High-Resolution Genome-Wide Maps Reveal Widespread Presence of Torsional Insulation
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eLife Assessment
The Twin Domain model proposed by Lui and Wang proposing that twin supercoiling domains of DNA emerge during transcription were first described decades ago, but direct experimental evidence has been challenging to obtain. Here, the authors make a fundamental contribution by directly measuring DNA torsion in cells using a photoactivatable intrastrand cross-linker compared to controls. They gather compelling data using this clever method, which provides direct evidence in support of the twin-supercoiled domain model, for torsional effects at transcription start and end sites, and thereby uncover novel features of higher order structure of chromatin in yeast. These data are exciting, and the tools will be of interest to anyone studying chromosome structure and gene regulation.
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Abstract
Torsional stress in chromatin plays a fundamental role in cellular functions, influencing key processes such as transcription, replication, and chromatin organization. Transcription and other processes may generate and be regulated by torsional stress. In the genome, the interplay of these processes creates complicated patterns of both positive (+) and negative (-) torsion. However, a challenge in generating an accurate torsion map is determining the zero-torsion baseline signal, which is conflated with chromatin accessibility. Here, we introduce a high-resolution method based on the intercalator trimethylpsoralen (TMP) to address this challenge. We describe a method to establish the zero-torsion baseline while preserving the chromatin state of the genome of S. cerevisiae . This approach enables both high-resolution mapping of accessibility and torsional stress in chromatin in the cell. Our analysis shows transcription-generated torsional domains consistent with the twin-supercoiled-domain model of transcription and suggests a role for torsional stress in recruiting topoisomerases and in regulating 3D genome architecture via cohesin. Significantly, we reveal that insulator sequence-specific transcription factors decouple torsion between divergent promoters, whereas torsion spreads between divergent promoters lacking these factors, suggesting that torsion serves as a regulatory mechanism in these regions. Although insulators are known to decouple gene expression, our finding provides a physical explanation of how such decoupling may occur. This new method provides a potential path forward for using TMP to measure torsional stress in the genome without the confounding contribution of accessibility in chromatin.
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eLife Assessment
The Twin Domain model proposed by Lui and Wang proposing that twin supercoiling domains of DNA emerge during transcription were first described decades ago, but direct experimental evidence has been challenging to obtain. Here, the authors make a fundamental contribution by directly measuring DNA torsion in cells using a photoactivatable intrastrand cross-linker compared to controls. They gather compelling data using this clever method, which provides direct evidence in support of the twin-supercoiled domain model, for torsional effects at transcription start and end sites, and thereby uncover novel features of higher order structure of chromatin in yeast. These data are exciting, and the tools will be of interest to anyone studying chromosome structure and gene regulation.
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Reviewer #1 (Public review):
Summary:
The manuscript by Hall et al reports a genome-wide map of supercoiling in yeast using psoralen as a probe that intercalates more effectively into underwound DNA and can then be fixed in place by UV-cross-linking. Sites of cross-linking are revealed by exonuclease digestion and sequencing. Cross-linking is compared with samples that are first fixed with formaldehyde, permeabilized, digested with Dpn II to release unrestrained torsion, and then crosslinked. The authors promote this "zero-torsion" approach as an improvement that corrects for nucleosomes (or binding by other macromolecules) that mask psoralen binding. The investigators then examine patterns of psoralen binding (and hence supercoiling) that are associated with promoter strength, promoter type (sequence-specific transcription factor …
Reviewer #1 (Public review):
Summary:
The manuscript by Hall et al reports a genome-wide map of supercoiling in yeast using psoralen as a probe that intercalates more effectively into underwound DNA and can then be fixed in place by UV-cross-linking. Sites of cross-linking are revealed by exonuclease digestion and sequencing. Cross-linking is compared with samples that are first fixed with formaldehyde, permeabilized, digested with Dpn II to release unrestrained torsion, and then crosslinked. The authors promote this "zero-torsion" approach as an improvement that corrects for nucleosomes (or binding by other macromolecules) that mask psoralen binding. The investigators then examine patterns of psoralen binding (and hence supercoiling) that are associated with promoter strength, promoter type (sequence-specific transcription factor dependent, insulator associated, or general TFs only) and gene length.
Strengths:
This is an interesting paper that reports an approach that reveals some new information about the relationship between torsional stress and gene activity in the yeast genome. The method is logical and interesting and provides evidence that spread of torsional stress through the genome is regulated.
Weaknesses:
The analysis is not entirely novel, and I believe that more valuable information can be culled from these datasets than is reported here.
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Reviewer #2 (Public review):
Summary:
This study describes a novel method for mapping torsional stress in the genome of Saccharomyces cerevisiae using trimethylpsoralen (TMP). It introduces a procedure to establish a zero-torsion baseline while preserving the chromatin state by treating cells with formaldehyde before releasing torsion with restriction enzyme digestion.
This approach allows foer more accurate differentiation between torsional stress effects and accessibility effects in the psoralen signal. The results confirm that psoralen crosslinking is strongly affected by accessibility of the DNA and to a much more limited extent by the torsional stress of the DNA. Subtracting the baseline signal (no torsion) from the total signal allows detecting torsional stress, although TMP accessibility is still affecting the read out. The …
Reviewer #2 (Public review):
Summary:
This study describes a novel method for mapping torsional stress in the genome of Saccharomyces cerevisiae using trimethylpsoralen (TMP). It introduces a procedure to establish a zero-torsion baseline while preserving the chromatin state by treating cells with formaldehyde before releasing torsion with restriction enzyme digestion.
