
This study aims to explore how different forms of "fragile nucleosomes" facilitate RNA Polymerase II (Pol II) transcription along gene bodies in human cells. The authors propose that pan-acetylated, pan-phosphorylated, tailless, and combined acetylated/phosphorylated nucleosomes represent distinct fragile states that enable efficient transcription elongation. Using CUT&Tag-seq, RNA-seq, and DRB inhibition assays in HEK293T cells, they report a genome-wide correlation between histone pan-acetylation/phosphorylation and active Pol II occupancy, concluding that these modifications are essential for Pol II elongation.
Strengths:
(1) The manuscript tackles an important and long-standing question about how Pol II overcomes nucleosomal barriers during transcription.
(2) The use of genome-wide CUT&Tag-seq …
Reviewer #1 (Public review):
Summary:
This study aims to explore how different forms of "fragile nucleosomes" facilitate RNA Polymerase II (Pol II) transcription along gene bodies in human cells. The authors propose that pan-acetylated, pan-phosphorylated, tailless, and combined acetylated/phosphorylated nucleosomes represent distinct fragile states that enable efficient transcription elongation. Using CUT&Tag-seq, RNA-seq, and DRB inhibition assays in HEK293T cells, they report a genome-wide correlation between histone pan-acetylation/phosphorylation and active Pol II occupancy, concluding that these modifications are essential for Pol II elongation.
Strengths:
(1) The manuscript tackles an important and long-standing question about how Pol II overcomes nucleosomal barriers during transcription.
(2) The use of genome-wide CUT&Tag-seq for multiple histone marks (H3K9ac, H4K12ac, H3S10ph, H4S1ph) alongside active Pol II mapping provides a valuable dataset for the community.
(3) The integration of inhibition (DRB) and recovery experiments offers insight into the coupling between Pol II activity and chromatin modifications.
(4) The concept of "fragile nucleosomes" as a unifying framework is potentially appealing and could stimulate further mechanistic studies.
Weaknesses:
(1) Misrepresentation of prior literature
The introduction incorrectly describes findings from Bintu et al., 2012. The cited work demonstrated that pan-acetylated or tailless nucleosomes reduce the nucleosomal barrier for Pol II passage, rather than showing no improvement. This misstatement undermines the rationale for the current study and should be corrected to accurately reflect prior evidence.
(2) Incorrect statement regarding hexasome fragility
The authors claim that hexasome nucleosomes "are not fragile," citing older in vitro work. However, recent studies clearly showed that hexasomes exist in cells (e.g., PMID 35597239) and that they markedly reduce the barrier to Pol II (e.g., PMID 40412388). These studies need to be acknowledged and discussed.
(3) Inaccurate mechanistic interpretation of DRB
The Results section states that DRB causes a "complete shutdown of transcription initiation (Ser5-CTD phosphorylation)." DRB is primarily a CDK9 inhibitor that blocks Pol II release from promoter-proximal pausing. While recent work (PMID: 40315851) suggests that CDK9 can contribute to CTD Ser5/Ser2 di-phosphorylation, the manuscript's claim of initiation shutdown by DRB should be revised to better align with the literature. The data in Figure 4A indicate that 1 µM DRB fully inhibits Pol II activity, yet much higher concentrations (10-100×) are needed to alter H3K9ac and H4K12ac levels. The authors should address this discrepancy by discussing the differential sensitivities of CTD phosphorylation versus histone modification turnover.
(4) Insufficient resolution of genome-wide correlations
Figure 1 presents only low-resolution maps, which are insufficient to determine whether pan-acetylation and pan-phosphorylation correlate with Pol II at promoters or gene bodies. The authors should provide normalized metagene plots (from TSS to TTS) across different subgroups to visualize modification patterns at higher resolution. In addition, the genome-wide distribution of another histone PTM with a different localization pattern should be included as a negative control.
(5) Conceptual framing
The manuscript frequently extrapolates correlative genome-wide data to mechanistic conclusions (e.g., that pan-acetylation/phosphorylation "generate" fragile nucleosomes). Without direct biochemical or structural evidence. Such causality statements should be toned down.
