Antisense non-coding transcription represses the PHO5 model gene at the level of promoter chromatin structure

  1. Ana Novačić
  2. Dario Menéndez
  3. Jurica Ljubas
  4. Slobodan Barbarić
  5. Françoise Stutz
  6. Julien Soudet
  7. Igor Stuparević

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Abstract

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis , and use of the CRISPRi system. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.

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  1. Version published to 10.1371/journal.pgen.1010432
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    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    Reply to the reviewers

    We wish to thank all three reviewers for their thorough examination of our manuscript and their constructive criticism that allowed us to increase its quality. You will see that, following their recommendations, we have included a good amount of new data in the manuscript. Specifically, we added a new figure with experiments proposed by the reviewers (now Fig. 4), as well as Figs. S3 and S4. In addition, we expanded one paragraph of our Discussion to comment on a very recent article published by Huang et al in Nature Structural and Molecular Biology with conclusions pertaining the interplay of Rpd3 and Gcn5 in PHO5 gene regulation. Below we include the point-by-point response (in blue) with the changes we have implemented to address their specific points. All the additions and changes in the manuscript are made in red.

    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Novačić et al., investigate into a mechanisms of the non-coding transcriptiondriven regulation of the phosphate-responsive PHO5 gene. The authors employ CRSPRi system to discern direct contribution of the antisense non-coding transcription (CUT025) expressed during phosphate -rich conditions to transcriptional repression of the yeast PHO5 gene and therefore challenging previous study from the Svejstrup's lab that proposed a positive role for non-coding transcription in control of PHO5 gene. They propose a model where non-coding transcription represses PHO5 by mediating recruitment of Rpd3 histone deacetylase leading to altered chromatin structure at PHO5 promoter due to reduced recruitment of the RSC chromatin remodelling complex. Overall, the data presented in the manuscript are of a good quality, experiments are well controlled and nicely presented. Manuscript is well written. My specific comments are below: 1. I am somewhat confused by the data presented in Figure 5. While there is similar impact on the chromatin structure seen in rrp6D and air1Dair2D strains (Fig 5C) that corresponds to more "closed" configuration of chromatin , it is not consistent with H3 ChIP data that show higher nucleosome occupancy across PHO5 UAS in rrp6D but loss of nucleosomes in the double mutant (or there is a mistake perhaps while plotting the data?)

    We now realize that the data was plotted confusingly, and we apologize for it. While doing the H3 ChIP experiment we only prepared the +Pi samples for the air1Δ air2Δ double mutant. In the figure we only included this one data point for the double mutant, which could lead to the false conclusion that at other timepoints there are no histones at its PHO5 promoter region. We decide to remove this data point from the figure to avoid the confusion and only keep the air1Δ air2Δ data for the ClaI assay. We believe that this should not be an issue as this data point is not critical for the conclusions we are making.

    1. To further explore direct link between nc transcription, Rpd3 and rrp6 mediated effect, I suggest to test the effect on PHO5 induction upon rpd3 and rrp6 deletions in CRISPRi CUT025 background.

    We performed this experiment and now include it as Fig. S3 in the manuscript. As expected, expressing the CRISPRi system only made difference when Rpd3 was present.

    1. It seems that most noticeable effect of blocking nc transcription by an elegant approach that utilizes CRISPRi system on the phosphatase activity is seen between 0-1.5h of induction. I suggest taking additional time points at 30-45 min.

    We took additional timepoints and the results were incorporated as the new Fig. 5E. The CRISPRi effect resulting in higher acid phosphatase activity was still most noticeable after 1,5 h of induction. This was mostly in line with the fact that the difference in PHO5 mRNA levels was most pronounced after 30 min of induction (Fig. 5D), as the time needed to achieve measurable protein level after induction can lag significantly for secretory proteins, such as acid phosphatase. Secretory proteins are cotranslationally translocated into the ER, after which they traverse the secretory pathway and undergo modifications before being finally exported to the periplasm where their activity can be measured. Consequently, the increase in acid phosphatase activity upon induction is only measurable after at least an hour.

    1. How do authors explain that the effect of the exosome mutations are reversed and phosphatase activity is increased at later time point (20 h, Fig 2A)? I suggest using more distinct colour for dis3 mutants.

