CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites
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Abstract
CUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (> 270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (< 120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.
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###Reviewer #2:
CUT&RUN, a recently developed method, is a convenient alternative to ChIP-seq. Because it generates a footprint of DNA protected from MNase digestion, it can potentially also provide more nuanced information than ChIP-seq. In this paper, CUT&RUN is applied to the mapping of RNA polymerase II (Pol II) binding sites in the genome of a human lung carcinoma cell line. A technical innovation in the current paper is that the authors bypass the attachment of cells to concanavalin A-magnetic beads for all steps from cell permeabilization on, and exploit the fact that the cells they use naturally adhere sufficiently well to the bottoms of multi-well plates that these steps can all be performed on the cell culture plates themselves.
In the original CUT&RUN paper, it was already pointed out that different size classes of protected …
###Reviewer #2:
CUT&RUN, a recently developed method, is a convenient alternative to ChIP-seq. Because it generates a footprint of DNA protected from MNase digestion, it can potentially also provide more nuanced information than ChIP-seq. In this paper, CUT&RUN is applied to the mapping of RNA polymerase II (Pol II) binding sites in the genome of a human lung carcinoma cell line. A technical innovation in the current paper is that the authors bypass the attachment of cells to concanavalin A-magnetic beads for all steps from cell permeabilization on, and exploit the fact that the cells they use naturally adhere sufficiently well to the bottoms of multi-well plates that these steps can all be performed on the cell culture plates themselves.
In the original CUT&RUN paper, it was already pointed out that different size classes of protected fragments might reveal different aspects of the biology of DNA bound factors. The authors of the current work extend this observation, and report two size classes of fragments that are produced by CUT&RUN applied to RNA polymerase II. They interpret the shorter fragments as marking Pol II sitting in a poised, compact state directly at the transcription start site (TSS), and the longer fragments downstream of the TSS as reflecting a less compact or larger, stalled Pol II complex after transcription has been initiated. This is consistent with what we know about regulation of nascent RNA elongation by Pol II shortly after transcription initiation, a phenomenon that has been known for individual genes since the 1980s, and that has first been documented genome-wide well over a decade ago.
In addition, the authors suggest that a substantial fraction of Pol II is also found in a paused/stalled/poised state upstream of the TSS. Unfortunately, it is unclear what the upstream signal reflects. E.g., is this pausing because of bi-directional transcription, or because of a separate pre-initiation complex or conformation? Without such insight, the observation does not add to our understanding of transcription initiation and elongation.
In aggregate, the authors present a simplification over conventional CUT&RUN for cell cultures, and they provide additional details for Pol II positioning near TSSs. While the work is technically well done, the technical improvements are relatively minor, and there are no principally new biological insights.
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###Reviewer #1:
The technical advance, which involves CUT&RUN on plates and doing paired end reads is modest. The main result of interest is the detection of a minor Pol II ChIP peak that maps around the transcriptional start site (TSS) as opposed to the major peak that corresponds to paused Pol II downstream from the TSS. The existence of the Pol II peak near the TSS is hardly surprising on first principles, and it is unknown what this peak corresponds to in terms of mechanism. The authors refer to this as "pre-initiation" and "poised", but there is no evidence for this. It is entirely possible (in my opinion more likely) that this peak corresponds to abortive initiation, a well-known step in the transcription cycle where Pol II makes short abortive transcripts that only occasionally get extended to longer products. It wasn't clear …
###Reviewer #1:
The technical advance, which involves CUT&RUN on plates and doing paired end reads is modest. The main result of interest is the detection of a minor Pol II ChIP peak that maps around the transcriptional start site (TSS) as opposed to the major peak that corresponds to paused Pol II downstream from the TSS. The existence of the Pol II peak near the TSS is hardly surprising on first principles, and it is unknown what this peak corresponds to in terms of mechanism. The authors refer to this as "pre-initiation" and "poised", but there is no evidence for this. It is entirely possible (in my opinion more likely) that this peak corresponds to abortive initiation, a well-known step in the transcription cycle where Pol II makes short abortive transcripts that only occasionally get extended to longer products. It wasn't clear what the CTD phosphorylation status of this TSS-linked Pol II is, but it seems like it was phosphorylated at serine 5 residues. If so, this would indicate that TFIIH had already mediated the phosphorylation, which would release Mediator and allow promoter escape. Whatever the explanation, the existence of the peak doesn't indicate anything about mechanism. Lastly, this TSS-linked peak has been seen by Erickson (2018) so the result per se isn't novel. The approach here is more physiological than Erickson, but this isn't a significant advance, especially since there is no mechanistic information.
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