Adenoviral chromatin organization primes for early gene activation

  1. Uwe Schwartz
  2. Tetsuro Komatsu
  3. Claudia Huber
  4. Floriane Lagadec
  5. Conradin Baumgartl
  6. Elisabeth Silberhorn
  7. Margit Nuetzel
  8. Fabienne Rayne
  9. Eugenia Basyuk
  10. Edouard Bertrand
  11. Michael Rehli
  12. Harald Wodrich
  13. Gernot Laengst

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Abstract

Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unknown. Here, we combined RNA-seq, MNase-seq, ChIP-seq and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the viral genome is arranged in precisely positioned nucleoprotein particles (Adenosomes), like nucleosomes. We identified 238 Adenosomes, being positioned by a DNA sequence code and protecting about 60 to 70bp of DNA. The incoming genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replace pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites, preceding transcriptional activation. Based on our results we propose a central role for the viral pVII nucleoprotein-architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.

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    Reply to the reviewers

    Manuscript number: RC- 2023-01819

    Corresponding author(s): Gernot Längst and Harald Wodrich

    Full revision of the manuscript

    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    2. Point-by-point description of the revisions

    Dear Reviewers, thank you very much for your appreciation of our study and your input. In this point-to-point response, we amended our text marked in blue colour.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The authors have addressed the nucleoprotein structure of human adenovirus during the very early stages of infection, and its relationship to onset of expression of viral genes, using a combination of RNA-seq, MNase-seq, ChIP-seq and single genome imaging. They show that in the virion and the newly-infecting DNA, protein VII is precisely position at specific sites on the viral DNA, with greater accessibility at early gene promoters compared to other regions. Nucleosomes containing H3.3 replace specific protein VII at distinct positions at the transcription start sites of genes, which are then acetylated. Association with histones and nucleosomes occurs prior to transcription. These studies confirm and greatly expand on results already in the literature, and also elucidate a novel role for protein VII in orchestrating positioning of nucleosomes prior to initiation of transcription.

    The authors provide excellent data in support of their conclusions and, in many instances, use alternative experiments (i.e. two different approaches) to support their claims. The details of methods are adequate (with small exceptions outlined below) and statistical methods appropriate.

    Minor comments:

    Line 561 "Protein VII molecules were exchanged for positioned nucleosomes at the +1 site of actively transcribed genes". This statement seems to suggest that the +1 position almost acts as a nucleating site, where replacement of a single, specific protein VII molecule at +1 is an initiating event, which then spreads from that site and into the rest of the gene. Data shown in Figure 6G and 6H shows that H3.3 appears to be found equally along the full length of E1A as early as 1 hr post infection (with no real "enhancement" at the +1 position), and that the overall levels simply increase over the next 4 hrs.

    As the reviewer pointed out, the histone ChIP-seq peaks are broader than the +1 nucleosome region, extending into the transcribed regions of the gene. This is expected, as the mean length of the immunoprecipitated DNA is about 400bp long. Still, ChIP-seq peaks are in proximity to the transcription start site and overlap with the position of the +1 nucleosome. As we do not have the required resolution, we toned done our statement. The text now reads as follows: “Protein VII molecules were exchanged for nucleosomes downstream of the transcription start site, overlapping the +1 nucleosome site, of actively transcribed genes“ (line 568 ff).

    Curiously, the authors chose not to use a wildtype virus for their studies - the virus contains a deletion in the E3 region. For clarity, I suggest that the authors should preferentially use an alternative designation for their virus rather than HAd-C5. Perhaps HAd-C5delE3 to differentiate this work from studies that truly use wildtype virus.

    As requested by the reviewer we have updated the nomenclature to HAd-C5dE3 throughout the text and the figures.

    The obvious limitation of the studies using the fluorescent TAF1-beta to label Ad genomes is that as protein VII is replaced by nucleosomes, the genomes would have declining detection by this method. Genomes devoid of protein VII would be "invisible".

    Our MNase data show that within the first 4h only a fraction of pVII is removed from the viral genome e.g. at early genes, while most of the genome remains bound by protein VII. This should provide enough binding sites for TAF1-beta to label Ad genomes without a significant drop in the signal. Furthermore, our recent work (PMID:29997215, Fig. 1D) compared the TAF1-beta labelling system with a second in vivo detection system (AnchOR3) that directly labels the viral DNA independently of protein VII in the same cells. This direct comparison of two technically non-related methods to detect individual incoming adenoviral genomes in living cells showed the equivalence of both methods, at least for the first hours of infection showing that partial removal of protein VII does not affect the fluorescent TAF1-beta staining.

    Line 275 "Interestingly, a central region of the viral genome (Late3) and a region between the E3 and E4 genes exhibited almost no peaks" for protein VII. The virus utilized in this study lacked at least part of the E3 region. Did this deletion "cause" this region to be devoid of protein VII? Is the same absence of protein VII peaks observed in a fully wildtype virus? Also, can the authors provide any speculation as to why the Late3 region also lacks protein VII?

    We confirm the reviewer's observation. The region marked as Late3 and the region between E3 and E4 is present in the genome and is, as the reviewer observed, not chromatinized in our analysis. At this point, we can only speculate. We have two not mutually exclusive hypotheses. First, both regions could be involved in the proper packaging of the viral genome into the capsid. Physical constraints during packaging may preclude this region from being packaged into pVII. Second, as we observed that pVII positioning correlates with distinct DNA sequence patterns (revised Fig.4 D and E, see response to reviewer 3 for details), it might be that the sequence composition at the pVII depleted regions disfavour pVII assembly to keep those regions available for cellular factors that drive processes post genome delivery, such as transcription. Our time-resolved MNase analysis shows that indeed post genome delivery, this site in the Late3 region becomes protected (Fig. 5C), suggesting the binding of one or more cellular factors. As shown in Figure S6 we find conserved binding sites for several transcription factors at this MNase protected site.

    Whether the chromatinization devoid regions would shift in position, remain in place or be chromatinized in a wildtype virus has to be addressed in the future and cannot be answered at this point. To address the comment, we have expanded the discussion (line 620 ff)

    Line 569 "Reasons could be that the few genomes undergoing nucleosome assembly and active transcription produce the replication enzymes, whereas the bulk of genomes enters replication without activation as an elegant way to avoid repeated chromatinization." This argument may make sense in the context of a high MOI infection, but would certainly limit virus function during normal, pathogenic infection where the MOI is likely extremely low. Essentially, the authors data predicts that 80% of normal, low MOI infections don't progress to gene expression (at least during the first 4 hrs analyzed in this study).

    We follow the argument of the reviewer. The high MOI in our study was necessary to perform the combined ‘omics’ approach to arrive at meaningful data within reasonable sequencing depth. To have equivalence we also used high MOI for the imaging approach. A detailed analysis for the effect of low MOI as well as positioning effects (see reviewer comment below) on transcriptional activation is an important question and will be addressed in future studies that require different techniques in addition. To address this comment, we have updated the discussion to emphasize the importance of MOI and positioning effects (line 587 ff).

    Line 576 "This observation is in agreement with recent pVII-ChIP experiments showing transcription and replication independent pVII removal in early infection (Giberson et al., 2018; Komatsu and Nagata, 2012; Komatsu et al., 2011)." The authors can also state that histone and nucleosome deposition is also independent of transcription and replication, as has been alluded to in the same cited studies but proven more directly in this study.