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Reviewer #2 (Public review):
Summary:
In this manuscript, the authors use various genomics approaches to examine nucleosome acetylation, phosphorylation, and PolII-CTD phosphorylation marks. The results are synthesized into a hypothesis that 'fragile' nucleosomes are associated with active regions of PolII transcription.
Strengths:
The manuscript contains a lot of genome-wide analyses of histone acetylation, histone phosphorylation, and PolII-CTD phosphorylation.
Weaknesses:
This reviewer's main research expertise is in the in vitro study of transcription and its regulation in purified, reconstituted systems. I am not an expert at the genomics approaches and their interpretation, and overall, I had a very hard time understanding and interpreting the data that are presented in this manuscript. I believe this is due to a problem with the …
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors use various genomics approaches to examine nucleosome acetylation, phosphorylation, and PolII-CTD phosphorylation marks. The results are synthesized into a hypothesis that 'fragile' nucleosomes are associated with active regions of PolII transcription.
Strengths:
The manuscript contains a lot of genome-wide analyses of histone acetylation, histone phosphorylation, and PolII-CTD phosphorylation.
Weaknesses:
This reviewer's main research expertise is in the in vitro study of transcription and its regulation in purified, reconstituted systems. I am not an expert at the genomics approaches and their interpretation, and overall, I had a very hard time understanding and interpreting the data that are presented in this manuscript. I believe this is due to a problem with the manuscript, in that the presentation of the data is not explained in a way that's understandable and interpretable to a non-expert. For example:
(1) Figure 1 shows genome-wide distributions of H3K9ac, H4K12ac, Ser2ph-PolII, mRNA, H3S10ph, and H4S1ph, but does not demonstrate correlations/coupling - it is not clear from these data that pan-acetylation and pan-phosphorylation are coupled with Pol II transcription.
(2) Figure 2 - It's not clear to me what Figure 2 is supposed to be showing.
(A) Needs better explanation - what is the meaning of the labels at the top of the gel lanes?
(B) This reviewer is not familiar with this technique, its visualization, or its interpretation - more explanation is needed. What is the meaning of the quantitation graphs shown at the top? How were these calculated (what is on the y-axis)?
(3) To my knowledge, the initial observation of DRB effects on RNA synthesis also concluded that DRB inhibited initiation of RNA chains (pmid:982026) - this needs to be acknowledged.
(4) Again, Figures 4B, 4C, 5, and 6 are very difficult to understand - what is shown in these heat maps, and what is shown in the quantitation graphs on top?
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Reviewer #3 (Public review):
Summary:
Li et al. investigated the prevalence of acetylated and phosphorylated histones (using H3K9ac, H4K12ac, H3S10ph & H4S1ph as representative examples) across the gene body of human HEK293T cells, as well as mapping elongating Pol II and mRNA. They found that histone acetylation and phosphorylation were dominant in gene bodies of actively transcribing genes. Genes with acetylation/phosphorylation restricted to the promoter region were also observed. Furthermore, they investigated and reported a correlation between histone modifications and Pol II activity, finding that inhibition of Pol II activity reduced acetylation/phosphorylation levels, while resuming Pol II activity restored them. The authors then proposed a model in which pan-acetylation or pan-phosphorylation of histones generates fragile …
Reviewer #3 (Public review):
Summary:
Li et al. investigated the prevalence of acetylated and phosphorylated histones (using H3K9ac, H4K12ac, H3S10ph & H4S1ph as representative examples) across the gene body of human HEK293T cells, as well as mapping elongating Pol II and mRNA. They found that histone acetylation and phosphorylation were dominant in gene bodies of actively transcribing genes. Genes with acetylation/phosphorylation restricted to the promoter region were also observed. Furthermore, they investigated and reported a correlation between histone modifications and Pol II activity, finding that inhibition of Pol II activity reduced acetylation/phosphorylation levels, while resuming Pol II activity restored them. The authors then proposed a model in which pan-acetylation or pan-phosphorylation of histones generates fragile nucleosomes; the first round of transcription is accompanied by pan-acetylation, while subsequent rounds are accompanied by pan-phosphorylation.
