Nucleosome Positioning Shapes Cryptic Antisense Transcription
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Abstract
Maintaining transcriptional fidelity is essential for precise gene regulation and genome stability. Despite this, cryptic antisense transcription, occurring opposite to canonical coding sequences, is a pervasive feature across all domains of life. How such potentially harmful cryptic sites are regulated remains incompletely understood. Here, we show that nucleosome arrays within gene bodies play a key role in suppressing cryptic transcription. Using the fission yeast Schizosaccharomyces pombe as a model, we demonstrate that CHD1-family chromatin remodelers coordinate with the transcription elongation machinery, specifically the PAF complex, to position nucleosomes at sites of cryptic transcription initiation within gene bodies. In the absence of CHD1, AT-rich sequences within gene bodies lose nucleosome occupancy, exposing promoter-like sequences that drive cryptic initiation. While cryptic transcription is generally detrimental, we identify a subset of antisense transcripts that encode critical meiotic genes, suggesting that cryptic transcription can also serve as a source of regulatory innovation. These findings underscore the essential role of nucleosome remodelers in maintaining transcriptional fidelity and reveal their broader contributions to cellular homeostasis and evolutionary adaptability.
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Referee #3
Evidence, reproducibility and clarity
Summary:
In the manuscript "Nucleosome positioning shapes cryptic antisense transcription", Kok and colleagues perform a characterization of nucleosome remodeling factors in S. pombe by assaying the impact of their deletion on antisense transcription and nucleosome organization. They find that deletion of Hrp3 leads to up-regulation of antisense RNA transcripts as well as disruption of phased nucleosomes in gene bodies. The authors then establish a catalogue of antisense transcripts in S. pombe using long read RNA sequencing, which they use to analyze the relationship between nucleosome positioning and antisense transcription. …
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Referee #3
Evidence, reproducibility and clarity
Summary:
In the manuscript "Nucleosome positioning shapes cryptic antisense transcription", Kok and colleagues perform a characterization of nucleosome remodeling factors in S. pombe by assaying the impact of their deletion on antisense transcription and nucleosome organization. They find that deletion of Hrp3 leads to up-regulation of antisense RNA transcripts as well as disruption of phased nucleosomes in gene bodies. The authors then establish a catalogue of antisense transcripts in S. pombe using long read RNA sequencing, which they use to analyze the relationship between nucleosome positioning and antisense transcription. Through this analysis, they associate nucleosome positioning with the initiation of antisense transcription and conclude that nucleosome positioning within gene bodies represses cryptic antisense transcription. They further support this observation by showing that the up-regulated genes in the Hrp3 knock-out are enriched for genes usually expressed in meiosis, which in S. pombe often occur as nested transcripts in reverse orientation. Using growth assays under various stress conditions, the authors narrow down the domain responsible for the phenotype to the C-terminal CHCT domain. To address how Hrp3 gains specificity, they perform an in-silico interaction prediction screen to identify Prf1 as a putative interactor of the CHCT domain. Using recombinant expression in bacteria followed by pulldowns from lysates, they confirm the interaction and introduce point mutants that abolish the interaction. The authors then link the interaction with Prf1 to transcriptional elongation, where they observe a correlation between Hrp3 presence and chromatin marks of transcription elongation, especially H2BK119ub, which is also reduced in the Hrp3 knockout. They further demonstrate that both gene body nucleosome phasing and antisense transcription are similarly affected in the prf1 knockout as well as the hrp1-hrp3-prf1 triple knock-out cells, which indicates that they affect the same pathway.
Major comments:
The manuscript is well-written and the claims are generally supported by the data. The authors demonstrate scientific rigor through comprehensive experiments using single and double knockouts. I have three main comments that can be addressed through additional analysis and limited experimentation:
The authors use the terms "Prf1" and "Paf1 complex" interchangeably multiple times in the manuscript (eg. Line 296). However, the experimental data presented only demonstrate a connection between Prf1 and Hrp3. Furthermore, published literature establishes that Prf1 and Paf1 represent distinct entities in S. pombe (Mbogning et al., 2013, PLoS Genetics 9(3): e1004029). The authors should clarify this distinction and use consistent, accurate terminology throughout the text. Reference: Mbogning, J., et al. (2013). The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast. PLoS Genetics, 9(3), e1004029. https://doi.org/10.1371/journal.pgen.1004029
The authors demonstrate that Hrp3 limits antisense promoter usage; however, the analysis lacks characterization of sequence composition, promoter classes (TATA-box versus TATA-less), or identification of enriched transcription factor motifs near these sites. A more thorough bioinformatic analysis would strengthen the paper and potentially reveal interesting biology, as the effect may be specific to certain transcription factors or promoter architectures.