    We have changed the text accordingly (line 576 and 598).

    Line 672 - the authors should be more definitive in the MOI that are used in all of their experiments. Line 672 states that an MOI of 3000 physical particles are applied per cell. There can be great variation between cell lines in how much virus binds to (and enters) a cell based on the surface levels of Ad receptors on different cell types. However, in general, 3000 is very high. Work by Wang et al. (PMID:24139403) showed that at an MOI of 200 or below most Ad will traffic correctly to the nucleus, whereas at an MOI above 200 there is a significant defect in Ad trafficking within the cell. How is this expected to affect all of the results in this study?

    We agree with this and the other reviewer that this is an important issue. The actual dose of virus that enters a given cell is dependent on the concentration of virus particles in the inoculum and the time and temperature this inoculum is in contact with the cells and the cells respective susceptibility to the virus. We applied an infection dose of 3000 physical particles per cell in a defined volume (1ml) at 37˚C for 30min followed by inoculum removal. We prefer this description because with these infection conditions, we find on average well below 100 virus particles that enter the cell (=> This is e.g., reflected in the number of accumulating genomes shown in figure 2A). In contrast, this permits to have enough viruses inside the cell to perform the different “omics” techniques applied in our study to obtain meaningful results at reasonable sequencing depths. This experimental setting was carefully chosen in full awareness of the work by Wang et al., cited by the reviewer, to avoid e.g., overloading the nuclear import rate. Thus, our experimental conditions do not exceed the “MOI of >200” that would affect nuclear import rates. The number (>200) in the Wang et al. study refers to the number of virus particles inside the cell, the infection condition used in the Wang study was an MOI of 30 bound to Hela cells in the cold for 30min and warmed for 150min which is significantly more virus than we have used in our study. We have expanded the information on the MOI used in the material and methods section to clarify this point (line 685 ff).

    Figure 5 is of low resolution and was difficult to read.

    We thank the reviewer for spotting it. It seems that the Figure quality was compromised during the PDF conversion. We updated the Figures and checked the resolution after PDF conversion.

    Figure S3 is missing a box from the top set of images indicating the region that is expanded in the detail picture.

    We updated Figure S3

    While I realize it is supplemental data, the difference in quality between the agarose gels shown in Figure S4A and S5A is shocking.

    The nature of the experiments is very different and therefore the expected MNase digestion profiles on agarose gels look different. In Figure S5 viral particles were digested with MNase, resulting in a smeary decrease in DNA size. This looks very different from the regular MNase pattern of whole cells that is dominated by the regularly spaced nucleosomes in the heterochromatic regions of the genome. As pVII protects only about 70bp of DNA and its spacing is not as homogenous as the nucleosomal spacing, the pictures shown in Figure S5A were expected as they are.

    Figure S7 is of low resolution.

    We updated the Figures and checked the resolution after PDF conversion.

    Reviewer #1 (Significance (Required)):

    At least in the field of adenovirus research, this is a very important study. There has been considerable debate in the field regarding the timing and degree of protein VII removal and histone deposition, and the necessity of active transcription for these two events. The data provided in this manuscript clearly shows that some protein VII is removed from early active genes and replaced by nucleosomes, and that these events occur prior to initiation of transcription. The authors speculate that the specific placement of protein VII, a protamine-like protein, on the Ad genome prescribes where nucleosomes are placed. This finding should be of interest to a broad general audience, as it provides novel information on chromatin assembly within mammalian cell. Key words for this reviewer: adenovirus research, HAdV nucleoprotein structure

    Reviewer #2 (Evidence, reproducibility and clarity (Required))

    The submitted manuscript presents a detailed and comprehensive analysis of the adenoviral nucleoprotein complexes as infection progresses, starting with the "adenosome" assembled with pVII which are then progressively replaced with H3.3.-containing nucleosomes as the infection progresses. The submission presents a combination of in situ and populational analyses of the viral DNA accessibility and complexes through infection. I brief, the infecting viral genomes are assembled in some 250 adenosomes with pVII, which become progressively replaced as infection progresses with nucleosomes containing H3.3 and acetylated H3K17, starting at the active promoters of the E genes. Chromatin remodeling precedes transcription, and the accessibility differs for genes of different kinetic classes at differ times after infection, although there is no correlation between accessibility and H3.3. or acetylation content. Only about 20% of the genomes become transcriptionally active, though, which somewhat complicates the analyses of the populational studies of accessibility and occupancy. Overall, the study is well conceived, performed and presented. A few issues that deserve further analyses and discussion, as described below.

    Major issues.

    As figure 2 nicely shows, only about 20% of the intranuclear genomes become transcriptionally active. However, MNase and ChIP analyses cannot differentiate these genomes from the 80% that are transcriptionally inactive. The interpretation of the positioning of pVII (figure 4) or the changes in compaction of the adenoviral chromatin at different loci (figure 5) does not appear to consider this heterogeneity other than for a brief comment about the stringent MNase digestion in page 11. The authors favor a model in which the changes in compaction shown in figure 5, at mild MNase digestions, directly correlate with transcription of the respective genes. This could well be correct, and in fact the correlation may be underestimated as 80% of the genomes may not undergo any changes, but it may also be incorrect. The analyses presented cannot differentiate whether the changes in chromatin compaction occur in only a subset of genomes or in all the genomes, regardless of whether they are transcribed or not, or even only in the non-transcribed genomes (which appears extremely unlikely). This intrinsic limitation to the methods used (and I know of no better alternative) should be acknowledged and discussed for the benefit of the reader. This limitation also impacts the analyses of the lack of correlation between H3.3 and acetylated H3K27 occupancy and compaction.

    A discussion is amended and located starting from line 571 in the text. “The heterogeneity of 80% inactive genomes and 20% activated genomes complicates the analysis of the MNase-seq data. High MNase concentrations do not differentiate between both states, and we suggest that low MNase conditions capture the dynamic viral proportion, changing and preparing its genome for gene activation. The data nicely suggest such a scenario, but there is the caveat that we catch an effect of the mixed population that we cannot differentiate.”

    The analysis of the histone ChIP is discussed below.

    Perhaps out of necessity to reach the required sensitivity, a high multiplicity of infection was used (although the actual moi is not stated, there are about 25-30 pVII foci/ per nuclei). The presentation, analyses and discussion of the results should emphasize this context. For example, one would presume that at low moi, when only one genome enters each cell, the percentage of transcriptionally active genomes in a given cell will be either 0 or 100%, but the "system" becomes saturated as more and more genomes enter the nucleus at higher moi resulting in only a subset of them being transcriptionally active. Along this line of reasoning, it is intriguing that the percentage of genomes estimated to be in nucleosomes at 4 hpi (14%) approaches the percentage of transcribed genomes.

    This issue was also raised by reviewer 1 (see detailed comment above). The reviewer is correct that we chose to use a higher MOI to reach the required sensitivity in our different “Omics” assays. The imaging approach was adapted to reach the morphological equivalence to fit this analysis. We agree that it would be interesting to also study the MOI effect on transcriptional activation (as well as positioning effects, see comment below) but this requires different approaches and will be addressed in a future study. To address this comment (and others in this review) we revised the text in the discussion to emphasize the importance of MOI and possible other effects such as positioning (line 587 ff).

