1. Chromosomes and Gene Expression
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Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

  1. Hajnalka L Pálinkás  Is a corresponding author
  2. Angéla Békési
  3. Gergely Róna
  4. Lőrinc Pongor
  5. Gábor Papp
  6. Gergely Tihanyi
  7. Eszter Holub
  8. Ádám Póti
  9. Carolina Gemma
  10. Simak Ali
  11. Michael J Morten
  12. Eli Rothenberg
  13. Michele Pagano
  14. Dávid Szűts
  15. Balázs Győrffy
  16. Beáta G Vértessy  Is a corresponding author
  1. Genome Metabolism Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungary
  2. Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Hungary
  3. Doctoral School of Multidisciplinary Medical Science, University of Szeged, Hungary
  4. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, United States
  5. Perlmutter Cancer Center, New York University School of Medicine, United States
  6. Howard Hughes Medical Institute, New York University School of Medicine, United States
  7. Cancer Biomarker Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungary
  8. Department of Bioinformatics and 2nd Department of Pediatrics, Semmelweis University, Hungary
  9. Genome Stability Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungary
  10. Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, United Kingdom
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Cite this article as: eLife 2020;9:e60498 doi: 10.7554/eLife.60498

Abstract

Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil incorporation contributing to their antiproliferative effect. Still, it is not yet characterized if uracil incorporations have any positional preference. Here, we aimed to uncover genome-wide alterations in uracil pattern upon drug treatments in human cancer cell line models derived from HCT116. We developed a straightforward U-DNA sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging. Using a novel robust analysis pipeline, we found broad regions with elevated probability of uracil occurrence both in treated and non-treated cells. Correlation with chromatin markers and other genomic features shows that non-treated cells possess uracil in the late replicating constitutive heterochromatic regions, while drug treatment induced a shift of incorporated uracil towards segments that are normally more active/functional. Data were corroborated by colocalization studies via dSTORM microscopy. This approach can be applied to study the dynamic spatio-temporal nature of genomic uracil.

Introduction

The thymine analogue uracil is one of the most frequent non-canonical bases in DNA appearing either by thymine replacing misincorporation or as a product of spontaneous or enzymatic cytosine deamination reaction (Krokan et al., 2002). Consequently, uracil in DNA is usually recognized as an error that is efficiently repaired by the multistep base excision repair (BER) pathway initiated by uracil-DNA glycosylases (UDGs) (Krokan and Bjørås, 2013; Wallace, 2014). In other respects, uracil in DNA is known to be involved in several physiological processes (e.g. antibody maturation [Liu and Schatz, 2009; Maul and Gearhart, 2010; Maul and Gearhart, 2014; Xu et al., 2012], antiviral response [Burns et al., 2015; Stenglein et al., 2010], insect development [Horváth et al., 2013; Muha et al., 2012]), however, the exact mechanism and regulation of uracil-DNA metabolism including the roles of UDGs need to be elucidated. There are four known members of the UDG family in humans: (i) the most active uracil-DNA glycosylase encoded by the ung gene (UNG1 mitochondrial and UNG2 nuclear isoform), (ii) the single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1), (iii) thymine DNA glycosylase (TDG specialized for repair of T:G and U:G) and (iv) methyl CpG binding domain protein 4 (MBD4 repairs U:G) (Visnes et al., 2009). UNG2 removes most of the genomic uracil from both single- and double-stranded DNA regardless of the uracil originating from mutagenic cytosine deamination or thymine replacing misincorporation (Kavli et al., 2002).

Thymine replacing uracil misincorporation is normally prevented by the tight regulation of the cellular dUTP/dTTP ratio maintained by two enzymes, the dUTPase and the thymidylate synthase. The dUTPase enzyme removes dUTP from the cellular pool by catalyzing dUTP hydrolysis into dUMP and PPi (Vértessy and Tóth, 2009). Lack or inhibition of dUTPase leads to increased dUTP levels and under such conditions, DNA polymerases readily incorporate uracil opposite to adenine. Similarly, several anticancer drugs (such as 5-fluorouracil (5-FU), 5-fluoro-2’-deoxyuridine (5FdUR), capecitabine, methotrexate, raltitrexed (RTX), pemetrexed) target the de novo thymidylate synthesis pathway via thymidylate synthase inhibition to perturb the tightly regulated dUTP/dTTP ratio, eventually triggering thymineless cell death (Blackledge, 1998; Requena et al., 2016; Wilson et al., 2014). Although the exact molecular mechanism is not yet fully understood, massive uracil misincorporation, hyperactivity of the repair process and/or stalling of the replication fork are all suggested to be involved in the process (Khodursky et al., 2015; Ostrer et al., 2015). UNG has been suggested to play a key role in this mechanism, as being responsible for the initiating step in uracil removal that may lead to futile cycles if the cellular dUTP/dTTP ratio is elevated. A quantitative insight into the magnitude and the pattern of uracil incorporation into genomic DNA as induced by these chemotherapeutic treatments is expected to contribute to a better understanding of the cell death mechanism induced by the respective drugs.

Direct observation of the uracil moieties incorporated upon drug treatments have been hampered by the efficient and fast action of UNG. To overcome this problem, we wished to counteract the action of UNG in human cells via introduction of the well characterized, specific UNG inhibitor, UGI (Luo et al., 2008; Mol et al., 1995) into the cellular milieu. It has already been shown that UGI expression does not affect either the cytotoxicity, or the DNA damage and cell cycle response upon RTX and 5FdUR treatment (Luo et al., 2008). Using UGI expressing cell lines, we aimed to reveal the nascent pattern of uracil moieties in DNA induced by perturbation of thymidylate metabolism both using genome-wide uracil-specific sequencing and in situ cellular imaging of uracils within human genomic DNA. Previously, we designed a uracil-DNA (U-DNA) sensor tailored from an inactive mutant of human UNG2 that was successfully applied in semi-quantitative dot blot analysis and direct immunocytochemistry (Róna et al., 2016). Some additional approaches have also been published to detect uracil-DNA within its genomic context such as (i) techniques focusing on specific, well-defined regions of the genome (qPCR [Horváth and Vértessy, 2010] and 3D-PCR [Suspène et al., 2005]), (ii) techniques that have been applied only to smaller sized genomes (Excision-seq [Bryan et al., 2014] and UPD-seq [Sakhtemani et al., 2019]), and (iii) techniques requiring labor-intensive isolation and multistep processing of genomic DNA samples (dU-seq [Shu et al., 2018]).

Here, we employ the U-DNA sensor in a DNA-IP-seq-like (DIP-seq-like) approach (termed as U-DNA-Seq) and develop a robust bioinformatic pipeline specifically designed for reliable interpretation of next generation sequencing (NGS) data for genome-wide distribution of uracil. We selected two drugs, RTX (raltitrexed, or tomudex) and 5FdUR that perturb thymidylate biosynthesis with different modes of action and analyzed their effects on genomic uracil distribution. These two drugs are frequently applied in treatment of colon cancers, therefore we chose a human colon carcinoma cell line, HCT116 and its mismatch repair (MMR) proficient variant as well-established and relevant cellular models (Koi et al., 1994; Meyers et al., 2001; Rashid et al., 2019). We show that drug treatment led to increased probability of uracil incorporation into more active chromatin regions in HCT116 cells expressing the UNG inhibitor protein UGI. In contrast, uracil was rather restricted to constitutive heterochromatic regions both in wild type cells and in non-treated UGI-expressing cells. Moreover, we further developed the U-DNA sensor-based staining method (Róna et al., 2016) that now uniquely allows in situ microscopic visualization of uracil in human genomic DNA. Confocal and super-resolution microscopy images and colocalization measurements strengthened the sequencing-based distribution patterns.

Results

Genome-wide mapping of uracil-DNA distribution patterns by U-DNA-Seq

We designed an adequate DNA immunoprecipitation method that can provide U-DNA specific genomic information by NGS. This method, termed U-DNA-Seq is based on the rationale of the well-established DIP-seq technology. Figure 1A presents the scheme of the protocol leading to an enriched U-DNA sample that was then subjected to NGS. Immunoprecipitation was carried out by applying the FLAG-tagged catalytically inactive ΔUNG sensor (described in Róna et al., 2016) to bind to uracil in purified and fragmented genomic DNA, followed by a pull-down with anti-FLAG agarose beads. All samples addressed by the U-DNA-Seq in the present study are summarized in Supplementary file 1-table 1.

Figure 1 with 2 supplements see all
U-DNA-Seq provides genome-wide mapping of uracil-DNA distribution.

(A) Schematic image of the novel U-DNA immunoprecipitation and sequencing method (U-DNA-Seq). After sonication, enrichment of the fragmented U-DNA was carried out by the 1xFLAG-ΔUNG sensor construct followed by pull-down with anti-FLAG agarose beads. U-DNA enrichment compared to input DNA was confirmed by dot blot assay before samples were subjected to NGS. (B) Immunoprecipitation led to elevated uracil levels in enriched U-DNA samples compared to input DNA in case of both 5FdUR (5FdUR_UGI) and RTX (RTX_UGI) treated, UGI-expressing HCT116 samples. For each treatment, the same amount of DNA was loaded as input and enriched U-DNA samples providing a correct visual comparison. Two-third serial dilutions were applied.

To allow better detection of nascent uracil, the UNG-inhibitor UGI was expressed in both MMR deficient and proficient HCT116 cells to prevent the action of the major uracil-DNA glycosylase. Besides transient transfection, stable UGI-expressing HCT116 cell lines were also established by retroviral transduction of human codon optimized UGI along with EGFP (Figure 1—figure supplement 1A). We proceeded to treat the UGI-expressing cells with either 5FdUR or RTX. Notably, this combination of UGI expression and drug treatment did not result in any observable cell death. As shown in Figure 1—figure supplement 1B–D, UGI expression and drug (5FdUR or RTX) treatment led to significantly increased uracil content in genomic DNA that is even more pronounced in case of the MMR proficient cells. It is important to note that either UGI expression or treatments with drugs targeting de novo thymidylate biosynthesis pathways on their own do not lead to elevated U-DNA level (Luo et al., 2008; Róna et al., 2016; Yan et al., 2016). Following U-DNA immunoprecipitation, successful enrichment of U-DNA could be confirmed by dot blot assay in the case of drug-treated cells (5FdUR_UGI or RTX_UGI, Figure 1B). To further confirm the capability of U-DNA-IP, uracil-containing spike-in DNA was combined with non-treated genomic DNA samples (Materials and methods). In these samples U-DNA-IP led to 4.5 fold enrichment of the uracil-containing spike-in DNA compared to the uracil-free spike-in as determined by qPCR. Specificity of U-DNA immunoprecipitation is also underlined by the fact that pull-down with empty anti-FLAG beads not containing the U-DNA sensor (i.e. negative control) resulted in negligible amount of DNA (less than 5%, Figure 1—figure supplement 2A, see also Supplementary file 1-table 1). Still, genome-wide sequencing data could be obtained from these negative control samples as well. We demonstrated that subtracting such control signals (for details see Supplementary file 1) will not affect the detected uracil distribution pattern regardless if the sample was drug-treated or not (Figure 1—figure supplement 2B–C). These control experiments provided confidence about the applicability and specificity of our U-DNA-IP method.

Then, enriched and input DNA samples both from treated (5FdUR_UGI, 5FdUR_UGI_MMR, RTX_UGI, and RTX_UGI_MMR) and non-treated (wild type (WT), NT_UGI, and NT_UGI_MMR) samples were subjected to library preparation and NGS. U-DNA-Seq was carried out in two independent biological replicates for each sample. We also performed U-DNA-Seq on non-treated wild type K562 cells in order to have a reference point to the published dU-seq data (Shu et al., 2018).

Sequencing data were analyzed using the herein developed computational pipeline shown in Figure 2 (for more details see the Figure 2—figure supplement 1 and Supplementary file 1-table 2). When reads were aligned to the reference GRCh38 human genome, only uniquely mapped reads were kept and regions suffering from alignment artefacts were excluded from the analysis by blacklisting (Figure 2—figure supplement 2). Statistics on pre-processing steps are shown in Supplementary file 1-table 3. Correlation among the samples at the level of cleaned aligned reads (bam files) was checked by Pearson correlation analysis (Figure 2—figure supplement 3, for details see Supplementary file 1). Here, a clear difference was found between the input and the enriched samples; input samples were more similar to each other regardless the applied treatment, while the enriched drug-treated and non-treated samples showed dramatic differences.

Figure 2 with 3 supplements see all
Data analysis pipeline.

Both input and enriched U-DNA samples were pre-processed the same way: initial trimming and alignment were followed by filtering for uniquely mapped reads and blacklisting of regions suffering from alignment artefacts, resulting in cleaned read alignments in the format of bam files. The key steps of our proposed data processing are (1) calculation of genome scaled coverage tracks (bigwig/bw files), (2) calculation of log2 (enriched coverage/input coverage) ratio tracks (bigwig/bw files), (3) extraction of interval (bed) files of uracil enriched regions from the corresponding log2 ratio tracks. To correlate the uracil enrichment profiles with other published data, first quick screens using interval files were done, and then detailed correlation analysis with a promising candidate of colocalizing genomic features was performed using coverage track files. GIGGLE search (Layer et al., 2018) and bedtools annotate (Quinlan and Hall, 2010) were used for scoring the similarities between query uracil-DNA and the database interval files. Genome segmentation analysis was performed on fold change over input bigwig files either from the ENCODE database, or our own ChIP-seq data and U-DNA profiles using Segway package (Chan et al., 2018; Hoffman et al., 2012). Figures corresponding to the different analysis steps are also indicated. A more detailed pipeline is shown in Figure 2—figure supplement 1, and the full methodology is described in the Supplementary file 1, 35.

There are two principal approaches to extract the signals of uracil enrichment from the cleaned aligned reads: (1) computing genome scaled coverage and log2 ratio tracks, and (2) peak calling that is conventionally used for ChIP-seq data analysis. Log2 ratio tracks provide more detailed information on the uracil-DNA distribution patterns, however, it is not compatible with efficient screening on large dataset (Figure 2 and Figure 2—figure supplement 1). Hence, we generated interval (bed) files from the log2 ratio tracks for each sample (Figure 3A) that contain simplified information on uracil enriched regions as described in the Supplementary file 1. Then, we evaluated both the regions derived from the log2 ratio tracks, and the peak calling results (Figure 3—figure supplement 1 and Figure 3—figure supplement 2). We found that the uracil enriched genomic regions are rather broad and much less intense than conventional peaks in ChIP-seq for transcription factors or even for histone modifications. This is somehow expected considering basically stochastic nature of uracil occurrence via both misincorporation and spontaneous cytosine deamination. In agreement with this, reliability and reproducibility of the peak calling approach (using MACS2 with ‘broad’ option) was found to be clearly suboptimal for determination of uracil distribution patterns (Figure 3—figure supplement 1 and Figure 3—figure supplement 2). Therefore, we decided to proceed with the coverage track approach rather than the peak calling. All of the main figures rely on analysis performed with either the log2 ratio tracks or the regions of uracil enrichment derived from the log2 ratio tracks.

Figure 3 with 4 supplements see all
Comparison of processed U-DNA-Seq data among samples.