This approach allows foer more accurate differentiation between torsional stress effects and accessibility effects in the psoralen signal. The results confirm that psoralen crosslinking is strongly affected by accessibility of the DNA and to a much more limited extent by the torsional stress of the DNA. Subtracting the baseline signal (no torsion) from the total signal allows detecting torsional stress, although TMP accessibility is still affecting the read out. The authors confirm the validity of the method by studying torsional stress in dependence of transcription levels, gene length and relative gene orientation. They propose that torsional stress may play a role in recruiting topoisomerases and regulating 3D genome architecture via cohesin. They also suggest that transcription factor binding might insulate negative supercoiling originated form transcription of neighboring divergent genes.
Strengths:
This paper offers a potentially interesting tool for future work.
Weaknesses:
The signal-to-background ratio, which represents the torsional fraction, appears to be quite limited relative to the overall signal (roughly 20x less, according to the scales in figs 2a and 2b, raising concerns about the robustness of the conclusions. It is clear from these figures, for instance, that a non-negligible fraction of the remaining signal is still dependent on DNA accessibility, revealing the nucleosomes footprints in spite of the fact that subtracting the zero-torsion signal should theoretically hinder the accessibility component. Because of this, some of the conclusions might be flawed, in that what is attributed to torsional stress might in reality be due, partially or fully, to accessibility issues.
Specific points:
Lines 226-227: "rotation may be more restricted with a lengthening in the RNA transcript, which is known to be associated with large machinery, such as spliceosomes". This argument is not appropriate to correlate torsional stress with gene length. Spliced genes are rare and generally short in yeast, generally in ribosomal proteins genes.
Lines 256-257 In discussing that torsional stress must hinder Pol II progression, the authors write: "Pol II has a minimal presence in the intergenic region between divergent genes and is enriched in the intergenic region between convergent genes, consistent with a previous finding that after termination, Pol II tends to remain on the DNA downstream of the terminator". The connection between Pol II distribution and torsional stress is unclear. Pol ii is depleted at promoters and is enriched at at 3'-end of convergent genes most likely because this ChIP signal is the sum of signals from the two convergent genes. The fact that positive torsional stress is observed in these region does not mean that polymerases accumulate because the torsional stress hinder Pol II progression. To claim elongation defects the authors should repeat the same analysis with stranded data (e.g. NET-seq or CRAC) and assess if polymerases transcribing these regions accumulate more when facing convergent genes compared to tandem genes. The claim that after termination the Pol II tends to remain on the DNA appears to be meaningless - the authors probably mean after RNA processing.
Lines 275-277: "These data provide evidence that the (+) supercoiling generated by transcription may facilitate genome folding in coordination with other participating proteins". This is an overstatement. It is known that cohesins accumulate between convergent genes. The fact that there is torsional stress in the same position does not imply that supercoiling participates in genome folding. These could be independent events, or even, supercoiling might depend on cohesins
Lines 289-290 "torsion generated from one gene can impact the expression of its neighboring gene, consistent with previous findings that the expression of these genes is coupled" the existence of negative torsional stress in a common intergenic region for two genes does not imply that torsion is causally associated to gene expression coupling
Lines 291-292: "Another large class of S. cerevisiae promoters (termed "TFO") are regulated by insulator ssTFs, such as Reb1 and Abf1, which decouple interactions between neighbouring genes" In these cases and others that depend on an activator binding the authors detect a region of accessibility interrupted by a valley, which they interpret as a topological insulator. However, the valley might be generated because of decreased TMP accessibility due to of TF binding.
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Reviewer #3 (Public review):
Summary:
The authors describe a new method for measuring DNA torsion in cells using the photoactivatable intrastrand cross-linker trimethyl psoralen (TMP). However, their method differs from previous TMP-based torsion mapping methods by comparing formaldehyde cross-linked and torsionally trapped chromatin to torsion-relieved (zero-torsion) chromatin in parallel. Comparison between the two datasets reveals a very slight difference, but enough to provide extremely high resolution genome-wide maps of torsion in the yeast genome. This direct comparison of the two maps confirms that blockage of TMP binding by nucleosomes and some DNA-binding proteins from TMP intercalation is a major complication of previous methods, and analysis of the data provides a glimpse of chromatin-based processes from within the DNA gyre.
Reviewer #3 (Public review):
Summary:
The authors describe a new method for measuring DNA torsion in cells using the photoactivatable intrastrand cross-linker trimethyl psoralen (TMP). However, their method differs from previous TMP-based torsion mapping methods by comparing formaldehyde cross-linked and torsionally trapped chromatin to torsion-relieved (zero-torsion) chromatin in parallel. Comparison between the two datasets reveals a very slight difference, but enough to provide extremely high resolution genome-wide maps of torsion in the yeast genome. This direct comparison of the two maps confirms that blockage of TMP binding by nucleosomes and some DNA-binding proteins from TMP intercalation is a major complication of previous methods, and analysis of the data provides a glimpse of chromatin-based processes from within the DNA gyre.
Strengths:
In addition to providing direct evidence for the twin-supercoiled domain model and for torsional effects at transcription start (TSS) and end (TES) sites, the authors' analyses reveal some novel features of yeast higher-order structure. These include the cohesin-dependent anchoring of DNA loops at sites of positive supercoiling and the insulation of torsion between closely spaced divergent genes by general transcription factors, which implies that these factors resist free rotation. The fact that method should be generalizable to complex eukaryotic cells with large genomes, and the implications for understanding how torsion impacts transcription and gene regulation will be of substantial interest to a broad community.
Weaknesses:
No serious weaknesses.
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