    The changes in chromatin compaction presented in figure 5 are in some respect puzzling. The compaction of most of the late genes increases as infection progresses, at least for the first four hours, as the authors discuss. However, the L genes appear to be at least as accessible as the E ones at the early times, when only the E are transcribed to high levels. This appears counterintuitive, and may not be consistent with the main conclusion that increase accessibility to a given gen directly correlates to its transcriptional activity level. The data presented in Figure 5C deserves a more nuanced analysis and discussion, parsing out the changes in accessibility to each given gene at different times from the different accessibility to the different genes at any given time. The later does not appear to support the main conclusion reached by the authors that accessibility to each individual gen correlates with its transcriptional level.

    We thank the reviewer for raising this point. While the viral genomes enter the nucleus, the viral chromatin structure is tightly condensed. Therefore, it is unlikely that after nuclear entry the viral chromatin undergoes further compaction. With our analysis, we expect to detect only decompaction of genomic sites relative to 0 hpi, when the virus has not entered the nucleus yet. At some sites and particularly at the Late genes the signal is decreasing, most likely due to normalization to sequencing depth and the variation in the number of viral genomes but not due to changes in compaction. We realized that the negative accessibility scores we used in the study are misleading and give a false impression. Therefore, we changed the analysis in that way, that negative values were not permitted and converted to zeros.

    Additionally, we raised the temporal resolution of the analysis and compared the accessibility at all available timepoints against 0 hpi as suggested by the reviewer. Now, we clearly observe, that most accessibility changes are accomplished rapidly after nuclear import, already at 1 hpi and do not change much after, until 4 hpi. Regions of decompaction coincide with early expressed genes and occur before transcription, underscoring the conclusions made in the study. Nevertheless, while most genomic regions covering late genes do not show decompaction, we observed some local sites showing a high accessibility score. As transcription at those sites appears later in the life cycle of the virus, we can only speculate about the function e.g. as enhancer elements.

    The Text and Figures were changed accordingly (line 347 ff).

    New legend:

    __C) __Profile illustrates HAd-C5dE3 genome coverage by low MNase-seq fragments. The average of two replicates is shown, except at timepoint 0 hpi where only one replicate was available. The accessibility score was calculated as the log(fold-change) between the indicated timepoint and 0 hpi. The score was assessed for each pVII peak (orange bars) and negative scores were set to 0. A new accessibility peak arising during infection in the Late3 region is marked by an asterisk. __D) __Boxplot showing the accessibility score distribution in each domain at each tested timepoint after infection.

    Minor comments

    The authors may wish to highlight in the discussion that the analyses are so far limited to a single adenovirus.

    We have taken up the suggestion of the reviewer and included it in the discussion part, starting at line 607:

    “The structural analysis is still limited to a single adenovirus genotype and it will be interesting to test whether these dynamic changes are conserved among other adenoviruses. Furthermore, reproducing such organization in adenoviral vectors could result in efficient and sustained transgene expression.”

    The y-axes in the transcriptome figures (figure 1 B, S2) could be presented in Log(2) scale, such that transcript levels at all times can be appreciated in the same graph (the earlier times are just not visible in a linear scale)

    As requested by the reviewer we changed the data to log2 scale. As there is no qualitative difference to the log10 scale, presented in the original version, we would like to keep the figure as it is. To highlight changes at early time points we generated the average expression of early genes in Fig1C.

    As an information for the reviewer, we provide here the data plotted as log2 scale.

    The (lack of) phenotype of the 24xMS2 binding site recombinant adenovirus used should be shown.

    We observed no difference in phenotype between the parental and the MS2 modified virus. We updated Figure S3 and included a gel analysis and specific infectivity data to show this absence of difference.

    The kymograph analyses presented in figure 3B appear to show that there are some sites of transcript accumulation sites which do not harbor viral genomes (i.e., green only tracks). Moreover, the interpretation of the TAF1beta-mCherry signal is complicated by the (fully expected) significant "background" signal. Although these results are consistent with those obtained by RNAscope/pVII staining, there appears to be intrinsic limitations to the system, which preclude reaching strong conclusions from it. These confirmatory analyses should probably be moved to the supplementary information section and removed from the main text and figures. The longer evaluation data mentioned as not shown in page 8 is critical to the conclusions and should be shown.

    Here we disagree with the reviewer and prefer to keep the data as main figure. All (immobile) transcript accumulation sites are identified by the kymograph analysis and coincide with a genome while free transcripts show a high mobility that is not picked up in the kymograph analysis. This is independently verified in the provided supplemental movies. Depending on the positioning of the genome inside the living cell, accumulating transcripts can appear adjacent to or on top of a genome. This explains the slight shift between RNA and DNA signal for some genomes in the merged image of the kymograph. This is expected as only fully transcribed transcripts and not nascent transcripts are marked by MS2 (the MS2 loops are positioned in the 3’UTR). Also, all genomes (transcribing and non-transcribing) can be identified in the kymograph above background level. To clarify the representation, we have added labels to the kymograph to show which signal is DNA and RNA and a merge respectively. We are convinced that this data set is in strong support of our study, as it is the only technique that permits the discrimination of transcribing and non-transcribing genomes in living cells at real time.

    As requested, we have also added two additional examples for a longer observation period (10min) into the supplemental data Fig. S3C.

    Although the plot of cleavage frequency presented in figure S5 is clear, it would be beneficial to the reader if the actual peaks were also presented to compare their distribution (if any) in gDNA and virus particle.

    In Figure S5 we wanted to test whether the regions lacking pVII peaks are resulting from the absence of pVII, protecting the DNA, and therefore being fully hydrolyzed by MNase, or whether this region is tightly packed by pVII thereby protecting DNA from MNase digestion. To test both possibilities we used a very limited MNase digestion approach, where even free DNA is not fully hydrolyzed, allowing the capture of DNA fragments. Therefore, the sequenced fragments comprise a mixture of protected and un-protected fragments. In this assay, the pVII protected fragments are not fully digested to the monomeric state, but a mix of mono-, di- and other multimers are present. As reflected by the fragment size distribution with the peak between 100-200 bp (Fig S5B), pVII dimers are predominantly enriched when compared to the high MNase digestion used to map pVII positions (compare to Fig4 B). Therefore, the peaks in the S5 data set have a low resolution and do not provide exact pVII positions (see below). Therefore, we would like to keep S5 as it is. We clarified this point in the text (line 279 ff)

    Legend:

    Fragment coverage plot of MNase digestions of gDNA (black) or Ad chromatin in virus particles (purple).

    The mRNA analyses of selected transcription factors provides little information, as there is no context, there is variability between experiments, and in most cases the changes appear modest. As these results are not critical to the conclusions or analyses, perhaps the authors may wish to remove them from the manuscript. Alternatively, more in-depth analyses would be required.

    We agree with the reviewer, that more information for the reader is needed. Therefore, we performed a statistical analysis of expression changes between 0 hpi and 4 hpi of the shown transcription factors using DESeq2. We added the corresponding log2(fold-change) and p-values to the figure. And adapted the text (line 471) and figure accordingly.