    That effect is indeed somewhat surprising. We hypothesize that the effects we are seeing after 20 h reflect the specific conditions of prolonged induction, i.e. keeping the chromatin open or semi-open for a very long period of time, which do not necessarily reflect the early gene induction period that we are using as a read-out of the effect of different mutations on acid phosphatase expression kinetics. We previously noticed a similar effect with chromatin remodeler-related mutants (e.g. rsc2Δ, unpublished result from S. Barbarić group), which speak in favour of the prolonged induction conditions resulting in a chromatin state with its own specialized cofactor requirements. We therefore consider the chromatin state after prolonged induction a topic for another study, however, we now comment on this effect in the manuscript. The dis3 mutants are now shown in more distinct colours.

    1. Figure 5A -label "H3 ChIP"

    The label was added.

    1. Error bars are quite high in Fig 1C, perhaps it is worth repeating the experiment

    Since significant differences in PHO5 mRNA levels can be seen between wt and rrp6Δ mutant cells at 0,75 and 3 h of induction, we feel that the higher error bars at 5 h of induction are not worth repeating the experiment – especially since the values are bound to converge to a similar one after a longer induction period, as demonstrated in Fig. 1D.

    Significance

    significant of interest for general audience

    Referee #2

    Evidence, reproducibility and clarity

    The authors study the PHO5 locus, which is known to a have antisense transcript and that has previously been shown the be important for activation of Pho5 sense transcription. The authors challenge the idea by an extensive analyses. They show the Pho5-AS represses sense transcription, and thus fits in the category as AS repressors instead of activators. They show a correlative data that when antisense goes down and sense goes up. They show that increase antisense levels leads to decrease sense levels. They use mutants of decay pathways to increase the levels antisense transcription. Moreover, they used crispri to repress the antisense transcript. Lastly, they show that histone deacetylation represses Pho5 sense. The data in the manuscript is convincing, and well presented. One thing that needs further clarification is the strategy to increase anti-sense levels by deletion mutants of decay or depletion of decay pathways. While it is clear that this stabilizes the pho5-AS and decrease pho5-sense, it is not clear that this causes an increase in transcription. Perhaps, it is possible that antisense transcript itself has a repressive effect. If one really wanted to increase antisense transcription than the antisense promoter should be increased in strength. On the other the CriprI experiment is very convincing. I am surprised how well the crisprI system works, it is thought to be not so efficient at blocking elongating polymerase and good at blocking initiation.

    We thank the reviewer for this feedback. We performed additional experiments which you will find described below. Based on the results, we would like to keep the point about AS transcription causing the effect.

    Major comments: - Are the key conclusions convincing? Perhaps, the conclusion that increased transcription leads to repression is not completely convincing. The authors use mutants in rrp6, exosome, and nrd1 to increase Pho5-AS transcription elongation. However, I am always under impression that these mutants stabilize the transcript. And the authors acknowledge this in their manuscript. So how do you discriminate between increased stability versus increased elongation? I support the conclusion that inhibition of Pho5-AS leads to increase Pho5-S. However, increase in elongation is not directly demonstrated. While still possible, it is equally possible that a more stable pho5-AS transcript has a repressive an effect on Pho5-AS. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? See above. Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. If the authors want to keep the message that increased transcription of Pho5-AS leads to more repression that may need to consider additional experiments. For example, increasing transcription from the antisense promoter.

    We performed the proposed experiment and now include it in the manuscript as Fig. 4AB. Briefly, we inserted the strong constitutive TEF1 promoter in the antisense configuration downstream of the PHO5 gene ORF, so that it drives AS transcription. The results of this experiment very clearly show the inverse relationship between PHO5 mRNA and AS transcripts levels at +Pi conditions. Importantly, this strong constitutive AS transcription had an even more pronounced effect on PHO5 gene expression than deletion mutant backgrounds (in which, like in wt cells, the AS promoter is presumably weak), and did not allow for full level of PHO5 gene expression to be reached. To verify that the AS RNA itself does not have a regulatory role, but rather the act of its transcription represses the corresponding gene, we performed an additional experiment with appropriate diploid strains. The design of this experiment is standardly used to test whether an AS transcript can work in trans (for example see Nevers et al. 2018 NAR Fig. 6). This experiment is now included as Fig. 4C. Together, the results of these experiments paint a clear picture of AS transcription, and not AS level/stability itself, driving the repression of the PHO5 gene.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. To me this is an optional experiment, but it would benefit the manuscript
    • Are the data and the methods presented in such a way that they can be reproduced? yes - Are the experiments adequately replicated and statistical analysis adequate? yes

    Minor comments: - Specific experimental issues that are easily addressable.