(A) Representative IGV view in genomic segment (chr2:64,500,000–89,500,001) shows log2 ratio signal tracks of enriched versus input coverage (log2, upper tracks) and derived regions of uracil enrichment (regions, bottom tracks) for non-treated: wild type (WT, red), UGI-expressing (NT_UGI, orange), and MMR proficient UGI-expressing (NT_UGI_MMR, yellow); and for treated: with 5FdUR (5FdUR_UGI, green; 5FdUR_UGI_MMR, light green) or raltitrexed (RTX_UGI, blue; RTX_UGI_MMR, cyan) HCT116 samples. Two replicates for each sample were merged before coverage calculation. Differences between treated and non-treated samples are clearly visible. Furthermore, 5FdUR and RTX treatments caused similar but not identical uracil enrichment profiles (differences are highlighted with yellow shade). The impact of the MMR status in case of the 5FdUR treated samples is highlighted with pink shade. (B) Comparison of log2 uracil enrichment profiles among samples was performed using multiBigwigSummary (deepTools) and Pearson correlation was plotted using plotCorrelation (deepTools). A heatmap combined with scatter plots is shown for the seven samples. Two replicates for each sample were merged before coverage calculation, and the same analysis for individual replicates are shown in Figure 3—figure supplement 3. (C) Histograms of log2 ratio profiles were calculated and plotted using R for the drug-treated samples. A sub-population of data bins with elevated log2 uracil enrichment signal is clearly visible (indicated with asterisk) in most cases, where high uracil incorporation was detected (Figure 1—figure supplement 1B–D). Thresholds applied in determination of uracil enriched regions are indicated with red line and also provided in Figure 3—source data 1 together with the histogram data.

Figure 3A shows the uracil distribution pattern in a selected chromosomal segment where an uneven distribution with variably spaced broad regions is observed (the same data for all the chromosomes are shown in Supplementary file 2). A clear difference between non-treated and drug-treated cells is already obvious from this view, and the correlations were also measured quantitatively on the whole log2 ratio tracks by Pearson correlation coefficients and related scatter plots (Figure 3B, for description of the samples see Supplementary file 1-table 1, for individual replicates see Figure 3—figure supplement 3). Interestingly, the impact of MMR proficiency on the uracil distribution pattern is obvious in case of the 5FdUR treatment, while RTX treated and especially the non-treated samples do not show notable differences compared to their MMR deficient counterparts.

The uracil-enrichment coverage tracks in Figure 3A and the related correlations in Figure 3B already revealed altered distribution of uracil-containing regions in the drug-treated as compared to the non-treated samples. This difference was further underlined in a histogram representation of uracil enrichment signal (Figure 3C and Figure 3—figure supplement 4) where drug treatment led to a higher number of genomic segments (more data bins) with increased uracil level. MMR proficiency in case of the 5FdUR treatment substantially changed this phenomenon. We investigated whether the uracil distribution patterns might show correlation to any previously determined genomic features. For this reason, we built a relevant database by collecting cell type specific ChIP-seq and DNA accessibility data (for details see Supplementary file 34).

Interrogation of the constructed specialized database with respect to the uracil-DNA distribution patterns was performed using interval (bed) files of uracil enriched regions (derived from log2 ratio track) for each U-DNA-Seq sample. To screen for similarity between sample and database interval (bed) files, we applied the GIGGLE search tool (for details see Supplementary file 3). GIGGLE scores measure the colocalization independently from the size of the compared intervals (Layer et al., 2018). Each interval file in the database corresponded to a ChIP-seq data with a given factor (e.g. histone markers, transcription factors, etc.). GIGGLE scores were then calculated pairwise (each sample to each database interval file), and plotted for the top ten factors corresponding to the highest scores (Figure 4A, full data are presented in Supplementary file 3-table 1). The similarity scores of the U-DNA-Seq data with regard to the different chromatin markers indicate that non-treated cells may possess uracils preferentially in the constitutive heterochromatin (high scores with H3K9me2 and H3K9me3 [Hyun et al., 2017; Saksouk et al., 2015]). On the other hand, drug treatment of the cells either with 5FdUR or RTX, induces uracil incorporation into more active genomic segments, which correlates with euchromatin histone marks (H3K36me3 [Becker et al., 2017; Hyun et al., 2017; Pfister et al., 2014], H3K4me1/3 [Hyun et al., 2017], H3K27ac [Creyghton et al., 2010], H3K9ac [Gates et al., 2017]), or factors associated to either activation or repression in a context dependent manner (SP1 [Doetzlhofer et al., 1999], H3K27me3 [Becker et al., 2017; Saksouk et al., 2015], H2AZ/AFZ [Giaimo et al., 2019]) (Figure 4A). Interestingly, MMR proficiency has an impact on this correlation in case of both drug-treated samples reflecting in decreased GIGGLE scores.

Figure 4 with 3 supplements see all
Characterization of U-DNA enrichment patterns.

(A) GIGGLE search was performed with interval (bed) files of uracil enriched regions on a set of HCT116 related ChIP-seq and DIP-seq experimental data (for details see Supplementary file 3). Factors corresponding to the top 10 hits for each sample were selected. GIGGLE scores between all seven samples and all experiments corresponding to these factors were plotted excluding those, where data were not informative (data are found in Supplementary file 3-table 1). Source data are available in Figure 4—source data 1. Histone marks and the transcription factors, SP1 and TCF7L2 are categorized depending on their occurrence in transcriptionally active or repressive regions. Notably, some of them have plastic behavior allowing either transcriptionally active or repressive function. U-DNA-Seq samples are as follows: non-treated wild type (WT, red), non-treated UGI-expressing (NT_UGI, orange), 5FdUR treated UGI-expressing (5FdUR_UGI, green) and RTX treated UGI-expressing (RTX_UGI, blue) HCT116 cells, and their MMR proficient counterparts (NT_UGI_MMR, yellow; 5FdUR_UGI_MMR, light green; RTX_UGI_MMR, light blue). GIGGLE scores are also indicated for our own H3K36me3 ChIP-seq experiments (RTX_UGI sample: empty squares, NT_UGI sample: empty triangles). The tendencies are even more pronounced if the RTX treated U-DNA-Seq is compared with the RTX treated ChIP-seq or if the non-treated U-DNA-Seq is compared with the non-treated ChIP-seq data. (B) Genome segmentation analysis was performed on signal tracks of 22 ChIP-seq data available for HCT116 cells in the ENCODE database, on our own ChIP-seq data for H3K36me3, and on the seven U-DNA enrichment profiles (bold). The Segway train was performed with 25 labels and the corresponding genomic segments were identified with Segway annotate (Chan et al., 2018). The signal distribution data were calculated using Segtools (Buske et al., 2011), and plotted using python seaborn/matplotlib modules (Hunter, 2007). Source data are available in Figure 4—source data 2. Details including the applied command lines are provided in Supplementary file 3. The color-code is applied for each factor (rows) independently, from the minimum to the maximum value as indicated. (C) Correlation with genomic features. Interval (bed) files of genomic features were obtained from UCSC, Ensembl, and ReplicationDomain databases (for details see Supplementary file 4-table 1), and correlation with interval files of uracil regions were analyzed using bedtools annotate software (details are provided in Supplementary file 4). Numbers of overlapping base pairs were summarized for each pair of interval files, and scores were calculated according the formula: (baseNo_overlap/baseNo_sample_file) * (baseNo_overlap/baseNo_feature_file) * 10000. Heatmap was created based on fold increase of the scores compared to the corresponding WT scores. Sizes of interval files in number of base pairs are also given in the second column and the second line. Upon drug treatments, a clear shift from non-coding/heterochromatic/late replicated segments towards more active/coding/euchromatic/early replicated segments can be seen. CDS, coding sequence; SINE, short interspersed element; LTR, long terminal repeat; LINE, long interspersed element; cytoBand, cytogenetic chromosome band negatively (gneg) or positively (gpos) stained by Giemsa; repl. timing, replication timing; DNaseHS, DNase hypersensitive site. (D) Correlation analysis with replication timing. Replication timing data (bigWig files with 5000 bp binsize) specific for HCT116 were downloaded from ReplicationDomain database (Weddington et al., 2008). Data bins were distributed to 10 equal size groups according to replication timing from early to late. Then log2 uracil enrichment signals for these data bin groups were plotted for each sample using R (Supplementary file 5). Source data are available in Figure 4—source data 3.

In order to decide whether drug treatments may cause any notable changes in the distribution pattern of epigenetic markers as compared to the normal patterns, we have performed a direct comparative ChIP-seq study on our UGI-expressing HCT116 cell line. For this, we have selected the H3K36me3 histone marker that gave the highest GIGGLE scores with the RTX treated U-DNA pattern. ChIP-seq for H3K36me3 was performed in both non-treated (NT_UGI_H3K36me3), and RTX treated (RTX_UGI_H3K36me3) UGI-expressing HCT116 cells (Materials and methods, for the details of the analysis see Supplementary file 3, for description of the samples see Supplementary file 1-table 1). Comparison of our H3K36me3 ChIP-seq data to those available within the ENCODE database is presented in Figure 4—figure supplement 1. These results reveal no substantial differences between RTX_UGI_H3K36me3 and NT_UGI_H3K36me3 samples suggesting no major chromatin rearrangement upon drug treatment. Moreover, our ChIP-seq data are similar to the corresponding ENCODE data. Furthermore, on Figure 4A, GIGGLE scores between the U-DNA patterns and our own ChIP-seq peaks are also indicated and these are in good agreement with the other corresponding scores in case of both the non-treated and RTX treated samples.

To understand broader genome-wide correlations, a genome segmentation approach was employed using Segway software (see details in Supplementary file 3). 22 independent, HCT116 related ChIP-seq experiments of the ENCODE database were selected for the analysis together with our U-DNA-Seq data and also our ChIP-seq data for H3K36me3 (NT_UGI_H3K36me3 and RTX_UGI_H3K36me3). 25 genomic segments were defined and identified with the signal distribution presented in Figure 4B. This analysis on one hand confirmed the correlations that had already been suggested by the GIGGLE search; on the other hand revealed that the histone markers are not the most correlating genomic features. The drug treatment induced shift towards the transcriptionally more active regions is also reflected in the segments 14 and 21, where treated samples show slightly increased U-DNA signal, in contrast to the definitely low signal in case of the non-treated samples. Moreover, it was also confirmed that the most correlating histone markers are the H3K36me3 and the H3K27me3 for the U-DNA pattern of the RTX (segments 19 and 8) and the 5FdUR (segments 17 and 4) treated samples, respectively. The differences between the two drug treatments (e.g. regarding the histone markers mentioned above), and also between the corresponding MMR deficient and proficient cells (e.g. MMR dependent decreased signal intensities in the segments (21 and 14) associated with active transcription), seem to be coherent with the GIGGLE analysis. Similarly, in the case of the non-treated samples, the H3K9me3 constitutive heterochromatin marker was confirmed to be the most correlating histone marker (segments 1 and 24). Nevertheless, the highest U-DNA signal segments are not matching with any of the investigated histone markers (see segments 18 for the non-treated; and 0 and 15 for drug-treated samples). The fact that the histone markers are not the most correlating genomic features prompted us to further search for potential correlating features.

Therefore, we investigated colocalization of U-DNA enriched regions with different coding properties, CpG islands, active regions based on DNase hypersensitivity, different types of repetitive segments, giemsa stained cytogenetic bands and different replication timing. Bedtools annotate software (Quinlan and Hall, 2010) was used to extract the number of overlapping bases. Scores measuring the colocalization are presented in Figure 4C for a systematic selection of the tested features. The results of the full analysis are provided in Supplementary file 4-table 1. The data suggest that uracil incorporation in transcriptionally active (e.g. active promoters, DNase hypersensitive sites) and potentially active genomic segments (CpG islands, genes, especially exons and CDS regions), is increased upon drug treatment, both in MMR deficient and proficient cells, although to different extents. The proposed uracil enrichment in transcriptionally active genomic regions is also in agreement with the colocalization with different repeat classes: the drug-treated samples show higher colocalization with short interspersed nuclear elements (SINEs [Kramerov and Vassetzky, 2005]) and long terminal repeats (LTRs [Kovalskaya et al., 2006]) which are known to be more frequently transcribed as compared to long interspersed nuclear elements (LINEs [Boissinot and Furano, 2005]) and satellite segments (López-Flores and Garrido-Ramos, 2012). It is interesting to note that MMR proficiency has an impact on this pattern also but only in case of the 5FdUR treatment.

The observed similarity between wild type uracil distribution and the patterns of histone markers associated with heterochromatin (Figure 4A–B) is further underlined by the positive correlation between U-DNA and cytogenetic chromosome G-bands (Figure 4C). Dark G-bands stained strongly by Giemsa were shown to correlate with AT-rich, heterochromatic, late replicating genomic segments (Gilbert, 2002; Holmquist et al., 1982). In contrast, negative G-bands are correlated better to the drug-treated uracil-DNA distribution pattern, also in agreement with our results from the comparison to histone markers (Figure 4A–B). Consistently, similar difference between patterns of U-DNA in non-treated versus drug-treated cells in early or late replicating genomic segments is also revealed. Late replicating regions are better correlated to the U-DNA distribution in non-treated cells, while the drug treatment induced U-DNA pattern is more similar to the early replicating segments (Figure 4C). Interestingly, in the 5FdUR treated samples, MMR proficiency led to a major decrease in the correlation between the U-DNA pattern and the early replicating segments, still the difference as compared to the non-treated samples remains. It is widely accepted that replication timing strongly correlates with chromatin structure, namely the open euchromatin and the condensed heterochromatin replicates in early and late S-phase, respectively (Gilbert, 2002). The correlation between U-DNA enrichment and replication timing was further analyzed using a better resolved time scale of replication (Figure 4D) which strengthened the initial observation. The correlations with G-banding and replication timing are also clearly visible on IGV views in Figure 4—figure supplement 2. Furthermore, colocalization with AT-rich heterochromatin for non-treated and GC-rich euchromatin for drug-treated samples is also reflected by the base composition of uracil enriched regions (Figure 3—figure supplement 2A). The surprisingly high correlation between uracil enrichment in drug-treated cells and CpG islands (Figure 4C) coincides with the elevated GC content of uracil enriched genomic regions in these samples. The replication timing correlation and the AT content were also calculated to the genomic segments identified by the Segway (cf. Figure 4B), and the above correlation was confirmed (Figure 4—figure supplement 3).

As the uracil distribution pattern in drug-treated cells shows correlation with the early replication timing, we wish to directly investigate if there is any cell cycle arrest occurring under our experimental conditions. Figure 5 shows characteristic scatter plots indicating an expected cell cycle arrest in the drug-treated cells, namely delayed S-phase entry and progression (Blackledge, 1998; Ding et al., 2019; Huehls et al., 2016; Yan et al., 2016; Zhao et al., 2016). In agreement with the literature (Luo et al., 2008), our data clearly show no observable cell cycle effect of UGI expression in our non-treated samples (Figure 5A). Our data also revealed that the MMR proficiency somewhat tempers the observed cell cycle arrest, especially in case of the 5FdUR treatment (Figure 5B). As expected (Meyers et al., 2001), 5FdUR and RTX treatments eventually lead to DNA double-strand breaks (DSBs) as measured by yH2AX staining (Figure 5—figure supplement 1). DNA damage induction by the drugs was similar in MMR deficient and proficient HCT116 cells.

Figure 5 with 1 supplement see all
Cell cycle analysis showing the impact of UGI expression with or without drug treatments in MMR deficient and proficient HCT116 cells.

Scatter plots represent the flow cytometric measurements of BrdU incorporation and propidium iodide (PI) DNA-staining. (A) Cell cycle distribution in non-treated, MMR deficient (WT), and UGI-expressing (NT_UGI); or in MMR proficient (NT_MMR), and UGI-expressing (NT_UGI_MMR) HCT116 cells. (B) Cell cycle differences caused by 5FdUR or RTX drug treatments in MMR deficient, UGI-expressing (5FdUR_UGI and RTX_UGI); or in MMR proficient, UGI-expressing (5FdUR_UGI_MMR and RTX_UGI_MMR) HCT116 cells.