    Legend:

    Gene expression changes of transcription factors over the infection time course. P-values and log2(fold-changes) from differential gene expression analysis between 4 hpi and 0 hpi using DESeq2 are indicated. ns = not significant

    It is unclear why the even distribution of H3.1-flag signal across the genome is considered indicative of no specific recruitment. The results presented are equally consistent with equal incorporation across the genome. Perhaps the authors have some additional information, such as an irrelevant antibody, input DNA, or the like, to support the conclusion. If so, that evidence should be presented and discussed. If not, the interpretation should be revisited. As an added complexity, endogenous H3.1 is normally expressed during S-phase. It is possible that Adenovirus infection may induce higher levels of expression of (untagged)endogenous H3.1, which would outcompete the tagged ectopically expressed histone. These analyses deserve a more nuanced and in-depth analysis.

    We have taken several measures in the study to address the concern of the reviewer. We consider timepoint 0 hpi as background control as the viral genome has not entered the nucleus yet. Consistently, we observe very few reads mapped to the Ad genome regardless of antibody and construct used (Fig 6B). Additionally, all samples at 0 hpi cluster together in PCA (Fig 6C) and correlation analysis (Fig S7D)

    H3.1 Flag tagged samples show at later timepoints (1 - 4hpi) slightly higher percentages of mapped reads to Ad, but plateau already at 1 hpi (Fig 6B) and cluster together in PCA (Fig 6C) and correlation analysis (Fig S7D) with 0 hpi samples. The low background signal starting at 1 hpi for H3.1 might arise due to the change of Ad genome location to the nucleus.

    Even though, the number of Ad mapped reads at later timepoints was low in H3.1 Flag tagged samples, it could still be that they accumulate at few sites on the Ad genome indicating a specific deposition. We tested this by plotting the signal across the whole Ad genome (Fig S7E) and zooming into the data (compare scale of H3.3 and H3.1 plot), but we could not detect any reproducible local enrichments. To enable the reader a better comparison between the levels of H3.1 incorporation with H3.3 we put now both on the same scale (Fig 6D and Fig S7D) clearly showing that we cannot detect H3.1 incorporation at Ad genomes in the first 4 hours of infection. The H3.1 signal corresponds to the background noise. We think for two reasons, that it is very unlikely that endogenous H3.1 outcompetes the tagged H3.1:

    • The time scale for the cells to transition into S-Phase and upregulate endogenous H3.1 would be only 1-2 hours in our timeseries experiments and therefore too short. To also show these experimentally we amended an experiment for the reviewer that is not included in the manuscript. The Western blots below show that the protein amount of H3 does not increase in the first 4hours of infection. Cells were infected and whole cell extracts were prepared 4hpi.
    • As most cells are not in S-phase in our experiments, the expression levels of H3.3 variant is higher than H3.1. With the Flag ChIPs we can clearly show that the tagged H3.3 are not outcompeted by endogenous H3.3. As there is a high sequence similarity between H3.3 and H3.1 it is very unlikely that they behave in that regard differently.

    It is highly unlikely that the somewhat higher H3K27ac signal observed in the H3.3 than in the H3.1 expressing cells may result from higher H3.3. occupancy in the viral genome as speculated in page 13. The total levels of H3.3. are unlikely to increase by the ectopically expressed one, and even if they did it is not likely that the occupancy of the viral genome would be limited by the levels of H3.3. This speculation should be removed.

    We removed the speculation.

    Materials and methods are too concise. A longer more detailed version, as supplementary information, would be highly desirable.

    We extended the materials and methods part.

    Reviewer #2 (Significance (Required)):

    The major strengths of this manuscript lie on its comprehensiveness, using several in situ and populational approaches to address biologically critical questions regarding the regulation of viral replication by chromatin and epigenetics. Experiments appears very well designed and performed and are mostly clearly presented. The interpretation analyses and discussion of the results may benefit from a more nuanced analysis of the issues posed by the existence of different populations of viral genomes in the cells infected at high moi and the accessibility across different genes at any given time versus the levels of transcription of the different genes, which appears not to be fully consistent with one of the main conclusions reached.

    This study makes a very significant contribution, describing the dynamic changes in the adenoviral nucleoprotein complexes at the early times of infection and providing a full description of both the adenosomes and the nucleosomes in more and less transcribed loci. The results are properly analyzed in context of what is known about the regulation of viral gene transcription by chromatin dynamics in other systems, including similarities and differences. This study is likely to be of high interest to a wide audience, ranging from virologist to epigeneticists, to those working in gene therapy and vectored vaccines.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The manuscript "Adenoviral chromatin organization primes for early gene activation" combines RNA-seq, MNase-seq, ChIP-seq, and single genome and transcript imaging (immunofluorescence, RNA-scope, and live cell techniques) during early Adenovirus infection in vitro to characterise the spatiotemporal dynamics of viral chromatin organisation and association with gene transcription. The manuscript is an interesting read and the authors have combined multiple complimentary techniques to make a substantial contribution to understanding the early events occurring after nuclear import of viral genomes. Adenoviruses are important causes of human and animal pathology, are a useful model of non-integrating extra-chromosomal DNA virus infection in mammalian cells, and are useful vectors for vaccination and the discoveries may influence gene therapy DNA vector design. The chromatin organisation in adenovirus infection is distinct from other DNA viruses, and is relatively poorly understood compared to, for example, SV40 or herpesviruses. The manuscript describes an early transition from purely viral chromatin with Adenovirus protein pVII packaging the virus in virions, to a viral-human hybrid chromatin pattern with apparently strategically positioned H3.3 nucleosomes and viral pVII "Adenosomes" in the early hours after nuclear import of the viral genome. The data shows that packaged Adenoviruses are in a transcriptionally accessible form and gene expression occurs rapidly after infection, the combination of the MNase-seq data with ChIP-seq data is particularly interesting demonstrating and average ~238 adenosomes positioned by specific DNA code protecting 60-70bp of DNA, and that the genome is accessible at loci that also decondense on infection, with adenosomes being replaced by cellular H3.3 containing nucleosomes at distinct sites. Particularly they show that +1 H3K27 acetylated nucleosomes are acquired at the TSS of key early genes. The authors argue that their spatiotemporal data imply that this chromatin transition "primes" for early gene transcription. The manuscript is well written, uncovers important viral chromatin biology by combining multiple experimental techniques, and the data is generally very clearly presented. A few comments follow. Major concerns: • Abstract and Title: o the abstract and title suggest that because the chromatin changes are observed coincidentally or before transcriptional changes, and that this means that these chromatin changes "prime" (title) or are "required" and play a "central role" (abstract) in early gene expression. The temporal relationship would be consistent with chromatin changes being required for transcriptional changes, but do not imply necessity. Experiments to demonstrate the necessity of these changes for early gene transcription are lacking, and I recommend amending the text or additional experiments to provide this evidence directly.

    We observe a clear timing of events, with chromatin opening, nucleosome assembly at the 5’ end of the gene followed by transcriptional activation, suggesting that these structural changes are essential for gene activation. Still, we cannot prove the direct dependency. Therefore we toned down the title of our manuscript and formulate the findings more conservatively.

