S-phase transcriptional buffering quantified on two different promoters

Transcriptional buffering enforced during DNA replication shows that histone acetylation governs the homeostasis process and can also restrict promoters from reaching maximum transcriptional potential

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Sincerely, Andrea Leibfried, PhD Executive Editor Life Science Alliance Meyerhofstr. 1 69117 Heidelberg, Germany t +49 6221 8891 502 e a.leibfried@life-science-alliance.org www.life-science-alliance.org Reviewer #1 (Comments to the Authors (Required)): In this MS, Shav-Tal and colleagues applied a system for in-vivo visualization of gene transcription to examine how cells adjust their gene expression following replication. Previous work by this group, as well as by other groups, have shown that cells attenuate transcription from each duplicated allele, thereby maintaining a constant ('homeostatic') level of gene expression despite the biased change in gene dosage caused by gene duplication. Previous studies in yeast also implicated a role for H3K56ac in enabling this buffering. The authors now establish that also in mammalian cells, expression buffering during S phase depends on histone acetylation. while they do not study a specific acetylation, they show convincingly that cells which are subject to the general TSA inhibitor, are not able to maintain buffering, but maintain high expression of both replicated allele. They further show that this inhibitor increases the overall level of transcription, suggesting that some limiting factor is released. The data is truly beautiful and convincing. the question examined is important, and the results interesting. i strongly recommend publication Reviewer #2 (Comments to the Authors (Required)): Understanding the transcriptional activity correlated to cell cycle and DNA packing states is important to the epigenetic field. Single molecule microscopy allowing high resolution and real-time measurement of gene transcription at single cell and single chromosome level is unique to study complicated biological systems. The manuscript "S-phase transcriptional buffering quantified on two different promoters" submitted by Yunger et. al. is an interesting application of the papers "Single-allele analysis of transcription kinetics in living mammalian cells" published in Nature Methods in 2010 and "Quantifying the transcriptional output of single alleles in single living mammalian cells" published in Nature Protocols in 2013 from the same lab. In this work, the authors quantitatively determined the transcriptional rate in promoter-, cell cycle-and DNA packingdependent manner. This paper is technically sound and suitable for publication in Life Science Alliance if the following issues are addressed. Figure 2A lead to bright nuclei and cytoplasm (instead of spotty-like patterns) in RNA FISH. Were these images used to count the number of cellular mRNAs ( Figure 2C)? If so, how many cellular RNAs were determined in these cells? 2. Figure 3: In the absence of TSA, the transcription rate of CCND1-MS2 at single site state is faster than the transcription rate at doublet states (8{plus minus} 2 vs. 4{plus minus} 3 transcripts/gene). However, it does not contribute to the total amount of CCND1-MS2 RNAs. The authors should discuss potential reasons in the manuscript. 3. Figure 5: It is not clear how these relative intensity plots were generated. 4. The terms describing cell cycle stage is not precise. The authors defined G1 stage by single transcription site and "after S" stage by duplicated transcription sites. However, the single transcription site can be found in both G1 and early S cell cycle stages (G1/early S). It is unclear what the "after S stage" means. 5. The authors claimed increased transcription caused by TSA treatment is dose-dependent, about 3~5 fold increase in nascent mRNA on the duplicated genes at different TSA concentration ( Figure  S2B). A common and sensitive approach (such as qRT-PCR) should be performed to confirm the sensitivity of imaging-based quantification. 6. While it might be obvious to FISH experts how the RNA FISH probes are labeled, it is not trivial for general readers who have little or no experience on FISH techniques. How to generate labeled FISH probes or where to purchase labeled FISH probes is important information to reproduce the experiments. Also, they used FISH probes conjugated with five fluorophores (three internal and one at each end). Was any control experiment (e.g., single molecule/dye bleaching steps) performed to demonstrate the average number of dyes incorporated into individual probes? Such information is important for quantification purpose.

The high concentrations of mRNAs in
Minor points: 7. Material and Method section should be revised with more details: (1) the source of original cell line (2) Filters/dichroic used in the microscope system? (3) At what temperature the immunofluorescence experiment was carried out? (4) Buffers used to make 4% PFA and 5% BSA in immunofluorescence experiments (5) The brand/name of the mounting medium (6) the version of Imaris software? (7) Were the cells tested for mycoplasma contamination and how? 8. Although insertion of MS2 cassettes is a well-known method to label mRNAs in living cells, modifications have been made to make MS2 aptamer and the coat protein suitable for different purposes. The authors should indicate which MS2 cassette was used in this study by either citing literature or providing the sequence information. 9. The authors never mention what TSA stands for.
1st Authors' Response to Reviewers: September 5, 2018 Points raised by reviewer 2: 1. The reviewer is correct. In the original figure we focused on showing the transcription sites and the differences in them between control and TSA-treated cells. We did not focus on the mRNA signals, and agree that the mRNAs should be clearly shown as well. We now provide images that reflect the mRNA signals too, in cells that were used in the actual quantifications. We also added the deconvolved images that are used in the process of quantification. The number of mRNAs is mentioned in the legend. 2. We have added text with an explanation regarding the total numbers of mRNAs in the cells, as follows: "We note that although there is a decrease in the transcriptional output of the alleles after replication from 8±2 nascent mRNAs for the single alleles in G1, to 4±3 nascent mRNAs for doublets after replication (before TSA treatment), the cellular levels of the mRNAs remain generally the same, since each of the two alleles is simultaneously producing half of the mRNAs that a single allele does." 3. We now refer to the protocol for generating the intensity plots where this is explained in detail, in the Methods section. 4. This is a valid point. In order to be precise we have modified the text in the appropriate places from "after S" to "after replication of the alleles". 5. As suggested we have added a qRT-PCR experiment (performed at least 3 times) in Fig. S2 and included information in the Methods section. 6. The information about the specific probe that we use, its sequence, labeling, and how we use it in detail, appear in our two very detailed protocol papers. Indeed, this was not clear enough in the Methods section of the manuscript and so we have now elaborated on where to find all the exhaustive information necessary to perform these experiments, in a way that will be clear also to people who are less knowledgeable with the FISH technique, such that anyone can repeat such experiments.
7. In continuation to the above we have added the requested details: (1) Source of original cell line -Flp-In HEK293 cells (Invitrogen,. (2) We listed the filters that we use on our microscope system. (3) IF was performed at room temperature. (4) PFA and BSA are dissolved in PBS.