In situ detection of U-DNA using super-resolution microscopy

We aimed to correlate genome-wide uracil distribution patterns in situ with chromatin architecture. Therefore, we further developed the U-DNA sensor constructs (Róna et al., 2016) to allow in situ detection of genomic U-DNA in complex eukaryotic cells using microscopy. Figure 6A shows a schematic representation of the U-DNA staining procedure. The U-DNA sensor constructs were fused to different tags allowing antibody-based or direct detection via fluorescence microscopy. In order to achieve a versatile labelling technique and to facilitate super-resolution imaging of U-DNA, we attached a SNAP-tag to the C'-terminal end of ΔUNG (FLAG-ΔUNG-SNAP), generating a novel sensor construct (Figure 6—figure supplement 1A). The SNAP-tag offers a flexible biorthogonal chemical labelling strategy as it reacts specifically and covalently with benzylguanine derivatives, permitting the irreversible labelling of SNAP fusion proteins with a wide variety of synthetic probes (Keppler et al., 2003). In order to check whether the functionality of this new construct is still preserved, we performed dot blot and staining experiments. Results shown in Figure 6—figure supplement 1B indicate that the FLAG-ΔUNG-SNAP construct is functional and shows similarly reliable U-DNA detection using dot blot approach, when compared to FLAG-ΔUNG-DsRed protein described previously (Róna et al., 2016). Figure 6—figure supplement 1C shows that the new labelling construct, FLAG-ΔUNG-SNAP, also recognizes the presence of extrachromosomal uracil enriched plasmid aggregates in the cytoplasm. These results confirmed that the FLAG-ΔUNG-SNAP construct is capable of U-DNA detection in dot blot assays and suitable for in situ staining applications.

Figure 6 with 1 supplement see all
In situ detection of the cellular endogenous U-DNA content.

(A). Scheme represents that genomic uracil residues can be visualized in situ using our further developed U-DNA sensor construct via immunocytochemistry (through FLAG-tag) or directly via SNAP-tag chemistry. (B) HCT116 cells expressing UGI and treated with 5FdUR show efficient staining with the uracil sensor compared to non-treated cells, detected by confocal microscopy. Uracil residues are labelled by our FLAG-ΔUNG-SNAP sensor protein visualized by the SNAP647 substrate. DAPI was used for DNA counterstaining. Our optimized staining method is capable of comparable, specific uracil detection in HCT116 cells even with paraformaldehyde (PFA) fixation compared to the Carnoy fixation applied previously (Róna et al., 2016). Scale bar represents 40 µm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest (Huehls et al., 2016; Yan et al., 2016).

Our goal was to use this new sensor to detect in situ endogenous uracils in human cells in a setup that also allows colocalization with other chromatin factors. For visualization of our sensor, photostable SNAP-tag substrates (here SNAP647 or SNAP546) were used. Figure 6B shows that drug treatment and the inhibition of cellular UNG enzyme by UGI lead to significantly increased uracil content in genomic DNA that is readily observable on conventional confocal microscopic images. Figure 6B also demonstrates that our FLAG-ΔUNG-SNAP sensor can be applied for straightforward staining of genomic uracil after either Carnoy (as used previously [Róna et al., 2016]) or PFA fixation. Unlike Carnoy, PFA fixative is compatible with most antibody-based staining procedures, thus it is suitable for multi-color imaging allowing colocalization studies. Next, we attempted to use super-resolution microscopy to have a better track of the uracil distribution pattern even in case of the low genomic uracil level found in the non-treated cells. Figure 7 compares confocal, STED and dSTORM microscopy techniques for U-DNA detection. The exquisite sensitivity of dSTORM is apparent from these experiments as it can detect the low level of genomic uracil in non-treated cells (Figure 7B). Importantly, we observed different heterogeneous staining in the nucleus for uracil in non-treated and drug-treated cells. Furthermore, images of drug-treated cells show uracil staining with signal enrichment at the nuclear membrane and areas surrounding the nucleoli. Movies in Figure 7—videos 14 (for the corresponding representative image see Figure 7—figure supplement 1) contribute to further visualization of uracil distribution captured by confocal and STED imaging.

Figure 7 with 5 supplements see all
The FLAG-ΔUNG-SNAP sensor enables super-resolution detection of genomic uracil by STED and dSTORM microscopy.

(A) U-DNA staining was performed on non-treated or 5FdUR treated HCT116 cells stably expressing UGI. Different SNAP-tag substrates, SNAP647 for confocal and SNAP546 for super-resolution imaging (STED) were used to label FLAG-ΔUNG-SNAP. Scale bar represents 20 µm for whole images and 10 µm for zoomed sections. (B) dSTORM imaging was performed on non-treated or drug-treated (5FdUR or RTX) HCT116 cells stably expressing UGI to compare the sensitivity of these imaging techniques. U-DNA staining shows a characteristic distribution pattern in cells with elevated uracil levels as compared to non-treated cells. SNAP647 substrate was used to label FLAG-ΔUNG-SNAP. Scale bar represents 10 µm for whole images and 2 µm for zoomed sections.

Based on the genome-wide sequencing data analysis, we proceeded to select cognate chromatin markers for colocalization studies. As shown in Figure 4A, the highest similarity (GIGGLE) scores corresponded to H3K36me3 and H3K27me3 for the RTX and the 5FdUR treated samples, respectively. Furthermore, Segway analysis strengthened that these two histone markers (from the 22 investigated factors) show the most similar signal distribution pattern to the U-DNA patterns of drug-treated samples (Figure 4B). Using the herein demonstrated immunofluorescence protocol we obtained co-stained images of uracil and these histone markers by both confocal and dSTORM microscopies (Figure 8A–B). Validating the U-DNA-Seq data, we found that U-DNA staining shows significant colocalization with staining for both chromatin markers; H3K36me3 and H3K27me3, which was quantified using a cross-pair correlation analysis of the dSTORM images as shown in Figure 8C–D. The rate of colocalization, as determined by the interaction factor (IF) value (Bermudez-Hernandez et al., 2017; Whelan et al., 2018), was statistically significant between the uracil signal and both chromatin markers in each case of drug treatment, when compared to the non-treated sample as well as to a generated set of random distribution patterns of these chromatin markers. The cross-pair correlation method probes the probability distributions across all possible pair-wise distances between two species, taking in account the number of foci for each species (Coltharp et al., 2014; Cutler et al., 2013; Veatch et al., 2012; Yin and Rothenberg, 2016). This normalization of the number of foci ensures that any increase in IF is specifically due to an increase in their co-localization probability density, and not due to the increase in the amount of either species.

Genomic uracil moieties colocalize with H3K36me3 and H3K27me3 analyzed by super-resolution microscopy.

Confocal and dSTORM imaging were performed on non-treated, 5FdUR or RTX treated HCT116 cells stably expressing UGI to compare the localization of genomic uracil residues (red) to histone markers, H3K36me3 (green) (A) or H3K27me3 (green) (B). Scale bar represents 5 μm. The graphs display the cross-pair orrelation analysis between U-DNA and H3K36me3 (C) or H3K27me3 (D) performed on dSTORM images. Overlap is defined as any amount of pixel overlap between segmented objects. Total numbers of analyzed nuclei for H3K36me3 staining (C) were the following: NT_UGI (n = 205), 5FdUR_UGI (n = 101) and RTX_UGI (n = 153) from two independent experiments. Total numbers of analyzed nuclei for H3K27me3 staining (D) were the following: NT_UGI (n = 154), 5FdUR_UGI (n = 151) and RTX_UGI (n = 107) from two independent experiments. Black line denotes the mean of each dataset, and error bars represent standard errors of the mean (SEM). The color code follows the one in Figure 3A. Source data are available in Figure 8—source data 1.

Discussion

Here we focus on the alteration of U-DNA distribution pattern upon treatment with drugs perturbing thymidylate biosynthesis. Towards this end, we combined two new applications of further developed U-DNA sensor that was originally described in Róna et al., 2016. On one hand, using a DNA-IP-seq like application, termed U-DNA-Seq, we provided genome-wide uracil distribution data that was compared to the patterns of different genomic features. On the other hand, in immunocytochemistry, the sensor was applied to detect colocalization of U-DNA and selected histone markers.

Using U-DNA-Seq, here we demonstrate that the distribution of uracil-containing regions is altered in the drug-treated (5FdUR or RTX, in combination with UGI) as compared to the non-treated (wild type and UGI-expressing) samples. We demonstrated that UGI expression did not cause any observable change either on cell cycle progression (Figure 5, and Luo et al., 2008) or uracil distribution pattern (Figure 3). We chose HCT116 cancer cell line that is deficient in mismatch repair (MLH1-/-), similarly to many types of cancer especially in colon cancer (Germano et al., 2018; Gupta and Heinen, 2019; Sekine et al., 2017). As its mismatch repair proficient counterpart is also available (Koi et al., 1994), we took the opportunity to address the impact of the MMR status on genomic uracil distribution. We found that the genomic uracil pattern is much more influenced by MMR proficiency in case of the 5FdUR treatment than in case of the RTX treatment (Figure 3). The genomic uracil distribution patterns either in non-treated or in drug-treated cells are found to be non-random: broad regions of uracil enriched genomic segments were detected. Within the third part of our pipeline (Figure 2—figure supplement 1 and Supplementary file 35), we also analyzed the distribution pattern of these broad peaks comparing them to a set of relevant and cell type-specific data of ChIP-seq experiments and other genomic features. In drug-treated cells, these broad segments showed the highest correlation with ChIP-seq-based patterns published for predominantly euchromatin and facultative heterochromatin markers (Figure 4A–B). Increasing evidence suggests that active and repressed chromatin states can be determined in a combinatorial fashion where simultaneous histone marks can efficiently shift gene expression from inactive to active states or vice versa (Gates et al., 2017; Hyun et al., 2017). Hence, it is of special interest to note that our colocalization data show similarity scores not just for one but for a variety of factors. Such combinatorial behavior was further demonstrated by the genome segmentation analysis using the Segway package (Figure 4B) that also pointed to the fact that the distribution of histone markers are not fully matched with the detected U-DNA pattern. Hence further genomic features were also studied (Figure 4C). Importantly, regarding these factors and additional features, our results are highly coherent. Namely, the outstanding correlation of uracil-DNA patterns in drug-treated samples with active promoters, CpG islands, early replicating segments and DNase hypersensitive sites, all of which are published for normally cycling cells, highly supports the above conclusion. Euchromatin was shown to imply early replicating genomic regions, whereas heterochromatin replicates in late S-phase (Black et al., 2012). Accordingly, we report that the drug treatment induced U-DNA pattern is more similar to the early replicating segments, whereas U-DNA distribution in non-treated (wild type and UGI-expressing) cells shows simultaneous association with both heterochromatin markers and late replicating regions (Figure 4C–D, also supported by Figure 4—figure supplement 3). It has to be noted that MMR proficiency leads to a major decrease in the correlation with early replication timing in case of the 5FdUR treated sample (Figure 4C–D), and a smaller decrease in the correlation with transcriptionally active regions in case of both treatments (Figure 4A–C). Still, difference between the uracil-DNA patterns of drug-treated and non-treated samples remains unambiguous, regardless the MMR status (Figures 3 and 4).

Taken together, in the non-treated cells, where the level of genomic uracil is low, we show that uracil is preferentially located in the constitutive heterochromatin, which can be explained by the fact that heterochromatin is generally highly condensed and thus less accessible for DNA repair and replicative DNA synthesis. In contrast, in the open, more frequently transcribed euchromatin, DNA repair can efficiently correct uracils in the presence of a balanced dNTP pool. The low amount of genomic uracil in non-treated cells might remain from either cytosine deamination or thymine replacing misincorporation that escaped DNA repair. However, drug (5FdUR or RTX) treatments perturb the cellular nucleotide pool, and consequently highly increase the rate of thymine replacing uracil misincorporation events overwriting the background uracil pattern of non-treated cells’ genome. Uracil appearance via thymine replacing misincorporation implies prior DNA synthesis involved in either replication, or transcription-coupled DNA repair, or epigenetic reprogramming (e. g. erasing the methyl-cytosine epigenetic mark). Importantly, we found that uracil pattern showed the highest correlation with the features (early replication, active promoters and DNase hypersensitive sites, and CpG islands) linked exactly to these processes (Figure 4C). This is further supported by the fact that in MMR proficient drug-treated samples higher U-DNA content was measured as compared to the MMR deficient ones (Figure 1—figure supplement 1C–D). This observation in MMR proficient cells might be explained by either longer segments synthesized during the MMR process (Bowen et al., 2013), or the less tight control on cell cycle arrest (Figure 5) allowing more extended replicative synthesis.

Our data showing that under normal conditions, that is in lack of drug treatment, localization of human genomic uracils can be associated with the heterochromatic regions which is in agreement with the recent study by Shu et al., 2018. We propose that this pattern may reflect less efficient DNA repair in the heterochromatin. In accordance, it was shown that mutation rate within the later replicating heterochromatin is markedly increased (Stamatoyannopoulos et al., 2009). Interestingly, uracil distribution in bacterial and yeast genomes was found to be mostly excluded from the earliest as well as from the latest replicating segments (Bryan et al., 2014), suggesting a partially different pattern as compared to what is observed in human cells. In yeast cells it was also shown that transcription coupled repair synthesis might result in elevated uracil incorporation into actively transcribed regions under normal conditions (Kim and Jinks-Robertson, 2009; Owiti et al., 2018). However, both yeast and bacteria show major differences in both mechanisms of dNTP pool regulation (Mathews, 2014) and the set of available UDGs, as they do not encode the SMUG1 enzyme which is an important backup of the UNG2 in human (Elateri et al., 2003; Kavli et al., 2002). These differences may account for the alterations found in the genomic uracil distribution patterns.The antifolate or nucleotide-based thymidylate synthase inhibitors, such as 5-FU, RTX or 5FdUR are known to lead to cell cycle arrest, as it is confirmed in our experimental system (Figure 5) and is also reflected in the detected uracil-DNA pattern that strongly correlates with the early replicating segments in case of both drug treatments. The two drugs caused similar, but not equivalent uracil-DNA pattern. On the one hand, the correlations with the H3K36me3 marker as well as with the early replicating segments are both markedly stronger with the RTX treated sample as compared to the 5FdUR treated sample (Figure 4). On the other hand, the correlation of uracil accumulation with the H3K27me3 marker and with the CpG islands is stronger in the 5FdUR treated sample. Moreover, the MMR status has markedly different influence on the resulting U-DNA pattern in case of the two drugs (Figures 34). Such differences might correspond to drug-specific mechanism of action, involving alterations in signaling processes, transcription regulation and the timing of cell cycle arrest (Van Triest et al., 2000). Details of these mechanisms remain obscure in the literature. Still, it is well-known that both drugs inhibit thymidylate synthase thereby facilitating dUMP incorporation into DNA, while the nucleotide analogue 5FdUR also leads to direct incorporation of 5-fluorodeoxyuridine monophosphate (FdUP) into the DNA (Longley et al., 2003; Pettersen et al., 2011). Genomic uracil and fluorouracil might have different effects on transcription and epigenetic regulation processes that could also contribute to the observed differences of the two U-DNA patterns. It should be noted that our method detects both uracil and also fluorouracil within the DNA, since the UNG enzyme binds to fluorouracil as well (Pettersen et al., 2011). Phenotypic differences in cell cycle progression upon the two drug treatments were also reported. The 5FdUR treatment was shown to cause an S-phase arrest in the second cycle (Huehls et al., 2016; Yan et al., 2016), while the actual time point of cell cycle arrest upon RTX treatment is still controversial (Blackledge, 1998; Ding et al., 2019; Zhao et al., 2016). Similarly, we also detected slightly altered cell cycle distribution patterns in case of the two drug treatments, which were differently influenced by the MMR status (Figure 5). In case of the 5FdUR treatment, MMR proficiency seems to lead to a weaker S-phase arrest. This might correspond to the observed decrease in the correlation of U-DNA pattern and early replication timing (Figure 4C). However, equally induced DNA damage response (reported by γH2AX) was detected upon both drug treatments (Figure 5—figure supplement 1). Consistently with our observations, Weeks et al recently showed that treatment with the antifolate pemetrexed in UNG -/- human colon cancer cells led to preferential enrichment of double-strand breaks (DSBs) within highly accessible euchromatic regions, like transcription factor binding sites, origins of replication, DNase hypersensitivity regions and CpG islands (Weeks et al., 2014). This study did not directly address the occurrence of uracil moieties but caught the process initiated by uracil incorporation at a later stage. Still, the distribution pattern of the resulting DSBs showed similarities to our U-DNA-Seq data.