    The title now reads: “Changes in adenoviral chromatin organization precede early gene activation”

    Results:o The IF data in Fig S1 is convincing, showing viral particles are accessible quickly in the nucleus. Although no statistics are provided for S1B and C, pVII foci appear at 0.5hpi and appear to mostly accumulate between 0.5hpi and 1hpi with further import between 1hpi and 4hpi. Can the authors be sure that a single pVII IF focus represents a single genome? If genomes tend to aggregate as they accumulate the number of foci per nucleus may not increase linearly with the number of genomes imported. Have the authors considered analysing the intensity of the individual pVII foci over the time points? A related question is whether the authors assume that all packaged virions contain intact complete viral genomes? Many viruses comprise some mixture of complete and incomplete packaged genomes, and the subsequent analyses determine the proportion of transcriptionally active copies with RNA-Scope to a single transcript E1A which lies at one end of the viral genome. Please comment explicitly on whether this is assumed and whether this assumption is realistic in light of known Adenovirus biology.

    We appreciate the reviewer's concern. Several studies in the adenovirus field have shown equivalence between protein VII nuclear foci and individual genomes, including our own (PMID: 26332038). Probably the most accurate study was performed by Daniel Engels lab PMID: 19406166, who used nuclear protein VII foci to titrate viral as well as vector genomes. In contrast, a different study from Patrick Hearings lab PMID: 21345950 showed that past 4hpi, the number of nuclear protein VII foci gradually declines. Based on our experience and because our study is limited to 4 hpi we are confident that protein VII foci accurately reflect individual viral genomes.

    Concerning genome packaging, adenovirus particles contain a single viral genome that is protected at each end by a covalently attached protein preventing its degradation. The packaging of adenoviruses is extremely efficient and only complete genomes are packaged into fully assembled particles. All viruses used in this study have been purified by double CsCl gradient purification. This density gradient based purification protocol removes all particles that are either empty or damaged or would contain partial genomes.

    o The RNA-Seq data in Fig 1 and Fig S2 and Table S1 demonstrates transcription of early genes is barely observable at 1hpi but is observable by 2hpi and is clearly much increased by 4hpi. Fig 2C, visualising pVII foci directly within single cells, suggests that approximately 80% of foci are observed by 1hpi and a further 20% between 1hpi and 2hpi and little thereafter. These data convincingly demonstrate that nuclear import is rapid, typically occurring in the first hour. The E1A RNA-Scope data in figure 2, visualising individual mRNA transcripts of E1A, is more sensitive than the bulk RNA-Seq, and shows transcripts at 1hpi with clearly discernible transcription by 2hpi (2A&D) which suggests that transcription occurs early, by 2hpi. Thus transcription lags nuclear genome import by approximately one hour by these methods. However, the conclusions of the subsequent analyses depend on the chromatin changes clearly preceding, rather than being approximately coincident with transcription, therefore transcription being evident by 2hpi is relevant as figure 6A and D suggest that the chromatin remodelling is subtle before 2hpi on the bulk sequencing analyses. The authors should comment on this given the importance to their argument.

    As stated by the reviewer we observe a clear lag between nuclear import and transcriptional activation. And we do also observe the largest changes in nucleosome occupancy (ChIP-seq data) between 1 and 2 hpi (Fig6A and D). Compared to 0hpi, we observe the strongest increase of nucleosome occupancy between 1hpi and 2hpi (4-8fold effect), whereas depending on the area a 2-3fold increase in occupancy can be observed from 2hpi to 4hpi (Fig6D). An effect that one would expect with chromatin structure preceding gene activation. Furthermore, the timing of nucleosome assembly perfectly matches the increase of MNase accessibility at 1 hpi, supporting our conclusions.

    o The validation of the E1A probe specificity in Fig 2B looks convincing, but there are no data presented for multiple cells to reassure that this image is representative. The equivalent figure for 2D for the Ad5-GFP control would address this.

    We include a large field overview with multiple cells for virus and vector control as new supplemental figure S2B showing that the RNAscope detection of the E1A transcript is highly specific.

    o Figure 2E is presented as a colocalization analysis but appears to be a ratio of mRNA foci to pVII foci per cell. If this is an incorrect interpretation then some clarification in the figure legend would be helpful. If this interpretation of these data is correct, then it is not truly a colocalization analysis, as a single genome may give rise to multiple transcripts and so a ratio We apologize that this figure was not clear. The data are based on real colocalizations and represent the number of pVII dots positive for E1A normalized with the total number of nuclear pVII. We have clarified the figure legend accordingly.

    o The live cell imaging experiments are elegant and convincing, but the agreement in Fig 3D of the % colocalization in MS2-BP data with the RNA-scope data is potentially misleading for the reasons outlined in the prior comment. Is the data in Fig 2E the same as the data in the right hand panel of Fig 3D. If so please comment on the n discrepancy (n=30 in 2E vs n=22 in 3D). The observation that 20% of genomes are transcriptionally active, via bursting or otherwise, is interesting, and would be consistent with the Suomalainen et al reference. The authors discuss two hypotheses to explain these findings: transcriptional bursting or a subset ~20% of genomes being transcriptionally active. This is an interesting and begs the question as to why this may occur. Assuming all imported genomes are intact (previous comment), it appears from the presented images that the foci at the radial periphery of the nucleus may be more frequently transcriptionally active, despite the nuclear periphery being enriched for heterochromatin. The authors might consider analysing the radial position of their TAF1B-mCherry genomes (active and inactive) as this might support position effect variegation rather than bursting as an explanation and they appear to already have the data to perform such analyses.

    o In the presented images (Fig 3A and Fig S3) it appears a higher proportion of genomes than 20% appear to be transcriptionally active, particularly in the low MOI experiment. The authors may wish to comment on this and quantify whether the proportion of transcribing genomes was affected by the input MOI.

    This and the previous comment concerning the influence of MOI, transcriptional bursting and the positioning effect of the genome on the transcriptional activity have also been in part raised above. As stated in our response to reviewer 1 we have used a high MOI in our experiments to have equivalence between all experimental approaches. We agree with the reviewers that all aspects (dose, bursting and positioning) merit a detailed investigation, which we plan in future studies. To be consistent and comparable in our comprehensive approach we decided to not include such studies here as they would address a different question. Nevertheless, to address this (and the above) comments we now mention positioning effects in the results (line 214) and enlarged the discussion (line 587 ff) where we especially raised awareness that such pertinent questions can be addressed with the tools presented in our study.

    We also decided to visually separate the comparison of MS2 and RNAscope data to avoid misleading the reader. Furthermore, the RNAscope data have been replaced. The RNAscope data are indeed from Fig. 2. The difference in n was due to our mistake showing two different normalized data sets. Data were either normalized using total amount of nuclear protein VII (Fig. 2E) or the total amount of nuclear E1A signals (Fig 3D), which due to the more heterogenous signal did not include all cells. In the updated version both figures display data normalized by total amount of nuclear protein VII

    o Fig 4C suggests that there is a large GC preference (or bias) in the pVII occupied regions. The authors may wish to comment on this and present a track with Adenovirus GC composition in Fig 4D.

    We thank the reviewer for raising this point. As suggested by the reviewer we analysed the GC content under pVII peaks and in the linker DNA. Indeed, pVII occupied regions have a significant higher GC content indicating that pVII preferentially positions at GC rich regions. We included this analysis as an additional Figure 4E (line 302 f).

    Legend:

    Boxplot showing GC content of pVII occupied (pVII) or free (linker) regions. Two biological replicates are shown side by side and the p-value of a students t-test of the corresponding pairs is indicated above.

    o Figure 6 presents convincing data showing H3.3. nucleosome positioning and acetylation at E1A and the data is nicely presented showing these changes occur early being observable by 2 and 4 hpi. Again, these changes are not convincingly prior to early gene activation but are certainly occurring early, and may occur prior to early gene activation at the level of individual foci, however, this is not demonstrated definitively.