Strengths:
This study addresses a highly significant problem in gene regulation. The author provided riveting evidence that certain histone acetylation and/or phosphorylation within the gene body is correlated with Pol II transcription. The author furthermore made a compelling case that such transcriptionally correlated histone modification is dynamic and can be regulated by Pol II activity. This work has provided a clearer view of the connection between epigenetics and Pol II transcription.
Weaknesses:
The title of the manuscript, "Fragile nucleosomes are essential for RNA Polymerase II to transcribe in eukaryotes", suggests that fragile nucleosomes lead to transcription. While this study shows a correlation between histone modifications in gene bodies and transcription elongation, a causal relationship between the two has not been demonstrated.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study aims to explore how different forms of "fragile nucleosomes" facilitate RNA Polymerase II (Pol II) transcription along gene bodies in human cells. The authors propose that pan-acetylated, pan-phosphorylated, tailless, and combined acetylated/phosphorylated nucleosomes represent distinct fragile states that enable eFicient transcription elongation. Using CUT&Tagseq, RNA-seq, and DRB inhibition assays in HEK293T cells, they report a genome-wide correlation between histone pan-acetylation/phosphorylation and active Pol II occupancy, concluding that these modifications are essential for Pol II elongation.
Strengths:
(1) The manuscript tackles an important and long-standing question about how Pol II overcomes nucleosomal barriers during transcription.
(2) The …
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study aims to explore how different forms of "fragile nucleosomes" facilitate RNA Polymerase II (Pol II) transcription along gene bodies in human cells. The authors propose that pan-acetylated, pan-phosphorylated, tailless, and combined acetylated/phosphorylated nucleosomes represent distinct fragile states that enable eFicient transcription elongation. Using CUT&Tagseq, RNA-seq, and DRB inhibition assays in HEK293T cells, they report a genome-wide correlation between histone pan-acetylation/phosphorylation and active Pol II occupancy, concluding that these modifications are essential for Pol II elongation.
Strengths:
(1) The manuscript tackles an important and long-standing question about how Pol II overcomes nucleosomal barriers during transcription.
(2) The use of genome-wide CUT&Tag-seq for multiple histone marks (H3K9ac, H4K12ac, H3S10ph, H4S1ph) alongside active Pol II mapping provides a valuable dataset for the community.
(3) The integration of inhibition (DRB) and recovery experiments oFers insight into the coupling between Pol II activity and chromatin modifications.
(4) The concept of "fragile nucleosomes" as a unifying framework is potentially appealing and could stimulate further mechanistic studies.
Really appreciate the positive or affirmative comments from the reviewer.
Weaknesses:
(1) Misrepresentation of prior literature
The introduction incorrectly describes findings from Bintu et al., 2012. The cited work demonstrated that pan-acetylated or tailless nucleosomes reduce the nucleosomal barrier for Pol II passage, rather than showing no improvement. This misstatement undermines the rationale for the current study and should be corrected to accurately reflect prior evidence.
What we said is according to the original report in the publication (Bintu et al., Cell, 2012). Here is the citation from the report:
Page 739,(Bintu, L. et al., Cell, 2012)(PMID: 23141536)
“Overall transcription through tailless and acetylated nucleosomes is slightly faster than through unmodified nucleosomes (Figure 1C), with crossing times that are generally under 1 min (39.5 ± 5.7 and 45.3 ± 7.6 s, respectively). Both the removal and acetylation of the tails increase eFiciency of NPS passage:71% for tailless nucleosomes and 63% for acetylated nucleosomes (Figures 1C and S1), in agreement with results obtained using bulk assays of transcription (Ujva´ ri et al., 2008).”
We will cite this original sentence in our revision.
