Cell cycle-regulated transcriptional pausing of Drosophila replication-dependent histone genes

  1. James P. Kemp
  2. Mark S. Geisler
  3. Mia Hoover
  4. Chun-Yi Cho
  5. Patrick H. O’Farrell
  6. William F. Marzluff
  7. Robert J. Duronio

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Abstract

Coordinated expression of replication-dependent (RD) histones genes occurs within the Histone Locus Body (HLB) during S phase, but the molecular steps in transcription that are cell cycle regulated are unknown. We report that Drosophila RNA Pol II promotes HLB formation and is enriched in the HLB outside of S phase, including G1-arrested cells that do not transcribe RD histone genes. In contrast, the transcription elongation factor Spt6 is enriched in HLBs only during S phase. Proliferating cells in the wing and eye primordium express full-length histone mRNAs during S phase but express only short nascent transcripts in cells in G1 or G2 consistent with these transcripts being paused and then terminated. Full-length transcripts are produced when Cyclin E/Cdk2 is activated as cells enter S phase. Thus, activation of transcription elongation by Cyclin E/Cdk2 and not recruitment of RNA pol II to the HLB is the critical step that links histone gene expression to cell cycle progression in Drosophila .

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  1. Review Commons

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    I thank the Referees for their...

    Referee #1

    1. The authors should provide more information when...

    Responses

    • The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A).
    • Though this is not stated in the MS
    1. Figure 6: Why has only...

    Response: We expanded the comparison

    Minor comments:

    1. The text contains several...

    Response: We added...

    Referee #2

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Kemp et al. aimed to explore the transcriptional cell cycle regulation of replication-dependent (RD) histone genes at histone locus body (HLB) in Drosophila. They evaluate the accumulation of RNA pol II and RD histone transcripts at HLB during the cell cycle using live and fixed imaging of Drosophila tissues at different stages of development. They find that RNA pol II is enriched at HLB, not only during S phase when RD histone genes are transcribed but throughout the cell cycle. Outside of S phase, they detect short but not full-length RD histone transcripts suggesting a mechanism of RNA pol II pausing. Full length RD transcripts are only produced upon cyclin E/Cdk2 activation when cells enter S phase, arguing that Cyclin E/Cdk2 can activate transcription elongation. They propose that the elongation release triggered by Cyclin E/Cdk2 is the critical step linking RD histone gene expression and cell cycle progression rather than the recruitment of RNA pol II to HLB. The data are interesting and robust, however, additional experiments could reinforce the findings and the proposed model.

    Specific comments/concerns are listed below.

    1. In Figure 3, quantifications of the fluorescence at HLBs for mCherry-RBP1 and MXC-mScarlet should be provided.
    2. In Figure 5C, both 5' and 3' transcripts are observed in G214 cells. However, their accumulation in the cytoplasm is not visible. How do the authors explain this result? What happens in S14 cells?
    3. In Figure 6, the authors observed RD histone 3' transcripts only in replicating cells (EdU positive) while they detected 5' transcripts in both replicating and non-replicating cells. They argue that the appearance of 3' transcripts is due to the release from transcriptional pausing. To further support particular states in the transcriptional arrest, data by immunofluorescence using specific antibodies recognizing either RNA pol II ser5P or ser2P would determine whether the presence of 3' transcripts is associated with the accumulation at HLB of RNA pol II ser2P (elongating polymerase). Moreover, is there a correlation between P-MXC and RNA pol II ser2P?
    4. In Figure 7 panels C and D, the 5' transcripts should be shown. Although RD histone 3' transcripts accumulate in CyE+ embryonic cells, unfortunately, their presence at HLBs (pointed by arrows) is not visible in the image of panel E. To firm up conclusions quantifications of the 3' and 5' transcripts should be provided for CycE+ and CycEnull cells. In Hur et al., 2020, the authors looked at RD histone transcripts in WT embryo and CycE+/-/Cdk2+/- mutant. They found that the amount of H3 transcripts using a probe corresponding to the coding sequence is not changed in the mutant as compared to the WT. In contrast, they found that there is an increase of transcripts that are not correctly processed using probes downstream the stem-loop region. This seems inconsistent with the results presented here where a decrease of 3' transcripts is observed. This needs an explanation/discussion. Are such incorrectly processed transcripts observed in CycEnull mutant?
    5. The authors suggest that active Cyclin E/Cdk2 triggers the release of RNA pol II promoter-proximal pausing and therefore induces transcriptional elongation at RD histone genes when cells enter S phase. To further support this hypothesis, determining whether there is an enrichment of the elongation factor p-TEFb at HLB when Cyclin E/Cdk2 is active would help.
    6. Instead of using cycling E mutants, to determine whether it is the phosphorylation of MXC which directly impacts the elongation of RD histone genes, it would be interesting to generate phospho-null or phospho-mimetic mutant of MXC.
    7. In Suzuki et al., 2022, the authors described 3' RNA pol II pausing at RD histone genes. Although this study used human cells, it would be interesting to discuss that in addition to a promoter-proximal pausing that regulates transcription elongation, a 3' pausing could also regulate the transcription termination and 3' processing.
    8. In the discussion, the authors should point out some limitations of their studies linked to the method and could propose for the future that a more precise and molecular view of the pausing mechanism could be carried out using sequencing methods such as ChIP-seq of various isoforms of the RNA pol II (total, ser2P, ser5P) and elongation regulators (p-TEFb.....) and PRO-seq.