    This question belongs to the same context addressed by the reviewer above. Please refer to the answer given above.

    Minor comments:

    Introduction: o Paragraph 1 - Introduction for DNA viruses in general, but the authors appear to be talking about Adenoviruses specifically, "little is known about the structural organization of the genome" and "nuclear viral genomes could undergo different parallel fates", arguably these statements are not accurate for other DNA viruses (e.g. Epstein Barr Virus) suggest amending the wording for clarity.

    The manuscript text was updated as suggested.

    o paragraph 2 - Why do the authors say that Adenoviruses are prototypic DNA viruses?

    We removed the term prototypic.

    o Paragraph 3 - A recent study is referenced but multiple references are given.

    The references were updated

    o "Protein VII stays associated with the viral genome imported to the nucleus, while pV dissociates from the viral DNA following ubiquitylation (Puntener et al., 2011). The fate of the μ-peptide is not known". - The reference suggests that pV dissociates on entry to the cytoplasm and during capsid disassembly at the nuclear pore. I find this sentence confusing as it doesn't make it clear that pV is lost before nuclear entry which is important for interpreting the data.

    We clarified this in the manuscript text

    Results:

    o Figure 5 is almost unreadable due to low resolution.

    We updated the Figures and checked the resolution after PDF conversion.

    o Reference to Fig 4C in text comes after Fig4D.

    The order of Figure panels was changed accordingly.

    Reviewer #3 (Significance (Required)):

    The manuscript is well written, uncovers important viral chromatin biology by combining multiple experimental techniques, and the data is generally very clearly presented

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript "Adenoviral chromatin organization primes for early gene activation" combines RNA-seq, MNase-seq, ChIP-seq, and single genome and transcript imaging (immunofluorescence, RNA-scope, and live cell techniques) during early Adenovirus infection in vitro to characterise the spatiotemporal dynamics of viral chromatin organisation and association with gene transcription. The manuscript is an interesting read and the authors have combined multiple complimentary techniques to make a substantial contribution to understanding the early events occurring after nuclear import of viral genomes.

    Adenoviruses are important causes of human and animal pathology, are a useful model of non-integrating extra-chromosomal DNA virus infection in mammalian cells, and are useful vectors for vaccination and the discoveries may influence gene therapy DNA vector design. The chromatin organisation in adenovirus infection is distinct from other DNA viruses, and is relatively poorly understood compared to, for example, SV40 or herpesviruses. The manuscript describes an early transition from purely viral chromatin with Adenovirus protein pVII packaging the virus in virions, to a viral-human hybrid chromatin pattern with apparently strategically positioned H3.3 nucleosomes and viral pVII "Adenosomes" in the early hours after nuclear import of the viral genome. The data shows that packaged Adenoviruses are in a transcriptionally accessible form and gene expression occurs rapidly after infection, the combination of the MNase-seq data with ChIP-seq data is particularly interesting demonstrating and average ~238 adenosomes positioned by specific DNA code protecting 60-70bp of DNA, and that the genome is accessible at loci that also decondense on infection, with adenosomes being replaced by cellular H3.3 containing nucleosomes at distinct sites. Particularly they show that +1 H3K27 acetylated nucleosomes are acquired at the TSS of key early genes.

    The authors argue that their spatiotemporal data imply that this chromatin transition "primes" for early gene transcription. The manuscript is well written, uncovers important viral chromatin biology by combining multiple experimental techniques, and the data is generally very clearly presented. A few comments follow.

    Major concerns:

    • Abstract and Title:
      • the abstract and title suggest that because the chromatin changes are observed coincidentally or before transcriptional changes, and that this means that these chromatin changes "prime" (title) or are "required" and play a "central role" (abstract) in early gene expression. The temporal relationship would be consistent with chromatin changes being required for transcriptional changes, but do not imply necessity. Experiments to demonstrate the necessity of these changes for early gene transcription are lacking, and I recommend amending the text or additional experiments to provide this evidence directly.
    • Results:
      • The IF data in Fig S1 is convincing, showing viral particles are accessible quickly in the nucleus. Although no statistics are provided for S1B and C, pVII foci appear at 0.5hpi and appear to mostly accumulate between 0.5hpi and 1hpi with further import between 1hpi and 4hpi. Can the authors be sure that a single pVII IF focus represents a single genome? If genomes tend to aggregate as they accumulate the number of foci per nucleus may not increase linearly with the number of genomes imported. Have the authors considered analysing the intensity of the individual pVII foci over the time points? A related question is whether the authors assume that all packaged virions contain intact complete viral genomes? Many viruses comprise some mixture of complete and incomplete packaged genomes, and the subsequent analyses determine the proportion of transcriptionally active copies with RNA-Scope to a single transcript E1A which lies at one end of the viral genome. Please comment explicitly on whether this is assumed and whether this assumption is realistic in light of known Adenovirus biology.
      • The RNA-Seq data in Fig 1 and Fig S2 and Table S1 demonstrates transcription of early genes is barely observable at 1hpi but is observable by 2hpi and is clearly much increased by 4hpi. Fig 2C, visualising pVII foci directly within single cells, suggests that approximately 80% of foci are observed by 1hpi and a further 20% between 1hpi and 2hpi and little thereafter. These data convincingly demonstrate that nuclear import is rapid, typically occurring in the first hour. The E1A RNA-Scope data in figure 2, visualising individual mRNA transcripts of E1A, is more sensitive than the bulk RNA-Seq, and shows transcripts at 1hpi with clearly discernible transcription by 2hpi (2A&D) which suggests that transcription occurs early, by 2hpi. Thus transcription lags nuclear genome import by approximately one hour by these methods. However, the conclusions of the subsequent analyses depend on the chromatin changes clearly preceding, rather than being approximately coincident with transcription, therefore transcription being evident by 2hpi is relevant as figure 6A and D suggest that the chromatin remodelling is subtle before 2hpi on the bulk sequencing analyses. The authors should comment on this given the importance to their argument.
      • The validation of the E1A probe specificity in Fig 2B looks convincing, but there are no data presented for multiple cells to reassure that this image is representative. The equivalent figure for 2D for the Ad5-GFP control would address this.
      • Figure 2E is presented as a colocalization analysis but appears to be a ratio of mRNA foci to pVII foci per cell. If this is an incorrect interpretation then some clarification in the figure legend would be helpful. If this interpretation of these data is correct, then it is not truly a colocalization analysis, as a single genome may give rise to multiple transcripts and so a ratio <1 could be expected even if all pVII foci were transcribing genomes. In addition, the text suggests that because there is no statistically significant difference between 2hpi and 4hpi a plateau has been reached. However, the difference between 1h and 2h is barely significant (p=0.04) and the mean is increased between 2h and 4h, albeit non-significantly, so the plateau is not convincingly demonstrated. If the authors wish to perform a colocalization analysis rather than a ratio, they might assign each transcript to the nearest pVII IF focus within the nucleus and count the proportion of pVII foci with any transcripts assigned over time. Alternatively, they can amend the description of this analysis in the figure and text.
      • The live cell imaging experiments are elegant and convincing, but the agreement in Fig 3D of the % colocalization in MS2-BP data with the RNA-scope data is potentially misleading for the reasons outlined in the prior comment. Is the data in Fig 2E the same as the data in the right hand panel of Fig 3D. If so please comment on the n discrepancy (n=30 in 2E vs n=22 in 3D). The observation that 20% of genomes are transcriptionally active, via bursting or otherwise, is interesting, and would be consistent with the Suomalainen et al reference. The authors discuss two hypotheses to explain these findings: transcriptional bursting or a subset ~20% of genomes being transcriptionally active. This is an interesting and begs the question as to why this may occur. Assuming all imported genomes are intact (previous comment), it appears from the presented images that the foci at the radial periphery of the nucleus may be more frequently transcriptionally active, despite the nuclear periphery being enriched for heterochromatin. The authors might consider analysing the radial position of their TAF1B-mCherry genomes (active and inactive) as this might support position effect variegation rather than bursting as an explanation and they appear to already have the data to perform such analyses.
      • In the presented images (Fig 3A and Fig S3) it appears a higher proportion of genomes than 20% appear to be transcriptionally active, particularly in the low MOI experiment. The authors may wish to comment on this and quantify whether the proportion of transcribing genomes was affected by the input MOI.
      • Fig 4C suggests that there is a large GC preference (or bias) in the pVII occupied regions. The authors may wish to comment on this and present a track with Adenovirus GC composition in Fig 4D.
      • Figure 6 presents convincing data showing H3.3. nucleosome positioning and acetylation at E1A and the data is nicely presented showing these changes occur early being observable by 2 and 4 hpi. Again, these changes are not convincingly prior to early gene activation but are certainly occurring early, and may occur prior to early gene activation at the level of individual foci, however, this is not demonstrated definitively.