The Hrp3-Prf1 interaction is demonstrated solely through recombinant overexpression and pulldown assays, which carries the risk of detecting non-physiological interactions. While the authors use mutations to verify pulldown specificity, in vivo evidence for this interaction is absent. Given that the authors cite a recent preprint demonstrating sophisticated techniques to show S. cerevisiae Chd1-Prf1 interactions, I presume standard approaches such as co-immunoprecipitation followed by mass spectrometry or Western blot were attempted. Even negative results from such experiments should be reported, as readers will likely question the physiological relevance of the interaction. Additionally, establishing the hierarchy between Hrp3, Prf1, and H2BK119Ub is crucial. While the authors show that Hrp3 ChIP-seq signal correlates with gene expression levels, the proposed Prf1-Hrp3 interaction raises questions about recruitment specificity and hierarchy. The authors mention in lines 344-345: "...the CHCT domain of Hrp3 is critical for its association with transcription elongation along the gene body..." which requires support from experimental data. Testing Hrp3 ChIP-seq in Prf1-depleted conditions would clarify how specificity is achieved and substantiate the functional importance of this interaction. As the authors have all the required strains I would estimate around 1.5-2 months for data generation and analysis.
[Optional] Based on strucutre predictions the authors suggest that the interaction of of CHD1 and RTF1 is conserved in arabidopsis and mouse. This should be further supported by pulldown assays and also the pre-print (Reference nr. 99) should be cited as they show similar results using yeast-tow-hybrid assays
Minor comments:
Figure 1B: Grouping individual panels according to different paralog groups would make the figure more accessible.
Figure 1D: The display of antisense transcription is not accessible. Perhaps boxplots, like those in Figures 2B and 5D, would be easier to read.
Line 335: The transition is abrupt and would benefit from additional explanation. Why do the authors use Rtf1 instead of Prf1 here? Consistent nomenclature would improve clarity.
Line 352: For the phrase "significant loss," please provide a statistical test or omit the word "significant."
Figure 7F: The model presented in panel F suggests that there are two parallel routes that lead to nucleosome phasing; however, the authors state in the text (lines 363-364): "further supporting the idea that Hrp3 and Prf1 act together in the same pathway to control antisense transcription." The model and the text should align better.
Significance
In the study, the authors establish Hrp3, one of the fission yeast CHD1 remodelers, as a crucial regulator of antisense transcription within gene bodies, which they link to both fitness penalties and the regulation of genes typically expressed during meiosis. They further link the recruitment of Hrp3 at gene bodies to transcriptional elongation, which provides an interesting model for how antisense transcription is prevented in actively transcribed regions of the genome.
The study is overall very well executed and controlled and provides strong evidence for connecting Hrp3 with the repression of antisense transcription using adequate experiments and technologies. This provides novel insights into a widespread phenomenon present in many organisms. A point that needs further improvement is the suggested physical link between Hrp3 and Prf1. Despite potentially being challenging to address using molecular biology techniques, the authors can further improve the study by dissecting the genetic hierarchy of Hrp3 and Prf1 using accessible tools. This study will be of interest to a broad audience in basic research as it addresses the broad question of how antisense transcription is repressed and provides mechanistic insights into this process. Consequently, this study will be relevant for the broader field of transcriptional regulation and could provide entry points for studying the role of CHD remodelers in other organisms.
Field of expertise: chromatin biology, small RNA mediated heterochromatin formation
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Referee #2
Evidence, reproducibility and clarity
Kok et al. report on the role of the chromatin remodelers Hrp1 and Hrp3 in maintaining nucleosome positioning and preventing antisense transcription in Schizosaccharomyces pombe. As commented below, the main criticism of the manuscript is that the first half describes results that are very similar to those already reported by several other laboratories. Therefore, the main novel aspect of the work is the interaction between Hrp3 and the Prf1 subunit of the PAF complex.