    Minor points:

    1. In Figure 1, for panels B and D as well as for panels C and E, to falicitate comparison of the localization of the different proteins, it would help to show the same developmental stages and the same image scales.
    2. In Figures 3 and 7 (C-F), the developmental stages should be indicated on the images, as it is done in the other figures.
    3. In the legend of Figure 7, it is indicated (D) and (E) instead of (C) and (D) in the sentence: "Endocycling midgut cells in (D) contain cytoplasmic histone mRNA which is absent in (E) (boxed regions)."

    Significance

    Kemp et al. aimed to explore the transcriptional cell cycle regulation of replication-dependent (RD) histone genes at histone locus body (HLB) in Drosophila. They evaluate the accumulation of RNA pol II and RD histone transcripts at HLB during the cell cycle using live and fixed imaging of Drosophila tissues at different stages of development. They find that RNA pol II is enriched at HLB, not only during S phase when RD histone genes are transcribed but throughout the cell cycle. Outside of S phase, they detect short but not full-length RD histone transcripts suggesting a mechanism of RNA pol II pausing. Full length RD transcripts are only produced upon cyclin E/Cdk2 activation when cells enter S phase, arguing that Cyclin E/Cdk2 can activate transcription elongation. They propose that the elongation release triggered by Cyclin E/Cdk2 is the critical step linking RD histone gene expression and cell cycle progression rather than the recruitment of RNA pol II to HLB.

    The data are interesting and robust, however, additional experiments could reinforce the findings and the proposed model.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Kemp et al. seek to define the molecular interactions that limit replication-dependent histone gene transcription to S-phase of the cell cycle. They use the Drosophila model system and leverage live-imaging tools, such as tagged proteins and Jabba trap, and RNA FISH in several tissues to determine that RNA Pol II is enriched at the locus throughout the cell cycle and is paused outside of S-phase. Therefore, they conclude that it is not Pol II recruitment to the locus that couples histone transcription to S-phase, but release of Pol II pausing.

    Major comments:

    The data presented are clean and well-presented. The claims are supported by the data without exaggeration. It would be helpful to provide -omics support for this entirely image-based analysis (e.g. PRO- or GRO-seq data from synchronized, sorted Drosophila cells may already exist- OPTIONAL).

    A major requirement is that the authors make clear in Introduction and Discussion that the observation of Pol Ii pausing at RD histone genes is not novel. This requires, at minimum, a discussion of Liu (2024) and Suzuki (2022). This allows readers to focus on the advance novel to this work, which is specifically the cell cycle coupling of Pol II pausing.

    As the authors are claiming different dynamics between Spt6 and RPB1 in Figure 1, they should provide similarly-staged embryos for comparison. For example, the authors should show RPB1 in early/mid S of NC 14, as this is when they see Spt6 variability. In theory, this should be relatively easy as these are stills from the live videos.

    Minor comments:

    The use of Spt6 live imaging early on was slightly confusing. The authors should consider moving this data later in the results or providing more written justification for why they investigated Spt6 (further than "to further explore the regulation of RNA pol II dynamics... p6). Similarly, Spt6 is included in the model figure, which might be a stretch given the only Spt6 data involves the timing of Spt6 colocalization with Mxc during the cell cycle.