    • Are prior studies referenced appropriately? yes
    • Are the text and figures clear and accurate? Yes - Do you have suggestions that would help the authors improve the presentation of their data and conclusions? no

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. The manuscript challenges previous work where it was claimed that Pho5-AS is important for activation of Pho5-S. As such, it is important work. In the field of noncoding the transcription the Pho5-AS fits in a class of AS transcript that has been well described.
    • Place the work in the context of the existing literature (provide references, where appropriate). See above.
    • State what audience might be interested in and influenced by the reported findings. In researchers in field of transcription, chromatin, and more specifically in yeast gene regulation.
    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Chromatin, transcription, yeast.

    Referee #3

    Evidence, reproducibility and clarity

    Novačić et al present a manuscript entitled "Antisense non-coding transcription represses the PHO5 model gene via remodeling of promoter chromatin structure" which is a locus-specific follow up to previous studies from Soudet and Stutz groups on genome-wide analysis of transcription interference mediated by antisense transcripts in S cerevisiae. Critically, the authors here employ a CRISPRi approach to reduce antisense transcription from reaching the PHO5 promoter and in doing so show that kinetics of PHO5 induction are increased as would be predicted from their previous model. Additionally, they show predicted epistasis between rpd3 and rrp6 on PHO5 expression and gcn5 and rrp6 that are consistent with their model. Comments are relatively minor but should be addressed. Introduction p3. "This mechanism was subsequently explored genome-wide in yeast, which revealed a group of genes that in the absence of Rrp6 accumulate AS RNAs and are silenced in an HDACdependent manner (14)." This sentence appears awkward- perhaps move "in the absence of Rrp6" to after "AS RNAs"?

    Corrected as proposed.

    p3 "Under a high phosphate concentration Pho4 undergoes phosphorylation by the cyclindependent-kinase (Pho80-Pho85)" Since "the" is used, don't use parentheses around Pho80-Pho85

    Corrected as proposed.

    Methods Give amount/concentration of glycine used in quenching formaldehyde for ChIP. Give the exact wash conditions and buffers not "extensively"

    All of those details are now provided in the manuscript. Figure 4C.

    Describe schematic in legend

    It is now described.

    Figure 4D. Indicate time of induction in legend.

    This was lacking for Figs. 4B-C (now 5B-C) so we added it there.

    Figure 5A. air∆ data are missing from later time points?

    Please see our first response to Reviewer 1. We removed the air1Δ air2Δ double mutant data, as we only had one data point for it in this assay.

    Figure 6. Legend needs to indicate what Pi conditions are. Since PHO5 expressed, appears to be low Pi. An issue that needs to be discussed is that rpd3∆ appears to decrease expression of PHO5 AS. Is this simply because of increased PHO5 expression? Does rpd3∆ have any effects on AS in high Pi? This is important to interpret if effects of rrp6 and rpd3 are epistatic or additive.

    We thank the Reviewer for bringing this to our attention. To explore the effect of rpd3Δ on PHO5 AS level, we quantified the PHO5 AS transcript by RT-qPCR with cells grown in (chemically defined) high Pi medium, which we now include in Fig. 7A. We find that rpd3Δ mutation has practically no effect on PHO5 AS transcript level both in the wt and the rrp6Δ mutant background. This result speaks in favor of rrp6Δ and rpd3Δ being epistatic rather than additive.

    Figure 7. Sth1-CHEC data are hard to interpret. Some sort of quantification might be required as effects are not clear from the browser track nor is it clear from browser track that the results are reproducible. Examination of Sth1-AA effects in gcn5∆ background might be more compelling that the effect on RSC is via acetylation. Otherwise it is a bit hard to say as RSC could be functioning in parallel to the acetylation-dependent pathways implicated.