Specific points:
The articles of Hennig et al. (2012), Pointner et al. (2012) and Shim et al. (2012) are cited in the manuscript (line 119, Refs. 61-63) only as a …
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Referee #2
Evidence, reproducibility and clarity
Kok et al. report on the role of the chromatin remodelers Hrp1 and Hrp3 in maintaining nucleosome positioning and preventing antisense transcription in Schizosaccharomyces pombe. As commented below, the main criticism of the manuscript is that the first half describes results that are very similar to those already reported by several other laboratories. Therefore, the main novel aspect of the work is the interaction between Hrp3 and the Prf1 subunit of the PAF complex.
Specific points:
The articles of Hennig et al. (2012), Pointner et al. (2012) and Shim et al. (2012) are cited in the manuscript (line 119, Refs. 61-63) only as a confirmation of the minor effect of the absence of Hrp1 on nucleosome positioning and antisense expression. However, these three articles reached the same conclusion as Kok et al. that the absence of Hrp3 in S. pombe causes severe, genome-wide loss of nucleosome positioning and overexpression of antisense transcripts, whereas the absence of Hrp1 has a much weaker effect. These results were also discussed in a short review article (Touat-Todeschini et al. EMBO J. 2012. 31: 4371). Although Kok et al. analysed transcription at a higher resolution and mapped transcription initiation using Pro-Seq (Figures 1, 2 and 3), their results do not add much to what was already reported in these previous studies.
Several sites in the manuscript state that Hrp3 belongs to the SWI/SNF family of chromatin remodelers (for example, line 92). However, Hrp3 is a member of the CHD family, whose members have a very different structure and function (see, for example, Clapier et al. 2017. Nat Rev Mol Cell Biol 18: 407; Paliwal et al. 2024 TIGs 41:236).
The authors should indicate where the nucleosome remodelling activity of some of the proteins in Figure 1A like Irc20, Rrp1, Rrp2 and Mot1) has been reported.
The analysis of nucleosome positioning by aggregating thousands of genes, such as those shown in Figure 1B, has low resolution and can only detect gross alterations affecting many genes. Nevertheless, several mutants, such as swr1∆ and rrp1∆, also exhibit altered nucleosomal profiles in Figure 1B. In other cases, the occupancy of the first and second nucleosomes after the TSS is reduced relative to the wild type. Therefore, it cannot be concluded that "nucleosome arrays in wild type and most remodeller mutant cells were highly ordered and regular" (line 105).
Although it was previously reported that hrp3∆ mutants overexpress antisense transcripts (see point 1 above), it is unclear how this finding is represented in Figure 1D. Similarly, it not clear either why antisense transcription is undetectable in hrp1∆ relative to WT in Figure 1D, yet significantly higher than in WT in Figures 2B, 3A and 3B. Furthermore, sense transcription in the single and double mutants is comparable to WT in Figure 2A, yet much higher in Figure S3B.
Figure S3C claims that antisense transcription is higher in genes with greater nucleosome disruption in the double mutant hrp1∆hrp3∆. However, without a quantitative analysis, it is difficult to discern any significant differences in the degree of disruption across the four quartiles of antisense expression.
Figures 3D and S4C show that the TSS of antisense transcription colocalizes with a region resistant to MNase that is at least 300 bp wide. This size does not correspond to that occupied by a nucleosome and contrasts with the expected size of the four nucleosome peaks downstream from it.
In relation to the previous point, Figure S4C (bottom) shows that the centre of the region above the TSS is slightly displaced in the three mutants. This displacement corresponds to an increase in the G+C content of approximately 1.5% (Figure S4C top), equivalent to an increase of less than 2.5 Gs and Cs every 150 bp of nucleosomal DNA. Without some cause and effect experiments, it is difficult to attribute a functional significance to such a tiny difference. How repetitive is this difference in biological replicates?
The authors should also explain how the position of the dyads was estimated in the double mutant hrp1∆hrp3∆ in Figure S4B. The severe loss of nucleosomal positioning suggests that the dyads occupy different positions in different cells within the same population. While most of the remaining figures show data for the three mutants, this figure shows results for the double hrp1∆hrp3∆ mutant only.
Figures 3G and 3H show the analysis of the promoter activity of some regions upstream from antisense transcripts, achieved by replacing the endogenous ura4 gene promoter with these regions. This analysis lacks negative controls showing the level of transcription in the recipient strain following the removal of the endogenous ura4 promoter and its replacement for genomic regions not associated with the initiation of antisense transcription in the mutants. Furthermore, transcription should be measured by quantitative PCR of the ura4 mRNA rather than by the more indirect method of measuring OD600 in 384-well plates (line 708).