(2) Incorrect statement regarding hexasome fragility
The authors claim that hexasome nucleosomes "are not fragile," citing older in vitro work. However, recent studies clearly showed that hexasomes exist in cells (e.g., PMID 35597239) and that they markedly reduce the barrier to Pol II (e.g., PMID 40412388). These studies need to be acknowledged and discussed.
“hexasome” was introduced in the transcription field four decades ago. Later, several groups claimed that “hexasome” is fragile and could be generated in transcription elongation of Pol II. However, their original definition was based on the detection of ~100 bps DNA fragments (MNase resistant) in vivo by Micrococcal nuclease sequencing (MNase-seq), which is the right length to wrap up one hexasome histone subunit (two H3/4 and one H2A/2B) to form the sub-nucleosome of a hexasome. As we should all agree that acetylation or phosphorylation of the tails of histone nucleosomes will lead to the compromised interaction between DNA and histone subunits, which could lead to the intact naïve nucleosome being fragile and easy to disassemble, and easy to access by MNase. Fragile nucleosomes lead to better accessibility of MNase to DNA that wraps around the histone octamer, producing shorter DNA fragments (~100 bps instead of ~140 bps). In this regard, we believe that these ~100 bps fragments are the products of fragile nucleosomes (fragile nucleosome --> hexasome), instead of the other way around (hexasome --> fragile).
Actually, two early reports from Dr. David J. Clark’s group from NIH raised questions about the existence of hexasomes in vivo (PMID: 28157509) (PMID: 25348398).
From the report of PMID:35597239, depletion of INO80 leads to the reduction of “hexasome” for a group of genes, and the distribution of both “nucleosomes” and “hexasomes” with the gene bodies gets fuzzier (less signal to noise). In a recent theoretical model (PMID: 41425263), the corresponding PI found that chromatin remodelers could act as drivers of histone modification complexes to carry out different modifications along gene bodies. The PI found that INO80 could drive NuA3 (a H3 acetyltransferase) to carry out pan-acetylation of H3 and possibly H2B as well in the later runs of transcription of Pol II for a group of genes (SAGA-dependent). It suggests that the depletion of INO80 will affect (reduce) the pan-acetylation of nucleosomes, which leads to the drop of pan-acetylated fragile nucleosomes, subsequently the drop of “hexasomes”. This explains why depletion of INO80 leads to the fuzzier results of either nucleosomes or “hexasomes” in PMID: 35597239. The result of PMID: 35597239 could be a strong piece of evidence to support the model proposed by the corresponding PI (PMID: 41425263).
From a recent report: PMID:40412388, the authors claimed that FACT could bind to nucleosomes to generate “hexasomes”, which are fragile for Pol II to overcome the resistance of nucleosomes. It was well established that FACT enhances the processivity of Pol II in vivo via its chaperonin property. However, the exact working mechanism of FACT still remains ambiguous. A report from Dr. Cramer’s group showed that FACT enhances the elongation of regular genes but works just opposite for pausing-regulated genes (PMID: 38810649). An excellent review by Drs. Tim Formosa and Fred Winston showed that FACT is not required for the survival of a group of differentiated cells (PMID: 33104782), suggesting that FACT is not always required for transcription. It is quite tricky to generate naïve hexasomes in vitro according to early reports from the late Dr. Widom’s group. Most importantly, the new data (the speed of Pol II, the best one on bare DNA is ~27 bps/s) from the report of PMID: 40412388, which is much slower than the speed of Pol II in vivo: ~2.5 kbs/min or ~40 bps/s. From our recovering experiments (Fig. 4C, as mentioned by reviewer #3), in 20 minutes (the period between 10 minutes and 30 minutes, due to the property of CUT-&TAG-seq, of which Pol II still active after cells are collected, there is a big delay of complete stop of Pol II during the procedure of CUT&TAG experiments, so the first period of time does not actually reflect the speed of Pol II, which is ~5 kb/min), all Pol IIs move at a uniform speed of ~2.5 kbs/min in vivo. Interestingly, a recent report from Dr. Shixin Liu’s group (PMID: 41310264) showed that adding SPT4/5 to the transcription system with bare DNA (in vitro), the speed of Pol II reaches ~2.5kbs/min, exactly the same one as we derived in vivo. Similar to the original report (PMID: 23141536), the current report of PMID:40412388 does not mimic the conditions in vivo exactly.