As we demonstrated here, the genome-wide uracil distribution patterns have relevance for example in case of drug-treated cancer cells. Therefore, besides the global U-DNA quantification methods (MS based [Galashevskaya et al., 2013], and dot blot [Róna et al., 2016]), NGS-based techniques also have high impact.The presented new method, termed U-DNA-Seq is a direct, feasible alternative to the recently published UPD-Seq (Sakhtemani et al., 2019), Excision-seq (Bryan et al., 2014) or dU-seq (Shu et al., 2018) methods, all of which rely on indirect detection requiring one or more auxiliary chemical or enzymatic step(s). Only these three methods have the potential thus far to map genome-wide distribution of uracil within isolated genomic DNA based on NGS, and only dU-seq was used in the context of human genome. One advantage of our U-DNA-Seq is that it is a direct method employing U-DNA specific binding of catalytically inactive UNG-derived sensor constructs to pull-down uracil-containing genomic DNA-fragments. In terms of resolution, only the pre-digestion Excision-seq was shown to be able to provide single-base resolution in case of smaller size genomes with high uracil content (Bryan et al., 2014). The resolution of other methods including our new U-DNA-Seq is limited by the fragment size of the DNA library. Importantly, single-base resolution of uracil positions has decreased relevance in most cases, considering the basically stochastic nature of uracil appearance either by incorporation as a result of drug-treatment-induced dNTP pool perturbations during DNA synthesis due to insensitivity of the polymerases, or by spontaneous cytosine deamination. Due to the stochastic processes, the actual positions of uracils are expected to be variable in every single cell. Therefore, a statistical approach has higher descriptive value about the uracil distribution in these cases. Accordingly, we constructed a novel computational pipeline (Figure 2 and Figure 2—figure supplement 1) that is suitable for the description of this kind of uracil distribution patterns. We also demonstrated that the usual analysis methods designed for ChIP-seq experiment are suboptimal in this case (Figure 3—figure supplement 2). Moreover, re-analysis of the earlier published dU-seq data (Appendix 1—table 1) with the herein developed pipeline, showed very high correlation with our U-DNA-Seq data in case of comparable samples (non-treated K562 cells in both cases; and 5FdUR treated UGI expressing HCT116 vs 5FdUR treated UNG-/- HEK293T cells, Appendix 1—figure 12) confirming robustness and reliability of our method. However, our interpretation is markedly different regarding the preferential centromeric location of uracils that has been suggested by Shu et al., 2018. We analyzed the underlying reasons of this discrepancy in Appendix 1.

The new U-DNA-Seq method was shown to be reliable, robust and potent enough to gain systematic information on uracil-DNA metabolism upon drug treatments. Such information could essentially contribute to the future understanding of the mechanistic details either of cytotoxic effect induced by anti-cancer drugs, or other biological processes involving genomic uracil appearance. To this end, it is also of key importance to establish new visualization methods allowing colocalization measurements between U-DNA and other factors in highly complex eukaryotic cells.

Therefore, we further developed the U-DNA sensor to visualize genomic uracil in situ in human cells. The FLAG-ΔUNG-SNAP sensor construct and the optimized staining method presented here were successfully applied in confocal and super-resolution (STED or dSTORM) microscopies (see Figures 68). To our knowledge, there is no alternative technique published so far for in situ microscopic detection of mammalian genomic uracil. A recent paper was published reporting a similar approach, where uracil-DNA glycosylase UdgX was coupled to a fluorescent tag and applied for staining of uracils in E. coli DNA (Datta et al., 2019), however, in our previous study ΔUNG had already been proved to be potent for in situ uracil detection in the same organism (Róna et al., 2016). Still, the UdgX-based tool was not further extended for detection of uracils within the highly complex chromatin of human cells. Moreover, our detection method also allows simultaneous staining for other factors in colocalization experiments, potentially providing mechanistic insight into several important biological phenomena that involve uracil-DNA. For colocalization studies, two histone markers were selected based on the U-DNA-Seq results, namely H3K36me3 and H3K27me3, which were the strongest correlating factors for RTX_UGI and 5FdUR_UGI U-DNA patterns, respectively (Figure 4A–B). Using dSTORM super-resolution microscopy we could confirm significant correlation of genomic uracil with both selected histone markers in drug-treated (5FdUR or RTX), UGI-expressing cells (Figure 8). H3K36me3 was shown to associate with actively transcribed genes (Becker et al., 2017; Hyun et al., 2017; Pfister et al., 2014), while H3K27me3 is the most cited marker for facultative heterochromatin (Becker et al., 2017; Saksouk et al., 2015). Strikingly, we found that H3K27me3 shows even stronger colocalization with the U-DNA pattern in case of the RTX treated sample as compared to the 5FdUR treated one, which might be indicative for RTX treatment induced chromatin remodeling at least regarding this histone modification. It is important to note that our U-DNA-Seq was compared to published data that corresponds to ChIP-seq experiments performed in non-treated cells. However, during in situ cellular colocalization studies, the drug treatment is obviously applied to both patterns (i.e. U-DNA and histone marker). With the ChIP-seq experiment for H3K36me3 performed on both RTX treated and non-treated cells, we demonstrated that such treatment-induced chromatin remodeling is not a general phenomenon, but may rather confine to certain factors (Figure 4A–B, and Figure 4—figure supplement 1). Based on these observations, we can confirm that such in silico correlation studies has a predictive potential allowing qualitative characterization, and further independent techniques are required for detailed studies. In summary, co-staining of the selected histone markers and the genomic uracil in drug-treated cells via dSTORM reinforced the association between uracil occurrence and transcriptionally active regions.

It has been argued that uracil accumulation may play a more decisive role in genomic instability than the induced uracil-excision repair (Huehls et al., 2016; Yan et al., 2016). Uracil in DNA may therefore be used as a key marker for estimating efficiency of chemotherapeutic drugs targeting thymidylate biosynthesis. Our presented new techniques, namely the U-DNA-Seq and the related in situ U-DNA detection methods provide key insights into the mechanism of chemotherapeutic drugs. The combination of these methods might become a highly potent approach in the future, that is to investigate the complex pattern of intra-tumor heterogeneity that is closely related to cancer progression and drug-resistance (Stanta and Bonin, 2018), therefore may contribute to improving clinical practice.

Materials and methods

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene
(bacteriophage PBS2)
UGIMol et al., 1995UniProtKB
- P14739
UNG inhibitor protein
encoded in Bacillus subtilis
bacteriophage PBS2
AntibodyAnti-FLAG M2,
mouse monoclonal
SigmaF3165(1:10000)
Antibodyanti-H3K36me3,
rabbit monoclonal
Cell Signaling Technologies4909T(1:8000) for ICC
Antibodyanti-H3K27me3,
rabbit monoclonal
Cell Signaling Technologies9733T(1:6000) for ICC
Antibodyanti-γH2AX,
rabbit monoclonal
Sigma05–636(1:500) for measuring DSBs
in flow cytometry
Cell line (Homo sapiens)293TYvonne Jones (Cancer
Research UK, Oxford, UK)
maintained in Dulbecco’s
modified Eagle’s medium
completed with
PenStrep and FBS
Cell line (Homo sapiens)K562European Collection of
Cell Cultures
maintained in RPMI 1640
(GlutaMAX Supplement, HEPES)
Medium (Gibco), completed
with PenStrep and FBS
Cell line (Homo sapiens)HCT116European Collection of
Cell Cultures
maintained in McCoy’s 5A medium,
completed with PenStrep and FBS
Cell line (Homo sapiens)HCT116+ch3
sub-line
C. Richard Boland (Baylor
University, Dallas, Texas, US)
sub-line of HCT116: MLH1 restored,
MMR is functional
Cell line (Homo sapiens)HCT116+
hUGI/EGFP
This papersub-line of HCT116: stable
expressing UGI (Materials and
methods: Generation of
UGI-expressing stable cell lines)
Cell line (Homo sapiens)HCT116+ch3+
hUGI/EGFP
This papersub-line of HCT116+ch3: stable
expressing UGI (Materials and
methods: Generation of
UGI-expressing stable cell lines)
Strain, strain background
(Escherichia coli)
XL1-BlueStratagene
Strain, strain background
(Escherichia coli)
CJ236 [dut-, ung-]NEBE. coli strain for preparation
of the uracil-containing DNA
Strain, strain background
(Escherichia coli)
BL21(DE3) ung-151Samuel E Bennett (Oregon
State University, Corvallis, US)
E. coli strain for expression
of ΔUNG sensor constructs
Sequence-based reagentactin-forSigma-Aldrich, Ho et al., 20165’-CCTCATGGCCTTGTCACAC-3’
Sequence-based reagentactin-revSigma-Aldrich, Ho et al., 20165’-GCCCTTTCTCACTGGTTCTCT-3’
Sequence-based reagentpET15b-ForSigma-Aldrich5’-CATATGCTCGAGGATCCGGC-3’
Sequence-based reagentpET15b-RevSigma-Aldrich5’-TCATCGATAAGCTTTAATGCGGT-3’
Sequence-based reagentSpin-FwSigma-Aldrich5’- ACCGGCATAACCAAGCCTAT-3’
Sequence-based reagentSpin-RevSigma-Aldrich5’- ACAATGCGCTCATCGTCATC-3’
Recombinant
DNA reagent
pLGC-hUGI/EGFPMichael D Wyatt (South
Carolina College of
Pharmacy, University of
South Carolina, US)
for producing sub-lines
stably expressing UGI
Recombinant
DNA reagent
pSNAPfNEBN9183Sto clone the FLAG-ΔUNG-SNAP
construct
Peptide, recombinant
protein
FLAG-ΔUNG-SNAPThis paperproduced in E. coli BL21(DE3)
ung-151 (Materials and methods:
Plasmid constructs and
cloning of the FLAG-ΔUNG-SNAP
construct)
Peptide, recombinant
protein
1xFLAG-ΔUNGRóna et al., 2016produced in E. coli
BL21(DE3) ung-151
Peptide, recombinant
protein
3xFLAG-ΔUNGRóna et al., 2016produced in E. coli
BL21(DE3) ung-151
Peptide, recombinant
protein
FLAG-ΔUNG-DsRedRóna et al., 2016produced in E. coli
BL21(DE3) ung-151
Commercial assay, kitQuick-DNA Miniprep
Plus Kit
Zymo ResearchD4069for genomic DNA preparation
Commercial assay, kitNucleoSpin Gel and
PCR Clean-up Kit
MACHEREY-NAGEL
GmbH and Co. KG
740609.25for IP DNA purification
Commercial assay, kitNGS including library
preparation
NovogeneNovaseq 6000,
20 GB, 150 PE
as a service
Commercial assay, kit5-Bromo-2′-deoxy-uridine
(BrdU) Labeling and
Detection Kit I
Roche, Sigma11296736001
Chemical compound,
drug
Anti-FLAG M2
agarose beads
SigmaA2220for U-DNA-IP
Chemical compound,
drug
Pierce Protein A/G
Magnetic Beads
Thermo Fisher Scientific88802for ChIP
Chemical compound,
drug
5-fluoro-2′-deoxyuridine
(5FdUR)
SigmaF0503Thymidylate synthase inhibitor,
treatment: 20 µM, 48 hr
Chemical compound,
drug
raltitrexed (RTX)SigmaR9156Thymidylate synthase inhibitor,
treatment: 100 nM, 48 hr
Chemical compound,
drug
SNAP-Surface
Alexa Fluor 546
NEBS9132SSNAP substrate for
superresolution imaging
Chemical compound,
drug
SNAP-Surface
Alexa Fluor 647
NEBS9136SSNAP substrate for
superresolution imaging
Software, algorithmImageJ (Fiji)National Institutes
of Health
for densitometry,
and image processing
Software, algorithmHuygens STED
Deconvolution Wizard
Huygens Softwaresuperresolution image
analyzing software package
Software, algorithmBWALi and Durbin, 2010short sequencing read
aligner software
Softwares, algorithmdeepTools packageRamírez et al., 2016NGS data processing tools
Software, algorithmbedtools packageQuinlan and Hall, 2010tools for analyzing interval files
Software, algorithmGIGGLE searchLayer et al., 2018.search tool for similarity screening
in large set of interval files
Software, algorithmSegway software
package
Chan et al., 2018;
Hoffman et al., 2012
machine learning software for
genome segmentation
Software, algorithmIntegrated Genome
Viewer (IGV)
Thorvaldsdóttir et al., 2013.tool for visualisation of many
types of processed NGS data

Plasmid constructs and cloning of the FLAG-ΔUNG-SNAP construct

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The pLGC-hUGI/EGFP plasmid was kindly provided by Michael D. Wyatt (South Carolina College of Pharmacy, University of South Carolina, US). Generation of catalytically inactive U-DNA sensor proteins (1xFLAG-ΔUNG, 3xFLAG-ΔUNG, FLAG-ΔUNG-DsRed) was described previously (Róna et al., 2016). pSNAPf (New England Biolabs (NEB), Ipswich, Massachusetts (MA), US) was PCR amplified with primers SNAP-Fw (5’ – TAA TGG TAC CGC GGG CCC GGG ATC CAC CGG TCG CCA CCA TGG ACA AAG ACT GCG AAA TG - 3’) and SNAP-Rev (5’ – ATA TCT CGA GGC CTG CAG GAC CCA GCC CAG G - 3’). The resulting fragments were digested by KpnI and XhoI, and ligated into the KpnI/XhoI sites of the plasmid construct FLAG-ΔUNG-DsRed (in a pET-20b vector) yielding the FLAG-ΔUNG-SNAP construct. Scheme of the used constructs is shown in Figure 6—figure supplement 1A. Primers used in this study were synthesized by Sigma-Aldrich (St. Louis, Missouri, US), and all constructs were verified by sequencing at Microsynth Seqlab GmbH (Göttingen, Germany). All UNG constructs were expressed in the Escherichia coli BL21(DE3) ung-151 strain and purified using Ni-NTA affinity resin (Qiagen, Hilden Germany) as described previously (Róna et al., 2016).

DNA isolation and purification

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pEGFP-N1 plasmid (Clontech, Mountain View, California, US) was transformed into XL1-Blue [dut+, ung+] (Stratagene, San Diego, California (CA), US) or CJ236 [dut-, ung-] (NEB) E. coli competent cells. Cell cultures were grown for 16 hr in Luria broth (LB) media supplemented with 50 µg/ml kanamycin at 37°C. Plasmids used in this study were purified using PureYield Plasmid Midiprep Kit (Promega, Madison, Wisconsin, US) according to the instructions of the manufacturer. XL1-Blue and CJ236 E. coli strains were propagated in LB media at 37°C and were harvested at log phase. Genomic DNA of bacterial samples as well as eukaryote cells was purified using the Quick-DNA Miniprep Plus Kit (Zymo Research, Irvine, California, US) using the recommendations of the manufacturer.