    Misleading language/missed citations:

    p3: "600 kB array" is misleading. The whole locus is ~ 600 kB.

    p3: Mxc may remain at the locus throughout the cell cycle, so the whole HLB does not disassemble (Terzo, 2015).

    p4: H1-specific factors include cramped (Gibert and Karch, 2011; Bodner et al. 2024 bioRxiv)

    p4: Hodkinson, 2023 is not the correct reference. The correct reference is Hodkinson, 2024, Genetics.

    p5: The Drosophila HLB is detectable at NC 10 (White, 2011; Terzo, 2015) not White, 2007

    p5: A typo: "imagining"

    p7: The section title "RNA pol II is necessary for HLB assembly" is incorrect, as Figure 3 shows that pol II is NOT necessary for Mxc recruitment, but for HLB growth. Mxc, however, is necessary for pol II recruitment.

    p9: The authors should clarify what "HisC" means as this is the first usage.

    Figures/experiments:

    Fig 2: The authors should show the gating in Figure I that led to the three categories in Figure J. The legend/colors in Figure J are not necessary.

    An "easy" experiment would be to use the FUCCI cell lines and 5'/3' RNA FISH in combination (assuming fluorophores allow) - OPTIONAL

    Discussion:

    p13: The reference to the work of Gugliemlmi, 2013 should first come up in the Introduction, as it provides rationale.

    p13: "without engaging in transcription" is misleading, as pol II is transcribing, but paused.

    p15: It makes sense for pol II to pause at histone genes in G1, as they are preparing for the rapid burst of histone transcription needed in S phase. But what might be the functional rationale for pol II pausing in G2, if the HLB disassembles in M?

    Methods:

    It should be made clear how embryos were staged for live imaging, as it is likely by timing after cell cycle events. What is this timing? It would be best if this detail is not just mentioned in the methods, but also in the main text. This is especially important for readers not familiar with Drosophila embryogenesis. Please cite/acknowledge DGRC for Fly-FUCCI line (if appropriate)

    Significance

    This study provides convincing evidence that pol II is enriched at the histone locus and paused outside of S-phase. What limits the significance is that several prior studies concluded that Pol II is paused at the histone locus:

    Lu et al. bioRxiv 2024, "Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription"

    Suzuki et al. Nat Comm 2022, "The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies' association with histone locus bodies"

    Neither of these studies is discussed or even cited in the manuscript, which is disappointing. Therefore, the advance is limited to the cell-cycle coupling of pausing. This is still important, as a major knowledge gap as outlined by the authors is that it is not clear how histone transcription is coupled to S-phase and they rule out Pol II pausing as a possible mechanism, and point toward Pol II pausing release.

    Moreover, there is also evidence (from these authors) that Mxc phosphorylation is not always coupled to histone transcription in Drosophila ovaries. This work is also not discussed or cited:

    Potter-Birriel et al. J Cell Sci 2021, "A region of SLBP outside the mRNA-processing domain is essential for deposition of histone mRNA into the Drosophila egg"

    The current research may be of interest to the broad cell cycle field, but it may also be useful as a model for those conducting basic, foundational research who seek to describe how Pol II is released from pausing. The histone locus may be of interest as a novel, facile model for pausing.

    Reviewer expertise: Drosophila, chromatin, gene expression

  4. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Reply to the Reviewers

    I thank the Referees for their...

    Referee #1

    1. The authors should provide more information when...

    Responses

    • The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A).
    • Though this is not stated in the MS
    1. Figure 6: Why has only...

    Response: We expanded the comparison

    Minor comments:

    1. The text contains several...

    Response: We added...

    Referee #2

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Kemp et al. aimed to explore the transcriptional cell cycle regulation of replication-dependent (RD) histone genes at histone locus body (HLB) in Drosophila. They evaluate the accumulation of RNA pol II and RD histone transcripts at HLB during the cell cycle using live and fixed imaging of Drosophila tissues at different stages of development. They find that RNA pol II is enriched at HLB, not only during S phase when RD histone genes are transcribed but throughout the cell cycle. Outside of S phase, they detect short but not full-length RD histone transcripts suggesting a mechanism of RNA pol II pausing. Full length RD transcripts are only produced upon cyclin E/Cdk2 activation when cells enter S phase, arguing that Cyclin E/Cdk2 can activate transcription elongation. They propose that the elongation release triggered by Cyclin E/Cdk2 is the critical step linking RD histone gene expression and cell cycle progression rather than the recruitment of RNA pol II to HLB. The data are interesting and robust, however, additional experiments could reinforce the findings and the proposed model.

    Specific comments/concerns are listed below.