    Minor comments:

    • Introduction:
      • Paragraph 1 - Introduction for DNA viruses in general, but the authors appear to be talking about Adenoviruses specifically, "little is known about the structural organization of the genome" and "nuclear viral genomes could undergo different parallel fates", arguably these statements are not accurate for other DNA viruses (e.g. Epstein Barr Virus) suggest amending the wording for clarity.
      • paragraph 2 - Why do the authors say that Adenoviruses are prototypic DNA viruses?
      • Paragraph 3 - A recent study is referenced but multiple references are given.
      • "Protein VII stays associated with the viral genome imported to the nucleus, while pV dissociates from the viral DNA following ubiquitylation (Puntener et al., 2011). The fate of the μ-peptide is not known". - The reference suggests that pV dissociates on entry to the cytoplasm and during capsid disassembly at the nuclear pore. I find this sentence confusing as it doesn't make it clear that pV is lost before nuclear entry which is important for interpreting the data.
    • Results:
      • Figure 5 is almost unreadable due to low resolution.
      • Reference to Fig 4C in text comes after Fig4D.

    Significance

    The manuscript is well written, uncovers important viral chromatin biology by combining multiple experimental techniques, and the data is generally very clearly presented

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    Referee #2

    Evidence, reproducibility and clarity

    The submitted manuscript presents a detailed and comprehensive analysis of the adenoviral nucleoprotein complexes as infection progresses, starting with the "adenosome" assembled with pVII which are then progressively replaced with H3.3.-containing nucleosomes as the infection progresses. The submission presents a combination of in situ and populational analyses of the viral DNA accessibility and complexes through infection. I brief, the infecting viral genomes are assembled in some 250 adenosomes with pVII, which become progressively replaced as infection progresses with nucleosomes containing H3.3 and acetylated H3K17, starting at the active promoters of the E genes. Chromatin remodeling precedes transcription, and the accessibility differs for genes of different kinetic classes at differ times after infection, although there is no correlation between accessibility and H3.3. or acetylation content. Only about 20% of the genomes become transcriptionally active, though, which somewhat complicates the analyses of the populational studies of accessibility and occupancy. Overall, the study is well conceived, performed and presented. A few issues that deserve further analyses and discussion, as described below.

    Major issues.

    As figure 2 nicely shows, only about 20% of the intranuclear genomes become transcriptionally active. However, MNase and ChIP analyses cannot differentiate these genomes from the 80% that are transcriptionally inactive. The interpretation of the positioning of pVII (figure 4) or the changes in compaction of the adenoviral chromatin at different loci (figure 5) does not appear to consider this heterogeneity other than for a brief comment about the stringent MNase digestion in page 11. The authors favor a model in which the changes in compaction shown in figure 5, at mild MNase digestions, directly correlate with transcription of the respective genes. This could well be correct, and in fact the correlation may be underestimated as 80% of the genomes may not undergo any changes, but it may also be incorrect. The analyses presented cannot differentiate whether the changes in chromatin compaction occur in only a subset of genomes or in all the genomes, regardless of whether they are transcribed or not, or even only in the non-transcribed genomes (which appears extremely unlikely). This intrinsic limitation to the methods used (and I know of no better alternative) should be acknowledged and discussed for the benefit of the reader. This limitation also impacts the analyses of the lack of correlation between H3.3 and acetylated H3K27 occupancy and compaction.

    Perhaps out of necessity to reach the required sensitivity, a high multiplicity of infection was used (although the actual moi is not stated, there are about 25-30 pVII foci/ per nuclei). The presentation, analyses and discussion of the results should emphasize this context. For example, one would presume that at low moi, when only one genome enters each cell, the percentage of transcriptionally active genomes in a given cell will be either 0 or 100%, but the "system" becomes saturated as more and more genomes enter the nucleus at higher moi resulting in only a subset of them being transcriptionally active. Along this line of reasoning, it is intriguing that the percentage of genomes estimated to be in nucleosomes at 4 hpi (14%) approaches the percentage of transcribed genomes.

    The changes in chromatin compaction presented in figure 5 are in some respect puzzling. The compaction of most of the late genes increases as infection progresses, at least for the first four hours, as the authors discuss. However, the L genes appear to be at least as accessible as the E ones at the early times, when only the E are transcribed to high levels. This appears counterintuitive, and may not be consistent with the main conclusion that increase accessibility to a given gen directly correlates to its transcriptional activity level. The data presented in Figure 5C deserves a more nuanced analysis and discussion, parsing out the changes in accessibility to each given gene at different times from the different accessibility to the different genes at any given time. The later does not appear to support the main conclusion reached by the authors that accessibility to each individual gen correlates with its transcriptional level.

    Minor comments

    The authors may wish to highlight in the discussion that the analyses are so far limited to a single adenovirus.

    The y-axes in the transcriptome figures (figure 1 B, S2) could be presented in Log(2) scale, such that transcript levels at all times can be appreciated in the same graph (the earlier times are just not visible in a linear scale)

    The (lack of) phenotype of the 24xMS2 binding site recombinant adenovirus used should be shown.

    The kymograph analyses presented in figure 3B appear to show that there are some sites of transcript accumulation sites which do not harbor viral genomes (i.e., green only tracks). Moreover, the interpretation of the TAF1beta-mCherry signal is complicated by the (fully expected) significant "background" signal. Although these results are consistent with those obtained by RNAscope/pVII staining, there appears to be intrinsic limitations to the system, which preclude reaching strong conclusions from it. These confirmatory analyses should probably be moved to the supplementary information section and removed from the main text and figures. The longer evaluation data mentioned as not shown in page 8 is critical to the conclusions and should be shown.