Cell culture, transient transfection and treatment of cells

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The 293T cell line was a generous gift of Yvonne Jones (Cancer Research UK, Oxford, UK). The HCT116 and the K562 cell lines were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK). The HCT116+ch3 sub-line (a kind gift from C. Richard Boland (Baylor University, Dallas, Texas, US)) is complemented with chromosome three carrying the wild type gene for hMLH1 and is competent in MMR function. 293T cells were grown in Dulbecco’s modified Eagle’s medium (Gibco, Life Technologies, Carlsbad, CA, US), while HCT116 and K562 cells were maintained in McCoy’s 5A medium (Gibco) and RPMI 1640 (GlutaMAX Supplement, HEPES) Medium (Gibco), respectively. Media was supplemented with 50 µg/ml Penicillin-Streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were cultured at 37°C in a humidified incubator with 5% CO2 atmosphere. All cell lines used in this study were tested for mycoplasma contamination. HCT116 cells were transfected with FuGENE HD (Promega) according to the manufacturer’s recommendation. For immunocytochemistry, HCT116 cells were transfected with normal pEGFP-N1 (purified from XL1-Blue [dut+, ung+] E. coli cells) or uracil-rich pEGFP-N1 (purified from CJ236 [dut−, ung−] E. coli cells) vector as described previously (Róna et al., 2016). Forty hours after transfection with UGI expressing vectors, transiently transfected cells were grown for an additional 48 hr either in the absence or presence of 20 µM 5FdUR (Sigma) before collecting them for genomic DNA purification.

Generation of UGI-expressing stable cell lines

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Retroviral packaging and stable cell line generation were done as described in Rona et al., 2018. Briefly, 293T cells (1.5 × 106 cells in T25 tissue culture flasks) were transfected with 1.5 µg pLGC-hUGI/EGFP, 0.5 µg pCMV-VSV-G envelope and 0.5 µg pGP packaging plasmids using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, US) according to the manufacturer’s recommendation. The supernatant, containing lentiviral particles was collected and filtered through a 0.45 µm filter (Merck Millipore, Burlington, MA, US) 36 hr after the transfection. Successfully transduced MMR deficient and proficient HCT116 cells were collected by FACS sorting for GFP-positive cells using a BD FACSAria III Cell sorter (BD Biosciences, San Jose, CA, US). UGI-expressing cells were treated with 20 µM 5FdUR or 100 nM RTX (Sigma) for 48 hr before fixation for immunocytochemistry or collecting them for genomic DNA purification described above.

Dot blot measurements and analysis for quantification of U-DNA

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Detection of the genomic uracil content by dot blot measurements were carried out using 3xFLAG-ΔUNG construct, as described earlier (Róna et al., 2016). Dot blot assay was used for measuring genomic uracil levels of non-treated and drug (5FdUR or RTX) treated MMR deficient and proficient HCT116 cells expressing UGI (Figure 1—figure supplement 1B–D), or to confirm the successful enrichment of uracil-containing DNA (Figure 1B), and also to compare uracil recognition specificity of the FLAG-ΔUNG-DsRed and FLAG-ΔUNG-SNAP constructs (Figure 6—figure supplement 1B). Densitometry was done using ImageJ (Fiji) software (National Institutes of Health, US). Analysis of the data and the calculation of the number of deoxyuridine nucleotides in the unknown genomic DNA was described before (Molnár et al., 2018; Róna et al., 2016). Briefly, the number of uracil/million bases in the unknown samples were determined by interpolating their normalized intensities to the calibration curve of the standard. Statistical analysis of dot blot (Figure 1—figure supplement 1C–D) was carried out by Microsoft Excel using the non-parametric two-sided Mann-Whitney U test. Differences were considered statistically significant at p<0.005. Data presented are representative of six independent datasets (n = 6).

DNA immunoprecipitation

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After 48 hr treatment, the surface attached cells were harvested. Genomic DNA was purified by Quick-DNA Miniprep Plus Kit (Zymo Research) and eluted in nuclease-free water. 12 µg of genomic DNA was sonicated into fragments ranging between 100 and 500 base pairs (bp) (checked by agarose gel electrophoresis) with a BioRuptor (Diagenode, Liège, Belgium). 25% of the samples was saved as input, and the remaining DNA was re-suspended in the following IP buffer: 30 mM TRIS-HCl, pH = 7.4, 140 mM NaCl, 0.01% Tween-20, 1 mM ethylenediaminetetraacetic acid (EDTA), 15 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine. Immunoprecipitations were carried out with 15 µg of 1xFLAG-ΔUNG construct for 2.5 hr at room temperature with constant rotation. Anti-FLAG M2 agarose beads (Sigma) were equilibrated in IP buffer, and then added to the IP mixture for 16 hr at 4°C with constant rotation. Beads were washed three times for 10 min in IP buffer, and re-suspended in elution buffer containing 1% sodium dodecyl sulfate (SDS), 0.1 M NaHCO3. Elution of uracil sensor protein binding U-DNA was done by vortexing for 5 min with an additional incubation for 20 min with constant rotation. After centrifugation (13000 rpm for 3 min), supernatant was transferred to clean tubes. This procedure was repeated with the same amount of elution buffer, and protein/DNA eluted complexes were combined in the same tube. Samples were incubated with 150 µg/ml RNAse A (Epicentre, Paris, France) for 30 min, followed by the addition of 500 µg/ml Proteinase K (Sigma) for 1 hr at 37°C for removal of RNA and proteins. Immunoprecipitated DNA was purified with NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL GmbH and Co. KG, Düren, Germany) according to the manufacturer’s instructions. Densitometry analysis of agarose gel was done using ImageJ (Fiji) software for concentration calculation of fragmented DNA. Enrichment of uracil in the DNA samples was examined by dot blot assay. DNA libraries were created from the samples and then subjected to next-generation sequencing (NGS). Scheme of U-DNA-Seq is shown in Figure 1A.

Controls of U-DNA-IP method

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For positive control of the U-DNA-IP, uracil-containing 315 bp spike-in oligo was prepared by PCR amplification from pET15b in the presence of 0.02 mM dUTP, and 0.2 mM dNTP mix using TEMPase Hot Start DNA polymerase (VWR (Radnor, Pennsylvania, US)). Uracil-free oligo was also amplified under the same reaction conditions but in the absence of dUTP. Primer sequences are as follows: pET15b-For: 5’-CATATGCTCGAGGATCCGGC-3’; pET15b-Rev: 5’-TCATCGATAAGCTTTAATGCGGT-3’. Spike-in oligos were purified with NucleoSpin Gel and PCR Clean-up Kit. 2.5 nM uracil-containing or uracil-free spike-in DNA was added into 3 µg of sonicated genomic DNA from non-treated HCT116 cells, then DNA-IP was carried out as described above. Enrichment was measured by qPCR (on a QuantStudio 1 qPCR instrument (Thermo Fisher Scientific (Waltham, MA, US))) and calculation was based on the comparison of the Cq values for IP samples using uracil-containing and uracil-free spike-in oligos. Primer sequences are as follows: Spin-Fw: 5’- ACCGGCATAACCAAGCCTAT-3’; Spin-Rev: 5’- ACAATGCGCTCATCGTCATC-3’. For negative control of the U-DNA-IP, mock IP experiments were also performed using empty anti-FLAG beads not containing the U-DNA sensor on genomic DNA from non-treated (NT_UGI) and 5FdUR treated (5FdUR_UGI), UGI-expressing HCT116 cells, using the same protocol as described above. The amounts of pulled down DNA were much decreased in these control IPs as compared to their true IP counterparts, still NGS were performed (Figure 1—figure supplement 2, Supplementary file 1).

High-throughput DNA sequencing and data analysis

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Sequencing of input and enriched U-DNA samples were done on two independent biological replicates at BGI (China) generating 100 bp paired-end reads (PE) on a HiSeq 4000 instrument or at Novogene (China) using the Novaseq 6000 platform resulting in 150 bp PE reads. Analysis pipeline is summarized in Figure 2, and details including the applied command lines and scripts are found in the Supplementary file 1 and 35. Sequencing reads were aligned to the GRCh38 human reference genome (version GRCh38.d1.vd1) (Jensen et al., 2017) using BWA (version 0.7.17) (Li and Durbin, 2010). Aligned reads were converted to BAM format and sorted using samtools (version 1.9) (Li et al., 2009). Duplicate reads were marked using Picard Tools (version 1.95). As a part of pre-processing, blacklisting and filtering of ambiguously mapped reads were also performed (Supplementary file 1 and Figure 2—figure supplement 2; Amemiya et al., 2019). For data processing, to derive uracil distribution signal, first, normalized coverage signals were calculated and smoothened using bamCoverage from the deepTools package (Ramírez et al., 2016), which resulted in genome-scaled coverage tracks in bigWig format. Then, log2 ratio of the coverage tracks (enriched/input) were calculated with bigwigCompare. These bigwig files were compared using the multiBigwigSummary, Pearson correlations were calculated using the plotCorrelation tools also from the deepTools package (Figure 3B). From the log2 ratio tracks, interval (bed) files were derived using reasonable thresholds (for details see Supplementary file 1 and Figure 3—figure supplement 2A). Log2 ratio signal distribution (Figure 3C) was calculated using R. Peaks of coverage were also called using the MACS2 with broad option (version 2.1.2), a standard tool in chromatin marker ChIP-seq data analysis (Feng et al., 2011; Zhang et al., 2008). Results of peak calling and the regions derived from the log2 ratio tracks were compared (Figure 3—figure supplement 2). Hereafter, the two terms ’peaks’ and ’regions’ will be consequently applied for the results of the two approaches, respectively. For the negative control IP samples, genome-scaled coverage tracks were also calculated in the same way. Then normalized signal tracks were subtracted from their corresponding U-DNA-IP tracks, and combined with their input to calculate log2 enrichment tracks (Supplementary file 1 and Figure 1—figure supplement 2). Colocalization analysis of identified uracil enriched regions with other ChIP-seq and DNA accessibility data was performed on a dataset containing HCT116 specific or other relevant data only (for details see Supplementary file 3) using GIGGLE search tool (Layer et al., 2018). To plot results of GIGGLE search, OriginPro 8.6 was used (Figure 4A). Genome segmentation analysis on our U-DNA-Seq data, our H3K36me3 ChIP-seq data, and HCT116 specific ChIP-seq data from the ENCODE database was performed using Segway software package (Chan et al., 2018; Hoffman et al., 2012Supplementary file 3, and Figure 4B). Measuring overlaps with other genomic features (Figure 4C) was done using bedtools annotate tool (Quinlan and Hall, 2010) as it is described in Supplementary file 4. Replication timing scores and AT content were calculated on the genomic segments defined by the Segway analysis as described in Supplementary file 4 (Figure 4—figure supplement 3). Correlation analysis between uracil enrichment and replication timing (Figure 4D and Appendix 1—figure 2C) was done using R as it is described in Supplementary file 5. Sequencing data were visualized (Figure 3A, Figure 1—figure supplement 2, Figure 2—figure supplement 2, Figure 3—figure supplement 1, Figure 4—figure supplement 2, Supplementary file 2, Appendix 1—figure 1A, Appendix 1—figure 2A) using the IGV browser (Thorvaldsdóttir et al., 2013).

Chromatin immunoprecipitation and sequencing (ChIP-seq)

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Sub-confluent cultures of UGI-expressing (non-treated or treated with 100 nM RTX for 48 hr) cells were washed with phosphate-buffered saline (PBS) and cross-linked with 1% paraformaldehyde (PFA) for 10 min, then quenched with the addition of 0.15 M glycine. Cells then were rinsed with ice-cold PBS twice and lysed with buffer LB1 (50 mM TRIS, pH = 7.5, 140 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.25% Triton X-100, 10% glycerol, and protease inhibitor cocktail) for 10 min at 4°C, then in LB2 (10 mM TRIS, pH = 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and protease inhibitor cocktail) for 10 min at 4°C. Nuclei pellets were sonicated in LB3 (10 mM TRIS, pH = 7.5, 0.5% N-Lauroylsarcosine sodium salt, 1 mM EDTA, 0.5 mM EGTA, and protease inhibitor cocktail) in a BioRuptor, which yielded fragments between 100 and 500 bp. After centrifugation for 10 min at 4°C, supernatants were diluted in dilution buffer (50 mM TRIS, pH = 7.5, 0.5% NP-40, 1 mM EDTA, 150 mM NaCl) followed by pre-clearing of Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific) for 3 hr at 4°C. Immunoprecipitation was performed overnight at 4°C using anti-H3K36me3 (CST (Danvers, MA, US), cat.no.: 4909T) antibody following the supplier's recommendations. After immunoprecipitation, protein A/G magnetic beads (pre-cleared with IgG-free fetal bovine serum albumin (BSA, Jackson ImmunoResearch (Cambridgeshire, UK)), overnight at 4°C) were added for further 7 hr of incubation. Precipitates were washed sequentially for 10 min each with the 1:1 combination of dilution buffer and HS buffer (20 mM TRIS, pH = 8.0, 0.1% SDS, 1% NP-40, 2 mM EDTA, 500 mM NaCl), with HS buffer, and finally with dilution buffer. Precipitates were then washed with TE buffer (10 mM TRIS-HCl, pH = 7.5, 1 mM EDTA) and eluted two times with 1% SDS and 0.1 M NaHCO3. Eluates were pooled and heated overnight at 65°C to reverse the formaldehyde crosslinking. Samples were incubated with 100 µg/ml RNAse A for 30 min, then with 200 µg/ml Proteinase K for 1 hr at 37°C for removal of RNA and proteins. Immunoprecipitated DNA was purified with NucleoSpin Gel and PCR Clean-up Kit according to the manufacturer’s instructions. Quantitative PCR analysis for human β-actin was carried out to check the efficiency of the H3K36me3 IP using the following primer sequences: actin-for: 5’-CCTCATGGCCTTGTCACAC-3’; actin-rev: 5’-GCCCTTTCTCACTGGTTCTCT-3’ (Ho et al., 2016). DNA libraries were created from the samples and then subjected to NGS at Novogene using the Novaseq 6000 platform resulting in 150 bp PE reads. Data analysis were performed similarly to the U-DNA-Seq analysis (Figure 4—figure supplement 1), details are provided in Supplementary file 3.

Cell cycle analysis and γH2AX staining

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2D cell cycle analysis was performed using 5-Bromo-2′-deoxy-uridine (BrdU) Labeling and Detection Kit I (Roche, Sigma) and Propidium Iodide (PI, Sigma) staining (Figure 5). Non-treated or drug-treated (20 µM 5FdUR or 100 nM RTX for 48 hr) HCT116 cells were labelled with 10 µM BrdU for 20 min followed by trypsinization, PBS washing and overnight fixation in 70% ethanol at 4°C. DNA was denatured for 30 min with 2 M HCl, 0.5% Triton X-100. Cells were re-suspended in 0.1 M sodium tetraborate (pH = 8.5) for 10 min, and then washed with blocking buffer (1% BSA, 0.05% Tween-20 in PBS). Samples were incubated with anti-BrdU antibody (1:10) in blocking buffer for 30 min at room temperature. After washing, Ig fluorescein coupled (FITC) anti-mouse (1:10) secondary antibody was applied in blocking buffer for 30 min. Finally, after a washing step, cells were incubated with propidium iodide (10 µg/ml) and RNase A (20 µg/ml) for 30 min in PBS. Occurrence of DSBs was investigated by immunofluorescent staining of γH2AX (Figure 5—figure supplement 1). Briefly, non-treated or drug-treated cells were fixed in 70% ethanol (overnight at 4°C), then DNA was denatured for 30 min with 2 M HCl, 0.5% Triton X-100. After blocking, cells were stained with an antibody against γH2AX (1:500, Sigma, cat.no.: 05–636) overnight at 4°C. FITC anti-mouse secondary antibody (1:10) was added for 30 min. Cell cycle analysis and measurement of γH2AX levels were carried out by flow cytometry with a BD FACSCalibur Cell Analyzer.