    We agree that the presumption that histone acetylation recruits RSC to the PHO5 gene promoter had to be tested. We therefore include the experiment involving Sth1-AA depletion in the gcn5Δ background as Fig. 8A. This experiment was complicated by the fact that RSC is highly abundant (and at the same time essential for cell viability), but we resolved this by starting to deplete RSC two hours before gene induction. These results position RSC and Gcn5 in the same pathway. In contrast, more complete Sth1 depletion severely impaired viability of the rrp6Δ mutant, making it hard to interpret the effect, so we now include this result as Fig. S4.

    To show the effect of AS transcription on RSC recruitment to the PHO5 promoter more quantitatively, we re-analyzed the Sth1-CHEC data (for two independent biological replicates) and now include the log2 values for the changes in Sth1 binding in the text of the manuscript.

    Significance

    The work is focused and narrower in impact but important because direct tests of locus-specific effects are performed, validating models from previous genomic analyses. **Referees cross-commenting**

    I think the other reviews are very reasonable. I would just suggest to the authors that they think carefully about the reviews and decide what they think is most valuable to improving the work/presentation

  3. Review Commons

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

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    Referee #3

    Evidence, reproducibility and clarity

    Novačić et al present a manuscript entitled "Antisense non-coding transcription represses the PHO5 model gene via remodeling of promoter chromatin structure" which is a locus-specific follow up to previous studies from Soudet and Stutz groups on genome-wide analysis of transcription interference mediated by antisense transcripts in S cerevisiae. Critically, the authors here employ a CRISPRi approach to reduce antisense transcription from reaching the PHO5 promoter and in doing so show that kinetics of PHO5 induction are increased as would be predicted from their previous model. Additionally, they show predicted epistasis between rpd3 and rrp6 on PHO5 expression and gcn5 and rrp6 that are consistent with their model. Comments are relatively minor but should be addressed.

    Introduction

    p3. "This mechanism was subsequently explored genome-wide in yeast, which revealed a group of genes that in the absence of Rrp6 accumulate AS RNAs and are silenced in an HDAC-dependent manner (14)."

    This sentence appears awkward- perhaps move "in the absence of Rrp6" to after "AS RNAs"?

    p3 "Under a high phosphate concentration Pho4 undergoes phosphorylation by the cyclin-dependent-kinase (Pho80-Pho85)"

    Since "the" is used, don't use parentheses around Pho80-Pho85

    Methods

    Give amount/concentration of glycine used in quenching formaldehyde for ChIP. Give the exact wash conditions and buffers not "extensively"

    Figure 4C. Describe schematic in legend

    Figure 4D. Indicate time of induction in legend.

    Figure 5A. air∆ data are missing from later time points?

    Figure 6. Legend needs to indicate what Pi conditions are. Since PHO5 expressed, appears to be low Pi. An issue that needs to be discussed is that rpd3∆ appears to decrease expression of PHO5 AS. Is this simply because of increased PHO5 expression? Does rpd3∆ have any effects on AS in high Pi? This is important to interpret if effects of rrp6 and rpd3 are epistatic or additive.

    Figure 7. Sth1-CHEC data are hard to interpret. Some sort of quantification might be required as effects are not clear from the browser track nor is it clear from browser track that the results are reproducible. Examination of Sth1-AA effects in gcn5∆ background might be more compelling that the effect on RSC is via acetylation. Otherwise it is a bit hard to say as RSC could be functioning in parallel to the acetylation-dependent pathways implicated.

    Significance

    The work is focused and narrower in impact but important because direct tests of locus-specific effects are performed, validating models from previous genomic analyses.

    Referees cross-commenting

    I think the other reviews are very reasonable. I would just suggest to the authors that they think carefully about the reviews and decide what they think is most valuable to improving the work/presentation

  4. Review Commons

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

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    Referee #2

    Evidence, reproducibility and clarity

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    The authors study the PHO5 locus, which is known to a have antisense transcript and that has previously been shown the be important for activation of Pho5 sense transcription. The authors challenge the idea by an extensive analyses. They show the Pho5-AS represses sense transcription, and thus fits in the category as AS repressors instead of activators. They show a correlative data that when antisense goes down and sense goes up. They show that increase antisense levels leads to decrease sense levels. They use mutants of decay pathways to increase the levels antisense transcription. Moreover, they used crispri to repress the antisense transcript. Lastly, they show that histone deacetylation represses Pho5 sense.