There is an urgent need for a revisit of the current definition of “hexasome”, which is claimed to be fragile and could be generated during the elongation of Pol II in vivo. MNase is an enzyme that only works when the substrate is accessible. In inactive regions of the genome, due to the tight packing of chromatin, MNase is not accessible to individual nucleosomes within the bodies of a gene or upstream of promoters, which is why we only see phased/spacing or clear distribution of nucleosomes at the transcription start sites, but it becomes fuzzy downstream or upstream of promoters. On the other hand, for fragile nucleosomes, the accessibility to MNase should increase dramatically, which leads to the ~100 bps fragments. Based on the uniform rate (2.5 kbs/min) of Pol II for all genes derived from human 293T cells and the similar rate (2.5 kbs/min) of Pol II on bare DNA in vitro, it is unlikely for Pol II to pause in the middle of nucleosomes to generate “hexasomes” to continue during elongation along gene bodies. Similar to RNAPs in bacterial (no nucleosomes) and Archaea (tailless nucleosomes), there should be no resistance when Pol IIs transcribe along all fragile nucleosomes within gene bodies in all eukaryotes, as we characterized in this manuscript.
(3) Inaccurate mechanistic interpretation of DRB
The Results section states that DRB causes a "complete shutdown of transcription initiation (Ser5-CTD phosphorylation)." DRB is primarily a CDK9 inhibitor that blocks Pol II release from promoter-proximal pausing. While recent work (PMID: 40315851) suggests that CDK9 can contribute to CTD Ser5/Ser2 di-phosphorylation, the manuscript's claim of initiation shutdown by DRB should be revised to better align with the literature. The data in Figure 4A indicate that 1 M DRB fully inhibits Pol II activity, yet much higher concentrations (10-100 ) are needed to alter H3K9ac and H4K12ac levels. The authors should address this discrepancy by discussing the differential sensitivities of CTD phosphorylation versus histone modification turnover.
Yes, it was reported that DRB is also an inhibitor of CDK9. However, if the reviewer agrees with us and the current view in the field, the phosphorylation of Ser5-CTD of Pol II is the initiation of transcription for all Pol II-regulated genes in eukaryotes. CDK9 is only required to work on the already phosphorylated Ser5-CTD of Pol II to release the paused Pol II, which only happens in metazoans. From a series of works by us and others: CDK9 is unique in metazoans, required only for the pausing-regulated genes but not for regular genes. We found that CDK9 works on initiated Pol II (Ser5-CTD phosphorylated Pol II) and generates a unique phosphorylation pattern on CTD of Pol II (Ser2ph-Ser2ph-Ser5ph-CTD of Pol II), which is required to recruit JMJD5 (via CID domain) to generate a tailless nucleosome at +1 from TSS to release paused Pol II (PMID: 32747552). Interestingly, the report from Dr. Jesper Svejstrup’s group (PMID: 40315851) showed that CDK9 could generate a unique phosphorylation pattern (Ser2ph-Ser5ph-CTD of Pol II), which is not responsive to the popular 3E10 antibody that recognizes the single Ser2phCTD of Pol II. This interesting result is consistent with our early report showing the unique phosphorylation pattern (Ser2ph-Ser2ph-Ser5ph-CTD of Pol II) is specifically generated by CDK9 in animals, which is not recognized by 3E10 either (PMID: 32747552). Actually, an early report from Dr. Dick Eick’s group (PMID: 26799765) showed the difference in the phosphorylation pattern of the CTD of Pol II between animal cells and yeast cells. We have characterized how CDK9 is released from 7SK snRNP and recruited onto paused Pol II via the coupling of JMJD6 and BRD4 (PMID: 32048991), which was published on eLIFE. It is well established that CDK9 works after CDK7 or CDK8. From our PRO-seq data (Fig. 3) and CUT&TAG-seq data of active Pol II (Fig. 4), adding DRB completely shuts down all genes via inhibiting the initiation of Pol II (generation of Ser5ph-CTD of Pol II). Due to the uniqueness of CDK9 only in metazoans, it is not required for the activation of CDK12 or CDK13 (they are orthologs of CTK1 in yeast), as we demonstrated recently (PMID: 41377501). Instead, we found that CDK11/10 acts as the ortholog of Bur1 kinase from yeast, is essential for the phosphorylation of Spt5, the link of CTD of Pol II, and CDK12 (PMID: 41377501).