Immunofluorescent staining of uracil residues

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Detection of uracil residues was done in extrachromosomal plasmids after transfection (Figure 6—figure supplement 1C) or in genomic DNA of HCT116 cells (Figures 68). Staining of extrachromosomal DNA was done as described previously (Róna et al., 2016) with minor modifications for comparison of FLAG-ΔUNG-DsRed or FLAG-ΔUNG-SNAP sensor constructs. Briefly, uracil residues were visualized by applying 1.5 µg/ml of the FLAG-ΔUNG-DsRed or the FLAG-ΔUNG-SNAP, and then primary (anti-FLAG M2 antibody (1:10000, Sigma)) and secondary antibodies (Alexa 488 (1:1000, Molecular Probes, Eugene, Oregon, US)). For immunofluorescent staining of genomic uracil residues, control or HCT116 cells stably expressing UGI were seeded onto 24-well plates containing cover glasses or onto µ-Slides (or their glass bottom derivative) (ibidi GmbH, Germany) suitable for use in STED and single molecule applications, and treated as indicated. In case of dSTORM imaging, coverslips were coated with poly-D-lysine (Merck Millipore) before seeding the cells. Sub-confluent cultures of cells were fixed using 4% PFA (pH = 7.4 in PBS) or Carnoy’s fixative (ethanol: acetic acid: chloroform = 6:3:1) for 15 min. In case of dSTORM imaging, cells were pre-extracted with ice-cold CSK buffer (10 mM PIPES, pH = 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM EGTA, 3 mM MgCl2, 0.25% Triton X-100) containing protease and phosphatase inhibitor tablets (Roche, Basel, Switzerland) for 5 min before PFA fixation. After washing or rehydration steps (1:1 ethanol:PBS, 3:7 ethanol:PBS, PBS), epitope unmasking was done by applying 2 M HCl, 0.5% Triton X-100 for 30 min. DNA denaturation with HCl was required in order to increase DNA accessibility for efficient staining and to eliminate any potential interaction between the overexpressed UGI and the applied UNG sensor construct. After neutralization with 0.1 M Na2B4O7 (pH = 8.5) for 5 min followed by PBS washes, cells were incubated in blocking solution I (TBS-T (50 mM TRIS-HCl, pH = 7.4, 2.7 mM KCl, 137 mM NaCl, 0.05% Triton X-100) containing 5% non-fat dried milk) for 15 min, followed by incubation in blocking buffer I supplemented with 200 µg/ml salmon sperm DNA (Invitrogen) for an additional 45 min. Uracil residues were visualized by applying 4 µg/ml of the FLAG-ΔUNG-SNAP construct for 1 hr in blocking buffer I with 200 µg/ml salmon sperm DNA at room temperature. After several washing steps with TBS-T containing 200 µg/ml salmon sperm DNA, primary, then secondary antibodies were operated in blocking buffer II (5% fetal goat serum (FGS), 3% BSA and 0.05% Triton X-100 in PBS). Anti-FLAG M2 antibody (1:10000, Sigma), then Alexa 488 conjugated secondary antibody (1:1000, Molecular Probes) was applied for 1 hr in blocking buffer II, enabling visualization of FLAG epitope. SNAP-tag substrates were also used to label SNAP-tag fusion proteins when FLAG-ΔUNG-SNAP was applied as the uracil sensor protein. Cells were labelled with 2.5 µM (0.5 µM for dSTORM imaging) SNAP-Surface Alexa Fluor 546 or 647 (indicated as SNAP546 and SNAP647 in this study) (NEB) for 20 min, and optionally counterstained with 1 µg/ml DAPI (4’,6-diamidino-2-phenylindole, Sigma) nucleic acid stain, followed by several PBS washing steps before embedding in FluorSave Reagent (Calbiochem, Merck Millipore). For labelling of histone markers, anti-H3K36me3 (1:8000, CST, cat.no.: 4909T) or anti-H3K27me3 (1:6000, CST, cat.no.: 9733T) primary antibodies were used, then visualized by Alexa 568 conjugated secondary antibody (1:10000, Molecular Probes) in dSTORM or Alexa 555 conjugated secondary antibody (1:2000, Molecular Probes) in confocal imaging.

Confocal and STED imaging and analysis

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Confocal images were acquired on a Zeiss LSCM 710 microscope using a 20x (NA = 0.8) or a 63x (NA = 1.4) Plan Apo objective or a Leica TCS SP8 STED 3X microscope using a 100x (NA = 1.4) Plan Apo objective. STED images were acquired on the Leica TCS SP8 STED 3X microscope using 660 nm STED (1.5 W, continuous wave) laser for depletion (in combination with Alexa 546). The same image acquisition settings were applied on each sample for comparison. A moderate degree of deconvolution was applied to the recorded STED images using the Huygens STED Deconvolution Wizard (Huygens Software), based on theoretical point spread function (PSF) values. Fluorescence images were processed using ZEN and ImageJ (Fiji) software. 3D projection movies (Figure 7—videos 14) were constructed from Z-stack images captured by confocal or STED imaging.

dSTORM imaging and image reconstruction

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Super-resolution images were obtained and reconstructed as previously described (Rona et al., 2018). Briefly, dSTORM images were recorded using an in-house built imaging platform based around an inverted microscope. Two color imaging was carried out sequentially on samples labelled with SNAP-Surface Alexa Fluor 647 and Alexa Fluor 568. The imaging buffer, consisting of 1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 10% glucose, 100 mM mercaptoethylamine (MEA) in PBS, was mixed and added just before imaging. For display purposes, super-resolution images shown in the manuscript have been adjusted for brightness and smoothed; however, quantitative analysis were performed on images before being manually processed to avoid any user bias.

Interaction factor

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The interaction factor (IF) quantifies the colocalization of red and green foci within a cell nucleus by measuring the area of overlap between the two sets of foci (Bermudez-Hernandez et al., 2017; Whelan et al., 2018). The positions of the green foci are then randomized and the overlap between the two colors is measured again. This randomization is repeated 20 times, and the interaction factor is the ratio between the experimental overlap area and the mean of the randomized overlap areas. If the red and green foci were completely independent of each other, the IF value would equal one. A value greater than one signifies a higher degree of colocalization compared to a random sample. Non-parametric Mann-Whitney U test was used to calculate statistics on the graphs. Differences of the IF values were considered statistically significant at p<0.0001 as indicated in Figure 8C–D. Data are presented from two independent biological experiments.

Appendix 1

Re-analysis and reinterpretation of dU-seq data published in Shu et al., 2018

Shu and co-workers recently published the dU-seq method and reported centromeric location of genomic uracil in non-treated cells (Shu et al., 2018). In this study, dU-seq as a short-read sequencing technology was used to map centromeric localization of a genomic feature. Centromeres are known to be highly repetitive, poorly mappable regions of the human genome, even if centromeric model sequences (Miga et al., 2014) are implemented into the GRCh38 (hg38) assembly (Guo et al., 2017). Therefore, blacklisting and mappability filters are highly recommended to be used, especially if the analysis is focusing on such critical regions, like in this case.

The dU-seq data analysis pipeline, as it was published in the Shu et al, included the following pre-processing steps: (1) pre-alignment of the 150 bp paired-end sequencing data to the spike-in sequences applied in their experiments; (2) trimming the adaptors and the low quality segments; (3) alignment of the remaining reads to reference human genome GRCh38 (it is not clear, to which set exactly) using bowtie2. There was no mention either about (1) deduplication of the data, or (2) filtering for uniquely mapped reads, or (3) applying recommended blacklists. However, in widely accepted ChIP-seq pipelines, only uniquely mapped reads are considered as valid information (Qin et al., 2016).

The detection of uracil enrichment within these aligned reads was done by peak calling using MACS2, separately for the uracil ‘pull-down’ and the ‘control’ samples. It was not mentioned if the corresponding input was included as a control in the peak calling process, or not. It is also not clear what options and parameters were used in their MACS2 runs (e. g. broad, no-model or broad-cutoff). Then they subtracted the peaks detected in the ‘control’ from the ones called in the ‘pulled-down’ samples. We claim that this approach is clearly suboptimal considering the lower descriptive value and the lower reproducibility of peak calling for the uracil-DNA distribution as shown in Figure 3—figure supplements 12. To judge the reproducibility of peak calling in dU-seq data, is also not trivial because they did not uploaded all the peak data for their replicates (GSE99011). We calculated the Jaccard indices for their uploaded non-treated HEK293 (two replicates) and UNG-knock-out HEK293 (four replicates) data, and found really low values (0.063 for the two replicates, and 0.030, 0.059, 0.075, 0.029, 0.029, 0.055 in the pairwise comparison of the four replicates). Jaccard indices between the individual replicates and the united data of the non-treated HEK293 sample were 0.134 and 0.092. These values are even lower than in case of similar peak calling on our U-DNA-Seq data (Figure 3—figure supplement 2B).

The correlation with the CENPA bound genomic regions, published as a key point in their paper, is also questionable. Raw reads of CENPA ChIP-seq data (GSE45497) were downloaded and mapped to GRCh38. Details are not provided in the paper; however, they most probably used the same procedure as in case of the dU-seq data, namely, aligning reads without blacklisting and mapping quality filters, followed by peak calling. Moreover, the CENPA data were originally aligned to GRCh37 (hg19) reference genome using a more careful algorithm filtering out potential artefacts (Hayden et al., 2013). It would have been recommended to either follow their approach or simply lift over the original alignment from GRCh37 to GRCh38.

Unfortunately, Shu et al incompletely uploaded the peak calling results for the replicates to the GEO database. The uploaded peaks were not always correlating with the ones they published on the manuscript’s figures (Figure 2a, where they show a K562 peak on chromosome 21 that is actually not present in their uploaded peak data, and another peak on chromosome 16 that corresponds to aligned reads characterized with MAPQ = 0, at least in our alignment). Given missing data uploads, it is challenging to reproduce their results.

Still, we were curious, whether their dU-seq data itself (not the interpretation of that) correlates with our U-DNA-Seq data, or not. Therefore, we used our novel analysis pipeline described in the present study, to process and re-analyses raw data from Shu et al., 2018, notably the following data:

  • K562 „input": SRR5572773/GSM2630035 and SRR5572774/GSM2630036

  • K562 „control": SRR5572775/GSM2630037 and SRR5572776/GSM2630038

  • K562 „PD": SRR5572777/GSM2630039 and SRR5572778/GSM2630040

  • HEK293T UNGKO 5FU „input": SRR5998406/GSM2769605 and SRR5998407/GSM2769606

  • HEK293T UNGKO 5FU „control": SRR5998408/GSM2769607 and SRR5998409/GSM2769608

  • HEK293T UNGKO 5FU „PD”: SRR5998410/GSM2769609 and SRR6026694/GSM2769610

We remapped the fastq reads to the human reference genome GRCh38 (GRCh38.d1.vd1.fa, GDC reference, https://gdc.cancer.gov/about-data/data-harmonization-and-generation/gdc-reference-files) using BWA, filtered out ambiguously mapped reads, and applied a blacklist created as in case of our data (Figure 2—figure supplement 2). The statistics of this pre-processing are summarized in Appendix 1—table 1. The relative amount of the spike-in sequences is significantly higher in mock pull-down („control”) and the non-treated K562 pull-down samples, where the sample DNA itself was not labelled or was present at extremely low level, respectively. The overall depth of sequencing was lower in these cases, which decrease the reliability or reproducibility of further data analysis.

Appendix 1—table 1
Statistics on pre-processing of dU-seq data.

Samples from the study of Shu et al., 2018 (non-treated K562 (K562), and 5FdUR treated UNG-knock-out HEK293 cells (5FdUR_UNGKO HEK293)) are compared to our samples (5FdUR treated UGI-expressing HCT116 cells (5FdUR_UGI), and non-treated wild type K562 cells (K562), the same data as in Supplementary file 1-table 3). In case of dU-seq samples, inputs are genomic DNA fragmented and treated according to the dU-seq protocol, also containing additional spike-in sequences; controls are pulled-down in a mock experiment excluding UNG treatment; while PD means the pull-down samples according to the dU-seq protocol. Number of raw reads means read number before starting alignment (the sum of the mapped and unmapped reads). Uniquely mapped read means that MAPQ is not zero. The dU-seq and the U-DNA-Seq samples markedly differ in the ratio of mapped (*) and unmapped (*) reads due to the spike-in DNA applied in dU-seq only.

SampleReplicatesNumber of raw readsNumber of mapped* readsUnmapped* readsUniquely mapped readsUniquely mapped reads after blacklisting
Number%Number%Number%
K562 inputSRR55727739,59,22,0099,06,63,27252,58,7375.488,40,45,27887.628,21,92,35385.69
SRR55727749,50,30,6628,98,16,93052,13,7325.498,33,06,81087.668,14,57,57785.72
K562 ControlSRR55727757,63,94,8704,61,89,8673,02,05,00339.544,13,85,04254.174,04,05,24152.89
SRR55727767,89,62,2874,10,53,9943,79,08,29348.013,62,60,49745.923,53,93,51244.82
K562 PDSRR55727778,74,66,2765,41,13,8373,33,52,43938.134,84,46,02655.394,73,24,07554.11
SRR55727788,24,99,1555,29,29,8492,95,69,30635.844,75,55,69357.644,64,66,91556.32
5FdUR_UNGKOSRR599840612,56,31,38010,83,20,7831,73,10,59713.7810,10,27,42880.429,90,12,73078.81
HEK293 inputSRR59984077,03,49,1016,10,39,63893,09,46313.235,69,70,38480.985,58,07,07379.33
5FdUR_UNGKOSRR599840811,36,54,1346,26,79,2925,09,74,84244.855,53,33,96948.695,41,29,56947.63
HEK293 ControlSRR599840912,91,96,9405,80,03,2227,11,93,71855.104,97,06,84638.474,86,00,41837.62
5FdUR_UNGKOSRR59984108,00,35,7626,79,39,5581,20,96,20415.116,34,53,86679.286,21,84,49777.70
HEK293 PDSRR60266946,62,42,4835,63,03,83799,38,64615.005,26,53,80479.495,15,98,00777.89
5FdUR_UGI input5FdUR1_son12,87,06,89512,86,69,77037,1250.0312,24,76,76695.1611,85,58,59792.12
5FdUR1_son20,19,26,20320,15,60,6653,65,5380.1819,30,86,64395.6218,47,56,29791.50
5FdUR_UGI enriched5FdUR1_IP15,05,96,24215,05,22,52273,7200.0514,45,54,26995.9914,15,82,87494.01
5FdUR2_IP13,86,51,76013,84,10,8332,40,9270.1713,32,00,76196.0712,85,84,89492.74
K562 inputK562_son10,61,37,62210,58,75,4372,62,1850.2510,03,26,10594.529,75,04,87691.87
K562 enrichedK562_IP10,94,90,39310,93,06,8541,83,5390.1710,53,10,29696.1810,21,17,05593.27

dU-seq and U-DNA-Seq were performed in completely independent laboratories, even on different continents; applying different conditions; in case of drug-treated samples different cell lines; and obviously different experimental protocols. Still, the resulting log2 ratio tracks are in surprisingly good correlation, if we use our robust analysis pipeline. This is demonstrated in Appendix 1—figure 1 showing an IGV view, the Pearson correlation analysis, and the histograms of uracil enrichment signal distribution, following the scheme of Figure 3 for better comparison. The clear difference between drug-treated and non-treated samples is also obvious. Furthermore, we have demonstrated that the centromeric peaks published in Shu et al., 2018 localize in blacklisted area (Appendix 1—figure 2). However, the re-analyzed dU-seq data could confirm our interpretation on genomic uracil distribution in both non-treated and drug-treated cells, using the herein developed robust analysis pipeline.