    1. In Figure 3, quantifications of the fluorescence at HLBs for mCherry-RBP1 and MXC-mScarlet should be provided.
    2. In Figure 5C, both 5' and 3' transcripts are observed in G214 cells. However, their accumulation in the cytoplasm is not visible. How do the authors explain this result? What happens in S14 cells?
    3. In Figure 6, the authors observed RD histone 3' transcripts only in replicating cells (EdU positive) while they detected 5' transcripts in both replicating and non-replicating cells. They argue that the appearance of 3' transcripts is due to the release from transcriptional pausing. To further support particular states in the transcriptional arrest, data by immunofluorescence using specific antibodies recognizing either RNA pol II ser5P or ser2P would determine whether the presence of 3' transcripts is associated with the accumulation at HLB of RNA pol II ser2P (elongating polymerase). Moreover, is there a correlation between P-MXC and RNA pol II ser2P?
    4. In Figure 7 panels C and D, the 5' transcripts should be shown. Although RD histone 3' transcripts accumulate in CyE+ embryonic cells, unfortunately, their presence at HLBs (pointed by arrows) is not visible in the image of panel E. To firm up conclusions quantifications of the 3' and 5' transcripts should be provided for CycE+ and CycEnull cells. In Hur et al., 2020, the authors looked at RD histone transcripts in WT embryo and CycE+/-/Cdk2+/- mutant. They found that the amount of H3 transcripts using a probe corresponding to the coding sequence is not changed in the mutant as compared to the WT. In contrast, they found that there is an increase of transcripts that are not correctly processed using probes downstream the stem-loop region. This seems inconsistent with the results presented here where a decrease of 3' transcripts is observed. This needs an explanation/discussion. Are such incorrectly processed transcripts observed in CycEnull mutant?
    5. The authors suggest that active Cyclin E/Cdk2 triggers the release of RNA pol II promoter-proximal pausing and therefore induces transcriptional elongation at RD histone genes when cells enter S phase. To further support this hypothesis, determining whether there is an enrichment of the elongation factor p-TEFb at HLB when Cyclin E/Cdk2 is active would help.
    6. Instead of using cycling E mutants, to determine whether it is the phosphorylation of MXC which directly impacts the elongation of RD histone genes, it would be interesting to generate phospho-null or phospho-mimetic mutant of MXC.
    7. In Suzuki et al., 2022, the authors described 3' RNA pol II pausing at RD histone genes. Although this study used human cells, it would be interesting to discuss that in addition to a promoter-proximal pausing that regulates transcription elongation, a 3' pausing could also regulate the transcription termination and 3' processing.
    8. In the discussion, the authors should point out some limitations of their studies linked to the method and could propose for the future that a more precise and molecular view of the pausing mechanism could be carried out using sequencing methods such as ChIP-seq of various isoforms of the RNA pol II (total, ser2P, ser5P) and elongation regulators (p-TEFb.....) and PRO-seq.

    Minor points:

    1. In Figure 1, for panels B and D as well as for panels C and E, to falicitate comparison of the localization of the different proteins, it would help to show the same developmental stages and the same image scales.
    2. In Figures 3 and 7 (C-F), the developmental stages should be indicated on the images, as it is done in the other figures.
    3. In the legend of Figure 7, it is indicated (D) and (E) instead of (C) and (D) in the sentence: "Endocycling midgut cells in (D) contain cytoplasmic histone mRNA which is absent in (E) (boxed regions)."

    Significance

    Kemp et al. aimed to explore the transcriptional cell cycle regulation of replication-dependent (RD) histone genes at histone locus body (HLB) in Drosophila. They evaluate the accumulation of RNA pol II and RD histone transcripts at HLB during the cell cycle using live and fixed imaging of Drosophila tissues at different stages of development. They find that RNA pol II is enriched at HLB, not only during S phase when RD histone genes are transcribed but throughout the cell cycle. Outside of S phase, they detect short but not full-length RD histone transcripts suggesting a mechanism of RNA pol II pausing. Full length RD transcripts are only produced upon cyclin E/Cdk2 activation when cells enter S phase, arguing that Cyclin E/Cdk2 can activate transcription elongation. They propose that the elongation release triggered by Cyclin E/Cdk2 is the critical step linking RD histone gene expression and cell cycle progression rather than the recruitment of RNA pol II to HLB.

    The data are interesting and robust, however, additional experiments could reinforce the findings and the proposed model.

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Kemp et al. seek to define the molecular interactions that limit replication-dependent histone gene transcription to S-phase of the cell cycle. They use the Drosophila model system and leverage live-imaging tools, such as tagged proteins and Jabba trap, and RNA FISH in several tissues to determine that RNA Pol II is enriched at the locus throughout the cell cycle and is paused outside of S-phase. Therefore, they conclude that it is not Pol II recruitment to the locus that couples histone transcription to S-phase, but release of Pol II pausing.