(4) Insufficient resolution of genome-wide correlations
Figure 1 presents only low-resolution maps, which are Insufficient o determine whether pan-acetylation and pan-phosphorylation correlate with Pol II at promoters or gene bodies. The authors should provide normalized metagene plots (from TSS to TTS) across different subgroups to visualize modification patterns at higher resolution. In addition, the genome-wide distribution of another histone PTM with a diFerent localization pattern should be included as a negative control.
A popular view in the field is that the majority of genomes are inactive since they do not contain coding RNAs, which are responsible for ~20,000 protein candidates characterized in animals. However, our genomewide characterization using the four histone modification marks, active Pol II, and RNA-seq, shows a different story. Figure 1 shows that most of the human genome of HEK293T is active in producing not only protein-coding RNAs but also non-coding RNAs (the majority of them). We believe that Figure 1 could change our current view of the activity of the entire genome, and should be of great interest to general readers as well as researchers on genomics. Furthermore, it is a basis for Figure 2, which is a zoom-in of Figure 1.
(5) Conceptual framing
The manuscript frequently extrapolates correlative genome-wide data to mechanistic conclusions (e.g., that pan-acetylation/phosphorylation "generate" fragile nucleosomes). Without direct biochemical or structural evidence. Such causality statements should be toned down.
The reviewer is right, we should tone down the strong sentences. However, we believe that our data is strong enough to derive the general conclusion. The reviewer may agree with us that the entire field of transcription and epigenetics has been stagnant in recent decades, but there is an urgent need for fresh ideas to change the current situation. Our novel discoveries, for sure, additional supporting data are needed, should open up a brand new avenue for people to explore. We believe that a new era of transcription will emerge based on our novel discoveries. We hope that this manuscript will attract more people to these topics. As Reviewer #3 pointed out, this story establishes the connection between transcription and epigenetics in the field.
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors use various genomics approaches to examine nucleosome acetylation, phosphorylation, and PolII-CTD phosphorylation marks. The results are synthesized into a hypothesis that 'fragile' nucleosomes are associated with active regions of PolII transcription.
Strengths:
The manuscript contains a lot of genome-wide analyses of histone acetylation, histone phosphorylation, and PolII-CTD phosphorylation.
Weaknesses:
This reviewer's main research expertise is in the in vitro study of transcription and its regulation in purified, reconstituted systems.
Actually, the pioneering work of the establishment of in vitro transcription assays at Dr. Robert Roeder’s group led to numerous groundbreaking discoveries in the transcription field. The contributions of in vitro work in the transcription field are the key for us to explore the complexity of transcription in eukaryotes in the early times and remain important currently.
I am not an expert at the genomics approaches and their interpretation, and overall, I had a very hard time understanding and interpreting the data that are presented in this manuscript. I believe this is due to a problem with the manuscript, in that the presentation of the data is not explained in a way that's understandable and interpretable to a non-expert.
Thanks for your suggestions. You are right, we have problems expressing our ideas clearly in this manuscript, which could confuse. We will make modifications accordingly per your suggestions.
For example:
(1) Figure 1 shows genome-wide distributions of H3K9ac, H4K12ac, Ser2phPolII, mRNA, H3S10ph, and H4S1ph, but does not demonstrate correlations/coupling - it is not clear from these data that pan-acetylation and pan-phosphorylation are coupled with Pol II transcription.