Appendix 1—figure 1
Re-analysis of the published dU-seq data (Shu et al., 2018) reveals that corresponding samples from dU-seq and U-DNA-Seq show similar patterns of uracil distribution.

(A) IGV view of dU-seq data (non-treated K562 cells (K562_Shu, wine track), and 5FdUR treated UNG-knock-out HEK293 cells (5FdUR_UNGKO_Shu, olive track)), compared to our own U-DNA-Seq data (non-treated K562 cells (K562, brown track), and 5FdUR treated UNG inhibited HCT116 cells (5FdUR_UGI, green track)) on chromosome 1. Log2 ratio tracks and the derived regions of uracil enrichment are also indicated. The bottom track shows replication timing data (grey) for HCT116 downloaded from Replication Domain database (Weddington et al., 2008). (B) Pearson correlation among dU-seq and U-DNA-Seq log2 ratio tracks calculated from merged replicates. The drug-treated and non-treated samples are well separated again. Pearson correlation between corresponding dU-seq and U-DNA-Seq samples are unexpectedly high, especially considering the cell line difference in case of drug-treated cells, and the overall low signal intensity in case of non-treated K562. (C) Log2 ratio signal distribution of dU-seq and U-DNA-Seq data. The non-treated K562 samples result in a normal like distribution of uracil enrichment signals, while in case of 5FdUR treated cells, these distributions show asymmetry: either a clear shoulder (asterisk), or a more elongated tail towards increased signals in both U-DNA-Seq and dU-seq data, respectively. Source data are available in Appendix 1—figure 1—source data 1.

Appendix 1—figure 2
Reinterpretation of dU-seq data.

(A) Comparison of the re-analyzed and the published dU-seq data in a representative IGV view of chromosome 8 zoomed to the centromeric region (also Figure 3 in Shu et al., 2018). All centromeric peaks in K562 published for chromosome 8 in Shu et al are found in blacklisted regions (red box). Overall, 75% of their published peaks in K562 are overlapping with blacklisted regions determined by our protocol (Figure 2—figure supplement 2). Accordingly, no peaks were called in the presented region during the re-analysis of the sequencing data (red box). Similarly, in drug-treated samples, published centromeric peaks were not reproducible (red box), while other peaks outside of the centromeres were similar in the published and the re-analyzed data (green box). (B) dU-seq data shows similar correlation to genomic features as compared to the corresponding U-DNA-Seq data. Similarity were measured by bedtools annotate tool and the scores were calculated in the same way as it was in Figure 4C. For each sample, cell type (HCT116 or K562) specific DNase hypersensitive site data were used. For 5FdUR treated HEK293 cells, similarity to DNase hypersensitive site data was not addressed (grey). (C) Correlation between uracil distribution and replication timing were confirmed by dU-seq data as well, although this correlation is weaker than the U-DNA-Seq results (Figure 4D). Replication timing data (bigWig files with 5000 bp binsize) were downloaded from ReplicationDomain database: Int90617792 for HCT116; Int57383924 for HEK293; Int37482971 for K562. Source data are available in Appendix 1—figure 2—source data 1.

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Decision letter

  1. Nayun Kim
    Reviewing Editor; University of Texas Health Science Center at Houston, United States
  2. Jessica K Tyler
    Senior Editor; Weill Cornell Medicine, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Your manuscript describes the application of combination of novel approaches – one molecular and the other microscopic, to map the location of uracil in the human genome in cells treated with the drugs 5-FdUR and RTX. The significant finding showing the correlation between a specific set of epigenetic markers and the distribution of uracil should be of great interest to the field of DNA repair and genome instability. Additionally, these findings provide significant leads for the mechanism of how the nucleotide pool imbalance caused by chemotherapeutics such 5-FdUR or RTX leads to genotoxic effects.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for choosing to send your work, "Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments", for consideration at eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered at this moment for publication in eLife. However, we believe that this is a very interesting study that after further work would be appropriate for eLife. It reveals that uracil distribution in DNA after the treatment with inhibitors of thymidine synthesis pathway is non-random. The pattern of enrichment of uracils at actively transcribed regions is very intriguing and anticipates many interesting questions regarding the mechanism. The correlation between uracil distribution and the histone markers associated with active chromatin is also very interesting, but the epigenetic marks have to be determined again under the conditions of this experimental system. The technique used to confirm such correlation, high-resolution microscopy, is very novel and promising. However, as detailed in the reviews, many of the controls that would add rigor to such techniques and findings are lacking. With the sufficient controls to support the main findings of the paper, this work would be highly significant reporting of novel findings and approaches. Please note that our policy is to aim to publish articles with a single round of revision that would typically be accomplished within two months. In our judgment, this manuscript would need extensive additional work beyond such time limit. So we would invite to resubmit a properly revised version attending the comments of the 3 reviewers accompanying your submission with a rebuttal explaining point-by-point the comments of the reviewers.

Reviewer #1:

In this manuscript, a catalytically inactive form of uracil DNA glycosylase is used to probe the genome-wide distribution patter of uracil in human cells. The expression of mutant UNG construct to detect uracil in DNA has been previously reported by the same group in Rona et al., 2016. In the same previous paper, IF was carried out to show that this construct is capable of detecting uracil-containing DNA in E. coli cells or on plasmids in MEF. This manuscript goes further from the previous report by analyzing the uracil distribution pattern in human cells following treatment with drugs (5FdUR and RTX) that inhibit thymidylate synthase enzyme and thus disrupt the dUTP-dTTP balance. Several different variations of the mutant UNG construct are used in two distinct major approaches in this manuscript. The first is to carry out ChIP-seq with antibody against the mutant UNG protein that will bind to uracil in DNA. The second is to modify the construct for the super-resolution immunofluorescent experiments to visualize the regions of uracil in DNA. The first approach was used to show that U-DNA are enriched at transcribed regions and early replicating regions, among others. The pattern of U-DNA in the genome after treatment with 5FdUR or RTX was also compared to the distribution pattern of multiple transcription factors and histone modification markers. Similar conclusion was made that histone markers unique to actively transcribed regions showed most similar pattern of distribution compared to U-DNA after drug treatment. The latter approach with IF was used to confirm that uracil in genomic DNA co-localize with two histone markers H3K36me3 and H3K27me3. Also, the potential mechanism of such distribution pattern is discussed. These are important findings that can also add to the understanding of the mechanism of the cytotoxicity of TS-inhibiting drugs. Overall, since the very interesting technical approach largely overlaps with the previously published report, the significance of the current manuscript should be in the substantive support for the new and significant finding regarding the distribution pattern of uracil after the drug treatment. There are however a few major flaws that detracts from the solidness of the significance of those findings. Two major weak points that call for further experiments are detailed below.

1) Figure 4. For the GIGGLE analysis, the ChIP-seq data for the histone markers and transcription factors were assembled from previously published data. The data set were matched for the same cell type used by the authors for U-DNA sequencing (HCT116). However, there was no consideration given to the fact that the treatment with 5FdUR or RTX could alter the pattern of transcription factors/histone markers significantly, obscuring the GIGGLE analysis between these data sets. Although generating a complete set of ChIP-seq data for all the different factors/markers for the 5FdUR or RTX-treated HCT116 cells would be beyond the scope of this manuscript and is not expected, a confirmation of the analysis for a number of the top hits would make for a much stronger argument. Generation of ChIP-seq results for H3K36me3 in RTX treated cells would complement the IF co-localization data shown in Figure 7 and more importantly would add to convince that such IF co-localization could reach the resolution to state confidently that U-DNA and the region of H3K36me3-modification overlap.

2) Figure 7: For the co-localization assay, H3K36me3 and H3K27me3 were chosen for further study. The rationale given by the authors is "As shown in Figure 4A, the highest similarity (GIGGLE) score corresponded to H3K36me3 and H3K27me3 for the RTX and 5FdUR treated samples." But according to Figure 4A, the correlation appears to be clearer for the H3K27ac. Why H3K27ac was not chosen instead for further study by IF should be discussed. Most notably, the negative correlation with WT or untreated sample was not very distinctive for the H3K27me3, making the difference in GIGGlE scores between NT_UGI and RTX_UGI very small. This is somewhat conflicting with the significant difference noted in the "Overlap area IF" between NT_UGI and RTX_UGI in Figure 7D and requires explanation. In general, αU-DNA signal for the 5FdUR or RTX-treated samples are much stronger than non-treated samples and whether difference in the signal strength interferes with the calling of the co-localization (overlap) is not fully discussed or controlled for. For convincing argument that super-resolution imaging can be used to analyze the genomic distribution pattern of uracil, similar experiments with at least one negative marker (such as H3K9me3 in Figure 4A) should be carried out.

Reviewer #2:

This manuscript describes a promising new low-resolution method for the visualization and mapping of uracils in DNA. It uses UNG inhibitor UGI in combination with a TYMS inhibitor Raltitrexed (RTX) or 5FdUR to increase uracils in the genome and then uses a mutant UNG to pull-down and sequence the DNA fragments. The authors claim that their data suggest that the increased uracils are predominantly in euchromatin/early replicating regions based on epigenetic markers. While the method has several attractive features including an ability to visualize uracil foci (especially Figure 7), there are several serious shortcomings of the manuscript.

1) The use of HCT116 cell line and UGI are troubling. The lack of mismatch repair (MMR) in HCT116 may affect their results. It is possible that the lack of active MMR during early replication affects the removal of uracils and thymines misincorporated across guanines. While UGI binds UNG and inhibits its action, it is unknown whether it has other physiological effects in the cells. In the very least, the results of this study should be compared with those in which MMR+ derivatives of HCT116 cells are used and UNG KO cells lines are compared with their WT parents.

2) Their uracil mapping software lacks a proper negative control. They calculate log2 ratio and plot this ratio across the genome for treated and untreated cells. The input DNA has not undergone the pull-down and hence the DNA pulled down using anti-FLAG antibodies will always show a different genomic distribution than the input DNA. The U-DNA sequencing results from treated cells should be normalized with respect to U-DNA sequencing reads from the pull-down of untreated samples, not the input DNA.

3) This method is unable to pinpoint uracils in the DNA that is pulled down. This is because, the uracils are in U:A pairs and hence they cannot be distinguished from normal T:A pairs after pull-down and sequencing. This is in contrast with most of the other available methods that can map uracils to a specific base pair. For this reason, this is a low resolution alternative to existing uracil mapping methods.

4) The manuscript lacks a proper positive control. It may be useful for the authors to introduce uracils by other means such introduction of AID gene to introduce uracils in the Ig locus and then demonstrate that their method can detect such localization of uracils. In the absence of a positive control, it is hard to know whether their method works correctly. It is possible- for example- that the treatment of cells with RTX or 5FdUR arrests the cells in early S phase and that is why the uracil incorporation occurs in early replicating regions. In which case, the lack of uracil-enriched fragments from the late replicating heterochromatin regions is a somewhat trivial result.

5) The authors dismiss the dU-seq (Shu et al., 2018) rather lightly. Shu and colleagues verified their findings by enrichment of centromeric regions showed by LC-MS/MS that centromeric uracils increased in UNG-knockout cells. Finally, they reintroduced UNG in the cells and showed that this reversed the uracil localization to centromeres. As noted above, the authors of the current manuscript have not done such careful controls in their experiments.

In summary, this is a moderately useful new technique to map uracils that does not distinguish itself from the other existing techniques except in the area of direct visualization of uracils in cells. If the authors were to use this unique feature of their method to answer an existing question, it would be attractive.

Reviewer #3:

The authors develop a new approach to measure the location and density of incorporated uracils in the human genome. They identify an interesting pattern of uracil incorporation in the human genome under normal and thymidine-depleted (using TS inhibitors) conditions, and associate these patterns with specific chromatin features. They also generalize the tool to visualize uracil incorporation with fluorescence microscopy. Both of these are significant advances of use to the DNA repair community. Moreover, these data may eventually be used to probe molecular and cellular consequences caused by common chemotherapies.

1) This study would be improved by employing computational methods designed to identify patterns across multiple independent genomic signals. The one-by-one correlations the authors present provide some insight into general correlations between uracil density, replication timing, and histone modifications, but this ad hoc approach could miss important correlations across all these signals. The segway software (Hoffman, Noble, et al) was developed specifically for this purpose and could be applied to data they have in hand to understand broader genome-wide correlations.

2) More characterization of any DNA damage response in these cells would be helpful to understand whether human uracil incorporation patterns are a "neutral" pattern in the absence of DDR (e.g., similar to high levels of seemingly innocuous uracil incorporation found in dut ung E. coli and yeast) , or represent a stress-induced condition caused by uracil incorporation followed by some checkpoint activation. Some flow cytometry would be useful to see the effect of RTX on cycling of these cells, and H2AX phosphorylation could be measured in bulk by western and in the microscopy experiments upon treatment.

3) Given previous indications that replication timing is somehow linked to uracil incorporation, more discussion is warranted to reconcile similarities and differences in this relationship in E. coli, yeast, and human uracil incorporation patterns relative to replication timing and associated changes in nucleotide pools.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

In this paper, the authors probe the genome-wide distribution patter of uracil in human cells using two innovative approaches. First, a catalytically inactive form of uracil DNA glycosylase is expressed and subjected to ChIP-seq to generate the genomic map of uracil distribution. The mutant UDG is also used in a super-resolution immuno-fluorescence experiments for higher throughput confirmation of uracil distribution pattern. These two complimentary approaches represent significant and useful advances in understanding the genome instability induced by uracil in DNA. Here, the authors' specific interest is the uracil distribution pattern in human cells following treatment with drugs (5FdUR and RTX) that inhibit thymidylate synthase enzyme and thus disrupt the dUTP-dTTP balance. The major findings are that U-DNA are mostly enriched at transcribed regions and early replicating regions. The correlation between the U-DNA in 5FdUR- or RTX-treated cells with epigenetic markers unique to actively transcribed regions, first determined using the ChIP-seq data, was further confirmed through the super resolution IF experiments. Such correlation is significant because it can inform the potential mechanism of uracil incorporation into the genome in thymine-depleted cells and thereby add to the understanding of the mechanism of the cytotoxicity of TS-inhibiting drugs.

Revisions:

The following minor revisions are strongly recommended for acceptance to publish in eLife.

1) The criticism of other methods are given the disproportionate significance in the discussion. Although the effort to highlight the advance and innovation of the technique used in the current manuscript over other previously published methods is completely valid and helpful, overly long and highly critical discussion of other methods diminishes the meaningful discussion of the findings in this paper and becomes unnecessary distraction. There is also a risk of misstating what the other methods do or point out deficiencies that may have simple explanations. A separate review paper comparing various methods would better suit such discussion. I recommend that the authors reorganize the Discussion section so that it starts by describing what this manuscript has accomplished and briefly summarize the pros and cons of all the methods towards the end.