    Although the plot of cleavage frequency presented in figure S5 is clear, it would be beneficial to the reader if the actual peaks were also presented to compare their distribution (if any) in gDNA and virus particle.

    The mRNA analyses of selected transcription factors provides little information, as there is no context, there is variability between experiments, and in most cases the changes appear modest. As these results are not critical to the conclusions or analyses, perhaps the authors may wish to remove them from the manuscript. Alternatively, more in-depth analyses would be required.

    It is unclear why the even distribution of H3.1-flag signal across the genome is considered indicative of no specific recruitment. The results presented are equally consistent with equal incorporation across the genome. Perhaps the authors have some additional information, such as an irrelevant antibody, input DNA, or the like, to support the conclusion. If so, that evidence should be presented and discussed. If not, the interpretation should be revisited. As an added complexity, endogenous H3.1 is normally expressed during S-phase. It is possible that Adenovirus infection may induce higher levels of expression of (untagged)endogenous H3.1, which would outcompete the tagged ectopically expressed histone. These analyses deserve a more nuanced and in-depth analysis.

    It is highly unlikely that the somewhat higher H3K27ac signal observed in the H3.3 than in the H3.1 expressing cells may result from higher H3.3. occupancy in the viral genome as speculated in page 13. The total levels of H3.3. are unlikely to increase by the ectopically expressed one, and even if they did it is not likely that the occupancy of the viral genome would be limited by the levels of H3.3. This speculation should be removed.

    Materials and methods are too concise. A longer more detailed version, as supplementary information, would be highly desirable.

    Significance

    The major strengths of this manuscript lie on its comprehensiveness, using several in situ and populational approaches to address biologically critical questions regarding the regulation of viral replication by chromatin and epigenetics. Experiments appears very well designed and performed and are mostly clearly presented. The interpretation analyses and discussion of the results may benefit from a more nuanced analysis of the issues posed by the existence of different populations of viral genomes in the cells infected at high moi and the accessibility across different genes at any given time versus the levels of transcription of the different genes, which appears not to be fully consistent with one of the main conclusions reached.

    This study makes a very significant contribution, describing the dynamic changes in the adenoviral nucleoprotein complexes at the early times of infection and providing a full description of both the adenosomes and the nucleosomes in more and less transcribed loci. The results are properly analyzed in context of what is known about the regulation of viral gene transcription by chromatin dynamics in other systems, including similarities and differences. This study is likely to be of high interest to a wide audience, ranging from virologist to epigeneticists, to those working in gene therapy and vectored vaccines.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors have addressed the nucleoprotein structure of human adenovirus during the very early stages of infection, and its relationship to onset of expression of viral genes, using a combination of RNA-seq, MNase-seq, ChIP-seq and single genome imaging. They show that in the virion and the newly-infecting DNA, protein VII is precisely position at specific sites on the viral DNA, with greater accessibility at early gene promoters compared to other regions. Nucleosomes containing H3.3 replace specific protein VII at distinct positions at the transcription start sites of genes, which are then acetylated. Association with histones and nucleosomes occurs prior to transcription. These studies confirm and greatly expand on results already in the literature, and also elucidate a novel role for protein VII in orchestrating positioning of nucleosomes prior to initiation of transcription.

    The authors provide excellent data in support of their conclusions and, in many instances, use alternative experiments (i.e. two different approaches) to support their claims. The details of methods are adequate (with small exceptions outlined below) and statistical methods appropriate.

    Minor comments:

    1. Line 561 "Protein VII molecules were exchanged for positioned nucleosomes at the +1 site of actively transcribed genes". This statement seems to suggest that the +1 position almost acts as a nucleating site, where replacement of a single, specific protein VII molecule at +1 is an initiating event, which then spreads from that site and into the rest of the gene. Data shown in Figure 6G and 6H shows that H3.3 appears to be found equally along the full length of E1A as early as 1 hr post infection (with no real "enhancement" at the +1 position), and that the overall levels simply increase over the next 4 hrs.
    2. Curiously, the authors chose not to use a wildtype virus for their studies - the virus contains a deletion in the E3 region. For clarity, I suggest that the authors should preferentially use an alternative designation for their virus rather than HAd-C5. Perhaps HAd-C5delE3 to differentiate this work from studies that truly use wildtype virus.
    3. The obvious limitation of the studies using the fluorescent TAF1-beta to label Ad genomes is that as protein VII is replaced by nucleosomes, the genomes would have declining detection by this method. Genomes devoid of protein VII would be "invisible".
    4. Line 275 "Interestingly, a central region of the viral genome (Late3) and a region between the E3 and E4 genes exhibited almost no peaks" for protein VII. The virus utilized in this study lacked at least part of the E3 region. Did this deletion "cause" this region to be devoid of protein VII? Is the same absence of protein VII peaks observed in a fully wildtype virus? Also, can the authors provide any speculation as to why the Late3 region also lacks protein VII?
    5. Line 569 "Reasons could be that the few genomes undergoing nucleosome assembly and active transcription produce the replication enzymes, whereas the bulk of genomes enters replication without activation as an elegant way to avoid repeated chromatinization." This argument may make sense in the context of a high MOI infection, but would certainly limit virus function during normal, pathogenic infection where the MOI is likely extremely low. Essentially, the authors data predicts that 80% of normal, low MOI infections don't progress to gene expression (at least during the first 4 hrs analyzed in this study).
    6. Line 576 "This observation is in agreement with recent pVII-ChIP experiments showing transcription and replication independent pVII removal in early infection (Giberson et al., 2018; Komatsu and Nagata, 2012; Komatsu et al., 2011)." The authors can also state that histone and nucleosome deposition is also independent of transcription and replication, as has been alluded to in the same cited studies but proven more directly in this study.
    7. Line 672 - the authors should be more definitive in the MOI that are used in all of their experiments. Line 672 states that an MOI of 3000 physical particles are applied per cell. There can be great variation between cell lines in how much virus binds to (and enters) a cell based on the surface levels of Ad receptors on different cell types. However, in general, 3000 is very high. Work by Wang et al. (PMID:24139403) showed that at an MOI of 200 or below most Ad will traffic correctly to the nucleus, whereas at an MOI above 200 there is a significant defect in Ad trafficking within the cell. How is this expected to affect all of the results in this study?
    8. Figure 5 is of low resolution and was difficult to read.
    9. Figure S3 is missing a box from the top set of images indicating the region that is expanded in the detail picture.
    10. While I realize it is supplemental data, the difference in quality between the agarose gels shown in Figure S4A and S5A is shocking.
    11. Figure S7 is of low resolution.

    Significance

    At least in the field of adenovirus research, this is a very important study. There has been considerable debate in the field regarding the timing and degree of protein VII removal and histone deposition, and the necessity of active transcription for these two events. The data provided in this manuscript clearly shows that some protein VII is removed from early active genes and replaced by nucleosomes, and that these events occur prior to initiation of transcription.

    The authors speculate that the specific placement of protein VII, a protamine-like protein, on the Ad genome prescribes where nucleosomes are placed. This finding should be of interest to a broad general audience, as it provides novel information on chromatin assembly within mammalian cell.

    Key words for this reviewer: adenovirus research, HAdV nucleoprotein structure

  5. Version published to 10.1101/2020.07.07.190926v2 on bioRxiv
  6. Version published to 10.1101/2020.07.07.190926v1 on bioRxiv