Figure 1 shows the overall distribution of the four major histone modifications, active Pol II, and mRNA genome-wide in human HEK293T cells. It tells general readers that the entire genome is quite active and far more than people predicted that most of the genome is inactive, since just a small portion of the genome expresses coding RNAs (~20,000 in animals). Figure 1 shows that the majority of the genome is active and expresses not only coded mRNA but also non-coding RNAs. After all, it is the basis of Figure 2, which is a zoom-in of Figure 1. However, it is beyond the scope of this manuscript to discuss the non-coding RNAs.
(2) Figure 2 - It's not clear to me what Figure 2 is supposed to be showing.
(A) Needs better explanation - what is the meaning of the labels at the top of the gel lanes?
Figure 2 is a zoom-in for the individual gene, which shows how histone modifications are coupled with Pol II activity on the individual gene. We will give a more detailed explanation of the figure per the reviewer’s suggestions.
(B) This reviewer is not familiar with this technique, its visualization, or its interpretation - more explanation is needed. What is the meaning of the quantitation graphs shown at the top? How were these calculated (what is on the y-axis)?
Good suggestions, we will do some modifications.
(3) To my knowledge, the initial observation of DRB eFects on RNA synthesis also concluded that DRB inhibited initiation of RNA chains (pmid:982026) - this needs to be acknowledged.
Thanks for the reference, which is the first report to show the DRB inhibits initiation of Pol II in vivo. We will cite it in the revision.
(4) Again, Figures 4B, 4C, 5, and 6 are very difficult to understand - what is shown in these heat maps, and what is shown in the quantitation graphs on top?
Thanks for the suggestions, we will give a more detailed description of the Figures.
Reviewer #3 (Public review):
Summary:
Li et al. investigated the prevalence of acetylated and phosphorylated histones (using H3K9ac, H4K12ac, H3S10ph & H4S1ph as representative examples) across the gene body of human HEK293T cells, as well as mapping elongating Pol II and mRNA. They found that histone acetylation and phosphorylation were dominant in gene bodies of actively transcribing genes. Genes with acetylation/phosphorylation restricted to the promoter region were also observed. Furthermore, they investigated and reported a correlation between histone modifications and Pol II activity, finding that inhibition of Pol II activity reduced acetylation/phosphorylation levels, while resuming Pol II activity restored them. The authors then proposed a model in which panacetylation or pan-phosphorylation of histones generates fragile nucleosomes; the first round of transcription is accompanied by panacetylation, while subsequent rounds are accompanied by panphosphorylation.
Strengths:
This study addresses a highly significant problem in gene regulation. The author provided riveting evidence that certain histone acetylation and/or phosphorylation within the gene body is correlated with Pol II transcription. The author furthermore made a compelling case that such transcriptionally correlated histone modification is dynamic and can be regulated by Pol II activity. This work has provided a clearer view of the connection between epigenetics and Pol II transcription.
Thanks for the insightful comments, which are exactly what we want to present in this manuscript.
Weaknesses:
The title of the manuscript, "Fragile nucleosomes are essential for RNA Polymerase II to transcribe in eukaryotes", suggests that fragile nucleosomes lead to transcription. While this study shows a correlation between histone modifications in gene bodies and transcription elongation, a causal relationship between the two has not been demonstrated.
Thanks for the suggestions. What we want to express is that the generation of fragile nucleosomes precedes transcription, or, more specifically, transcription elongation. The corresponding PI wrote a hypothetical model on how pan-acetylation is generated by the coupling of chromatin remodelers and acetyltransferase complexes along gene bodies, in which chromatin remodelers act as drivers to carry acetyltransferases along gene bodies to generate pan-acetylation of nucleosomes (PMID: 41425263). We have a series of work to show how “tailless nucleosomes” at +1 from transcription start sites are generated to release paused Pol II in metazoans (PMID: 28847961) (PMID: 29459673) (PMID: 32747552) (PMID: 32048991). We still do not know how pan-phosphorylation along gene bodies is generated. It should be one of the focuses of our future research.
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