Figure F4 suggests that Hrp3 may regulate the expression of genes specific to meiosis by showing an anticorrelation between the expression levels of Hrp3 and a selection of genes that are upregulated during meiosis (MUGs) 5 hours after the onset of meiosis. While this is an interesting possibility, it will remain speculative until it is demonstrated that the level of Hrp3 protein is reduced at the same stage of meiosis, and that MUG overexpression is associated with reduced nucleosomal occupancy adjacent to their TSS at that stage.
The experiments in Figures 5 and 6, which describe the interaction between the Hpr3-specific CHCT domain and the Prf1 protein, are interesting and represent the main element of novelty of the manuscript. However, this interaction in figure 6D and 6E should be confirmed in vivo.
Kok et al. indicate that the triple prf1∆ hrp1∆ hrp3∆ mutant exhibits stronger growth defects than the single prf1∆ mutant. However, Figure S9F shows that no growth is detectable in the single prf1∆ mutant, a phenotype that cannot be exacerbated in the triple mutant. Perhaps the use of a prf1 mutant showing a less severe phenotype migh help.
Significance
As indicated in point 1, the first half of the manuscript describes results that are very similar to those already reported in the literature.
The interaction between Hrp3 and the Prf1 subunit is new and interesting, and could lead to further research and a new manuscript.
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Referee #1
Evidence, reproducibility and clarity
This is an excellent study that leverages the chromatin biology of Schizosaccharomyces pombe to uncover the central role of CHD1-family remodelers in maintaining nucleosome organisation and suppressing cryptic transcription. The work is carefully executed. In short, the authors show that Hrp3 is the primary CHD1-family remodeler responsible for maintaining nucleosome organisation over gene bodies. This represses antisense transcription from cryptic promoters in gene bodies. They provide evidence that Hrp3 is repressed in meiosis to allow the induction of meiotic genes. They further identified that a conserved domain, the CHCT …
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 #1
Evidence, reproducibility and clarity
This is an excellent study that leverages the chromatin biology of Schizosaccharomyces pombe to uncover the central role of CHD1-family remodelers in maintaining nucleosome organisation and suppressing cryptic transcription. The work is carefully executed. In short, the authors show that Hrp3 is the primary CHD1-family remodeler responsible for maintaining nucleosome organisation over gene bodies. This represses antisense transcription from cryptic promoters in gene bodies. They provide evidence that Hrp3 is repressed in meiosis to allow the induction of meiotic genes. They further identified that a conserved domain, the CHCT domain of Hrp3, is essential for its interaction with Prf1 (PAF complex subunit), which is critical for the chromatin organisation in gene bodies. This manuscript is of excellent quality and is an important contribution towards understanding how transcription initiation is repressed within gene bodies. I have small comments and suggestions for clarification.
Minor comments:
The study demonstrates that Hrp3 represses antisense transcription at meiotic genes, showing that Hrp3 is reduced in meiosis, which could facilitate the induction of meiotic genes. Is there a phenotype in the hrp3Δ or the hrp1Δ hrp3Δ mutant in relation to meiosis? E.g. do these strains enter meiosis uncontrolled?
Figure 3C - ORC4 Locus TSS presentation. The presented data do not show a well-defined TSS on the sense strand. For reference, it would be useful to show that sense TSS is not altered between the different strains.
The study focuses on antisense cryptic transcription, which is relatively easy to measure by RNA-seq. Often, however, cryptic transcription can also occur in the sense direction in gene bodies. Do the authors also find evidence of cryptic sense transcription in gene bodies (based on TSS-seq data)? This could be useful for completeness to report, as this could lead to aberrant protein-coding isoforms.
The manuscript alternates between "Prf1" (S. pombe) and "RTF1" (other eukaryotes). This is at times confusing. I recommend consistent use of gene nomenclature.
The authors show epistatic interaction for nucleosome spacing in Figure 7D for the prf1Δ and hrp1Δ hrp3Δ prf1Δ strains. It would be informative to have the hrp1Δ hrp3Δ data also included in Figure 7D, like in the other figure panels.
Significance
This is an excellent study that leverages the chromatin biology of Schizosaccharomyces pombe to uncover the central role of CHD1-family remodelers in maintaining nucleosome organisation and suppressing cryptic transcription. This manuscript is of excellent quality and is an important contribution towards understanding how transcription initiation is repressed within gene bodies.
I am an expert on transcription regulation and noncoding transcription.
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