    The data in the manuscript is convincing, and well presented. One thing that needs further clarification is the strategy to increase anti-sense levels by deletion mutants of decay or depletion of decay pathways. While it is clear that this stabilizes the pho5-AS and decrease pho5-sense, it is not clear that this causes an increase in transcription. Perhaps, it is possible that antisense transcript itself has a repressive effect. If one really wanted to increase antisense transcription than the antisense promoter should be increased in strength. On the other the CriprI experiment is very convincing. I am surprised how well the crisprI system works, it is thought to be not so efficient at blocking elongating polymerase and good at blocking initiation.

    Major comments:

    • Are the key conclusions convincing?

    Perhaps, the conclusion that increased transcription leads to repression is not completely convincing. The authors use mutants in rrp6, exosome, and nrd1 to increase Pho5-AS transcription elongation. However, I am always under impression that these mutants stabilize the transcript. And the authors acknowledge this in their manuscript. So how do you discriminate between increased stability versus increased elongation? I support the conclusion that inhibition of Pho5-AS leads to increase Pho5-S. However, increase in elongation is not directly demonstrated. While still possible, it is equally possible that a more stable pho5-AS transcript has a repressive an effect on Pho5-AS.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    See above.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    If the authors want to keep the message that increased transcription of Pho5-AS leads to more repression that may need to consider additional experiments. For example, increasing transcription from the antisense promoter.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    To me this is an optional experiment, but it would benefit the manuscript

    • Are the data and the methods presented in such a way that they can be reproduced?

    yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    yes

    Minor comments:

    • Specific experimental issues that are easily addressable.
    • Are prior studies referenced appropriately?

    yes

    • Are the text and figures clear and accurate?

    Yes

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    no

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    The manuscript challenges previous work where it was claimed that Pho5-AS is important for activation of Pho5-S. As such, it is important work. In the field of noncoding the transcription the Pho5-AS fits in a class of AS transcript that has been well described.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    See above.

    • State what audience might be interested in and influenced by the reported findings.

    In researchers in field of transcription, chromatin, and more specifically in yeast gene regulation.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Chromatin, transcription, yeast.

  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 #1

    Evidence, reproducibility and clarity

    In this manuscript, Novačić et al., investigate into a mechanisms of the non-coding transcription-driven regulation of the phosphate-responsive PHO5 gene. The authors employ CRSPRi system to discern direct contribution of the antisense non-coding transcription (CUT025) expressed during phosphate -rich conditions to transcriptional repression of the yeast PHO5 gene and therefore challenging previous study from the Svejstrup's lab that proposed a positive role for non-coding transcription in control of PHO5 gene. They propose a model where non-coding transcription represses PHO5 by mediating recruitment of Rpd3 histone deacetylase leading to altered chromatin structure at PHO5 promoter due to reduced recruitment of the RSC chromatin remodelling complex.

    Overall, the data presented in the manuscript are of a good quality, experiments are well controlled and nicely presented. Manuscript is well written. My specific comments are below:

    1. I am somewhat confused by the data presented in Figure 5. While there is similar impact on the chromatin structure seen in rrp6D and air1Dair2D strains (Fig 5C) that corresponds to more "closed" configuration of chromatin , it is not consistent with H3 ChIP data that show higher nucleosome occupancy across PHO5 UAS in rrp6D but loss of nucleosomes in the double mutant (or there is a mistake perhaps while plotting the data?)
    2. To further explore direct link between nc transcription, Rpd3 and rrp6 mediated effect, I suggest to test the effect on PHO5 induction upon rpd3 and rrp6 deletions in CRISPRi CUT025 background.
    3. It seems that most noticeable effect of blocking nc transcription by an elegant approach that utilizes CRISPRi system on the phosphatase activity is seen between 0-1.5h of induction. I suggest taking additional time points at 30-45 min.
    4. How do authors explain that the effect of the exosome mutations are reversed and phosphatase activity is increased at later time point (20 h, Fig 2A)? I suggest using more distinct colour for dis3 mutants.
    5. Figure 5A -label "H3 ChIP"
    6. Error bars are quite high in Fig 1C, perhaps it is worth repeating the experiment

    Significance

    significant

    of interest for general audience

  6. Version published to 10.1101/2022.02.21.481265v2 on bioRxiv
  7. Version published to 10.1101/2022.02.21.481265v1 on bioRxiv