    Major comments:

    The data presented are clean and well-presented. The claims are supported by the data without exaggeration. It would be helpful to provide -omics support for this entirely image-based analysis (e.g. PRO- or GRO-seq data from synchronized, sorted Drosophila cells may already exist- OPTIONAL).

    A major requirement is that the authors make clear in Introduction and Discussion that the observation of Pol Ii pausing at RD histone genes is not novel. This requires, at minimum, a discussion of Liu (2024) and Suzuki (2022). This allows readers to focus on the advance novel to this work, which is specifically the cell cycle coupling of Pol II pausing.

    As the authors are claiming different dynamics between Spt6 and RPB1 in Figure 1, they should provide similarly-staged embryos for comparison. For example, the authors should show RPB1 in early/mid S of NC 14, as this is when they see Spt6 variability. In theory, this should be relatively easy as these are stills from the live videos.

    Minor comments:

    The use of Spt6 live imaging early on was slightly confusing. The authors should consider moving this data later in the results or providing more written justification for why they investigated Spt6 (further than "to further explore the regulation of RNA pol II dynamics... p6). Similarly, Spt6 is included in the model figure, which might be a stretch given the only Spt6 data involves the timing of Spt6 colocalization with Mxc during the cell cycle.

    Misleading language/missed citations:

    p3: "600 kB array" is misleading. The whole locus is ~ 600 kB.

    p3: Mxc may remain at the locus throughout the cell cycle, so the whole HLB does not disassemble (Terzo, 2015).

    p4: H1-specific factors include cramped (Gibert and Karch, 2011; Bodner et al. 2024 bioRxiv)

    p4: Hodkinson, 2023 is not the correct reference. The correct reference is Hodkinson, 2024, Genetics.

    p5: The Drosophila HLB is detectable at NC 10 (White, 2011; Terzo, 2015) not White, 2007

    p5: A typo: "imagining"

    p7: The section title "RNA pol II is necessary for HLB assembly" is incorrect, as Figure 3 shows that pol II is NOT necessary for Mxc recruitment, but for HLB growth. Mxc, however, is necessary for pol II recruitment.

    p9: The authors should clarify what "HisC" means as this is the first usage.

    Figures/experiments:

    Fig 2: The authors should show the gating in Figure I that led to the three categories in Figure J. The legend/colors in Figure J are not necessary.

    An "easy" experiment would be to use the FUCCI cell lines and 5'/3' RNA FISH in combination (assuming fluorophores allow) - OPTIONAL

    Discussion:

    p13: The reference to the work of Gugliemlmi, 2013 should first come up in the Introduction, as it provides rationale.

    p13: "without engaging in transcription" is misleading, as pol II is transcribing, but paused.

    p15: It makes sense for pol II to pause at histone genes in G1, as they are preparing for the rapid burst of histone transcription needed in S phase. But what might be the functional rationale for pol II pausing in G2, if the HLB disassembles in M?

    Methods:

    It should be made clear how embryos were staged for live imaging, as it is likely by timing after cell cycle events. What is this timing? It would be best if this detail is not just mentioned in the methods, but also in the main text. This is especially important for readers not familiar with Drosophila embryogenesis. Please cite/acknowledge DGRC for Fly-FUCCI line (if appropriate)

    Significance

    This study provides convincing evidence that pol II is enriched at the histone locus and paused outside of S-phase. What limits the significance is that several prior studies concluded that Pol II is paused at the histone locus:

    Lu et al. bioRxiv 2024, "Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription"

    Suzuki et al. Nat Comm 2022, "The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies' association with histone locus bodies"

    Neither of these studies is discussed or even cited in the manuscript, which is disappointing. Therefore, the advance is limited to the cell-cycle coupling of pausing. This is still important, as a major knowledge gap as outlined by the authors is that it is not clear how histone transcription is coupled to S-phase and they rule out Pol II pausing as a possible mechanism, and point toward Pol II pausing release.

    Moreover, there is also evidence (from these authors) that Mxc phosphorylation is not always coupled to histone transcription in Drosophila ovaries. This work is also not discussed or cited:

    Potter-Birriel et al. J Cell Sci 2021, "A region of SLBP outside the mRNA-processing domain is essential for deposition of histone mRNA into the Drosophila egg"

    The current research may be of interest to the broad cell cycle field, but it may also be useful as a model for those conducting basic, foundational research who seek to describe how Pol II is released from pausing. The histone locus may be of interest as a novel, facile model for pausing.

    Reviewer expertise: Drosophila, chromatin, gene expression

  7. Version published to 10.1101/2024.12.16.628706 on bioRxiv