2) Clarification is required regarding the replication scores derived from Weddington et al. Is the distribution of scores correlated with timing (negative = early, positive = late?). It's unfortunate that ENCODE Repli-seq data is not available for the HCT116 cell line; this would have been a better comparison. Please add discussion or speculate on the possible mechanism regarding why uracil enrichment upon treatment both appear at similar replication timing, relative to the control samples (Figure 4—figure supplement 3)?

https://doi.org/10.7554/eLife.60498.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

It reveals that uracil distribution in DNA after the treatment with inhibitors of thymidine synthesis pathway is non-random. The pattern of enrichment of uracils at actively transcribed regions is very intriguing and anticipates many interesting questions regarding the mechanism. The correlation between uracil distribution and the histone markers associated with active chromatin is also very interesting, but the epigenetic marks have to be determined again under the conditions of this experimental system.

We performed a pair of ChIP-seq experiments for H3K36me3 epigenetic mark (as reviewer #1 suggested) in non-treated and RTX-treated UGI-expressing HCT116 cells. We found that there are no major differences in the genome-wide distribution of this factor, or in its correlation with the U-DNA-Seq patterns upon the applied treatment conditions. Results are presented in Figure 4—figure supplement 1, and Figure 4A-B. Corresponding texts are inserted to the respective figure legends (4A: “GIGGLE scores are also indicated for our own H3K36me3 ChIP-seq…”, 4B: “…our own ChIP-seq data for H3K36me3…”); to the Results (a paragraph started with “In order to decide whether drug treatments may cause any notable changes…”); to the Materials and methods (a paragraph with the title: “Chromatin immunoprecipitation and sequencing (ChIP-seq)”), to the Discussion (“With the ChIP-seq experiment for H3K36me3…”); and to the Supplementary file 3 (a section with the title: “ChIP-seq for H3K36me3 histone marker in non-treated and RTX treated UGI-expressing HCT116 cells.” including Supplementary file 3—table 2). ChIP-seq peak results are also uploaded to the GEO GSE126822.

The technique used to confirm such correlation, high-resolution microscopy, is very novel and promising. However, as detailed in the reviews, many of the controls that would add rigor to such techniques and findings are lacking. With the sufficient controls to support the main findings of the paper, this work would be highly significant reporting of novel findings and approaches.

In the revised manuscript, we provide a positive control experiment for the U-DNA-Seq, namely, a uracil-containing spike-in sequence was added to genomic DNA fragments, then the U-DNA-IP was performed and the enrichment of the spike-in was followed by qPCR. Corresponding sentences are inserted to the Results (“To further confirm the capability of U-DNA-IP, uracil-containing spike-in DNA was combined with…”), and to the Materials and methods (a paragraph with the title: “Controls of U-DNA-IP method”).

In the submitted manuscript, we have already mentioned a negative control experiment for the U-DNA-Seq: “Specificity of U-DNA immunoprecipitation is also underlined by the fact that pull-down with empty anti-FLAG beads not containing the U-DNA sensor resulted in negligible amount of DNA (less than 5%).” During the revision, we have obtained the full genome sequencing data for this negative (empty bead) control, and we provide the corresponding data in Figure 1—figure supplement 2, and Supplementary file 1—table 1. The main text was also modified accordingly, see text segment: “Still, genome-wide sequencing data could be obtained from these negative control samples as well. We demonstrated that subtracting such control signals (for details see Supplementary file 1) will not affect the detected uracil distribution pattern regardless if the sample was drug-treated or not (Figure 1—figure supplement 2B-C). These control experiments provided confidence about the applicability and specificity of our U-DNA-IP method.”. Corresponding description was added to the Materials and methods (a paragraph with the title: “Controls of U-DNA-IP method” and “High-throughput DNA sequencing and data analysis”).

Reviewer #1:

In this manuscript, a catalytically inactive form of uracil DNA glycosylase is used to probe the genome-wide distribution patter of uracil in human cells. The expression of mutant UNG construct to detect uracil in DNA has been previously reported by the same group in Rona et al., 2016. In the same previous paper, IF was carried out to show that this construct is capable of detecting uracil-containing DNA in E. coli cells or on plasmids in MEF. This manuscript goes further from the previous report by analyzing the uracil distribution pattern in human cells following treatment with drugs (5FdUR and RTX) that inhibit thymidylate synthase enzyme and thus disrupt the dUTP-dTTP balance. Several different variations of the mutant UNG construct are used in two distinct major approaches in this manuscript. The first is to carry out ChIP-seq with antibody against the mutant UNG protein that will bind to uracil in DNA. The second is to modify the construct for the super-resolution immunofluorescent experiments to visualize the regions of uracil in DNA. The first approach was used to show that U-DNA are enriched at transcribed regions and early replicating regions, among others. The pattern of U-DNA in the genome after treatment with 5FdUR or RTX was also compared to the distribution pattern of multiple transcription factors and histone modification markers. Similar conclusion was made that histone markers unique to actively transcribed regions showed most similar pattern of distribution compared to U-DNA after drug treatment. The latter approach with IF was used to confirm that uracil in genomic DNA co-localize with two histone markers H3K36me3 and H3K27me3. Also, the potential mechanism of such distribution pattern is discussed. These are important findings that can also add to the understanding of the mechanism of the cytotoxicity of TS-inhibiting drugs. Overall, since the very interesting technical approach largely overlaps with the previously published report, the significance of the current manuscript should be in the substantive support for the new and significant finding regarding the distribution pattern of uracil after the drug treatment. There are however a few major flaws that detracts from the solidness of the significance of those findings. Two major weak points that call for further experiments are detailed below.

1) Figure 4. For the GIGGLE analysis, the ChIP-seq data for the histone markers and transcription factors were assembled from previously published data. The data set were matched for the same cell type used by the authors for U-DNA sequencing (HCT116). However, there was no consideration given to the fact that the treatment with 5FdUR or RTX could alter the pattern of transcription factors/histone markers significantly, obscuring the GIGGLE analysis between these data sets. Although generating a complete set of ChIP-seq data for all the different factors/markers for the 5FdUR or RTX-treated HCT116 cells would be beyond the scope of this manuscript and is not expected, a confirmation of the analysis for a number of the top hits would make for a much stronger argument. Generation of ChIP-seq results for H3K36me3 in RTX treated cells would complement the IF co-localization data shown in Figure 7 and more importantly would add to convince that such IF co-localization could reach the resolution to state confidently that U-DNA and the region of H3K36me3-modification overlap.

We agree to the possibility that treatment with 5FdUR or RTX could alter the pattern of transcription factors/histone markers. In our manuscript, we use the comparison between the uracil-DNA distribution pattern in normal and drug-treated cells and the pattern of the epigenetic markers reported in the databases for the same cell line (HCT116) under normal conditions as a potential indication that need to be investigated further by independent experiments. We use super-resolution microscopy (dSTORM) as a relevant and highly potent further independent technique. We found that dSTORM qualitatively confirmed the positive correlation between the epigenetic markers and the U-DNA patterns (reported in the GIGGLE scores). We also agree that determination of the epigenetic marker distribution under drug treatment would be very interesting and informative and this is one of the directions we plan to continue our further research.

We agree with the reviewer’s comment that “generating a complete set of ChIP-seq data for all the different factors/markers for the 5FdUR or RTX-treated HCT116 cells would be beyond the scope of this manuscript”. Instead, the reviewer suggested to generate ChIP-seq data for H3K36me3 in RTX treated cells, as an example to “complement the IF co-localization data”. We agree to this suggestion and in our revision, we have performed the required ChIP-seq experiments for non-treated UGI-expressing and RTX-treated UGI-expressing cells. We found that there are no major differences in the genome-wide distribution of this factor, or in its correlation with the U-DNA-Seq patterns upon the applied treatment conditions. Results are presented in Figure 4—figure supplement 1, and Figure 4A-B. Corresponding texts are inserted to the respective figure legends (4A: “GIGGLE scores are also indicated for our own H3K36me3 ChIP-seq…”, 4B: “…our own ChIP-seq data for H3K36me3…”); to the Results (a paragraph started with “In order to decide whether drug treatments may cause any notable changes…”); to the Materials and methods (a paragraph with the title: “Chromatin immunoprecipitation and sequencing (ChIP-seq)”), to the Discussion (“With the ChIP-seq experiment for H3K36me3…”); and to the Supplementary file 3 (a section with the title: “ChIP-seq for H3K36me3 histone marker in non-treated and RTX treated UGI-expressing HCT116 cells.” including Supplementary file 3—table 2). ChIP-seq peak results are also uploaded to the GEO GSE126822.

2) Figure 7: For the co-localization assay, H3K36me3 and H3K27me3 were chosen for further study. The rationale given by the authors is "As shown in Figure 4A, the highest similarity (GIGGLE) score corresponded to H3K36me3 and H3K27me3 for the RTX and 5FdUR treated samples." But according to Figure 4A, the correlation appears to be clearer for the H3K27ac. Why H3K27ac was not chosen instead for further study by IF should be discussed. Most notably, the negative correlation with WT or untreated sample was not very distinctive for the H3K27me3, making the difference in GIGGlE scores between NT_UGI and RTX_UGI very small. This is somewhat conflicting with the significant difference noted in the "Overlap area IF" between NT_UGI and RTX_UGI in Figure 7D and requires explanation. In general, αU-DNA signal for the 5FdUR or RTX-treated samples are much stronger than non-treated samples and whether difference in the signal strength interferes with the calling of the co-localization (overlap) is not fully discussed or controlled for. For convincing argument that super-resolution imaging can be used to analyze the genomic distribution pattern of uracil, similar experiments with at least one negative marker (such as H3K9me3 in Figure 4A) should be carried out.

The main reason for our selections of the H3K36me3 and the H3K27me3 markers for IF colocalization measurements was that these factors produced the highest scores for RTX and 5FdUR treatments, respectively, as reviewer #1 also wrote. We agree that we could have selected other factors like H3K27ac, H3K9ac, H3K4me3, or even the transcription factor SP1, where both treatments resulted in high GIGGLE scores and these are well separated from the non-treated cases. However, with the choice of H3K36me3 and the H3K27me3, we sought to cover better the spectra from the actively transcribing euchromatin to the facultative heterochromatin. Furthermore, with this choice, we could also address putative differences between the two drug treatments. A new genome segmentation analysis performed in our revision upon the request of the reviewer #3, also supports our choice (cf. Figure 4B in the revised manuscript, and inserted text to the Results (“Furthermore, Segway analysis strengthened…”)).

Regarding the reviewer’s comments that “the negative correlation with WT or untreated sample was not very distinctive for the H3K27me3…”, and that “this is somewhat conflicting with the significant difference noted in the "Overlap area IF" between NT_UGI and RTX_UGI in Figure 7D…”, we provide the following explanation. On the one hand, as the reviewer also pointed out (point #1), the two experimental setup has a clear difference, namely in GIGGLE analysis we compare our uracil patterns in non-treated and drug-treated cells with non-treated ChIP-seq data from databases, while in dSTORM microscopy the overlap of the two features can be calculated in the same treated cells. On the other hand, the available ChIP-seq data are from different experiments and experimental backgrounds, hence show quite big deviation among themselves. Therefore, such in silico correlation analysis can provide potential indications, but not strict rules for the choice of markers to be addressed in colocalization studies by dSTORM microscopy. Still, the in silico analysis gave meaningful prediction, as co-staining of the selected histone markers and the genomic uracil in drug-treated cells reinforced the association between uracil occurrence and transcriptionally active regions. Moreover, with the H3K36me3 ChIP-seq (cf. point #1), we also demonstrated that the treatment induced chromatin remodeling is not a general phenomenon, but may rather confines to certain histone marks. The apparent conflict between our results from these different techniques is further explained now in the Discussion of the revised manuscript (“Strikingly, we found that H3K27me3 shows even stronger colocalization with the U-DNA pattern in case of the RTX treated sample…”).

Regarding the possible influence of the overall signal strength on the calculation of IF (overlap), we have inserted a detailed explanation to the Results: “The cross-pair correlation method probes the probability distributions across all possible pair-wise distances between two species, taking in account the number of foci for each species (PMIDs: 25179006, 23717596, 22384026 and 27545293). This normalization of the number of foci ensures that any increase in IF is specifically due to an increase in their co-localization probability density, and not due to the increase in the amount of either species.”

Regarding the suggestion to stain for another marker (such as H3K9me3) that is supposed to negatively correlate with genomic uracils based on the GIGGLE scores and also on the genome segmentation results (cf. Figure 4A-B), we agree that it would be interesting to perform such an experiment. However, both Figure 4 and Figure 4—figure supplement 3 show that the distribution pattern of the genomic uracils is not fully represented by any of the histone markers. The best correlating features are the early replication timing and AT rich heterochromatin in case of the drug-treated and non-treated samples, respectively. As a negative control for the drug-treated samples, it would be required therefore to identify a true heterochromatin (or late replication) marker that might be challenging. We also wish to point out that due to the current pandemic situation of COVID-19, the laboratory at the New York University (School of Medicine) – where these studies are performed – is hardly available. We cannot define any clear schedule for performing additional dSTORM experiments, and this situation would lead to an unpredictable and disproportionate delay in the publication process.

Reviewer #2:

This manuscript describes a promising new low-resolution method for the visualization and mapping of uracils in DNA. It uses UNG inhibitor UGI in combination with a TYMS inhibitor Raltitrexed (RTX) or 5FdUR to increase uracils in the genome and then uses a mutant UNG to pull-down and sequence the DNA fragments. The authors claim that their data suggest that the increased uracils are predominantly in euchromatin/early replicating regions based on epigenetic markers. While the method has several attractive features including an ability to visualize uracil foci (especially Figure 7), there are several serious shortcomings of the manuscript.

1) The use of HCT116 cell line and UGI are troubling. The lack of mismatch repair (MMR) in HCT116 may affect their results. It is possible that the lack of active MMR during early replication affects the removal of uracils and thymines misincorporated across guanines. While UGI binds UNG and inhibits its action, it is unknown whether it has other physiological effects in the cells. In the very least, the results of this study should be compared with those in which MMR+ derivatives of HCT116 cells are used and UNG KO cells lines are compared with their WT parents.

The HCT116 cell line has been widely used as a model cell line in colon cancer research over the course of almost four decades (PMID: 7214343, 6956756, 22955616), for which numerous published results – including epigenomic data – are available e.g. in the ENCODE database. Colon cancer is frequently associated with deficiency in mismatch repair (MMR) (PMID: 30959407, 30442708, 28548127), and thymidylate synthase inhibitors represent a possible option as first-line treatment in the clinical practice (PMID: 24732946). Based on these data, we propose that the choice of HCT116 cell line was relevant in our study for revealing how TS inhibitors may affect uracil distribution patterns. We agree that the potential contribution of the mismatch repair to the mechanism of the effect of these drugs is a highly intriguing question that might have impact also on clinical prognosis and/or the choice of treatments. Initially, we felt that such an analysis is beyond the focus of this manuscript, but prompted by the request of the reviewer, we decided to include comparative studies on the MMR proficient version of HCT116 cells (created by the reintroduction of chromosome 3 (PMID: 8044777)), as well. For this, 12 new U-DNA-Seq experiments were performed on MMR proficient samples (shown in modified Figure 2—figure supplement 3, Figure 3, Figure 3—figure supplement 3, and the new Figure 3—figure supplement 4), we have carried out the corresponding correlation analysis (shown in modified Figure 4, Figure 4—figure supplement 2, and the new Figure 4—figure supplement 3), as well as measurement of uracil-content by dot blot (shown in new Figure 1—figure supplement 1 panel D) were also performed. Further experiments suggested by reviewer #3, namely cell cycle analysis (shown in new Figure 5) and γH2AX staining in flow cytometry (shown in new Figure 5—figure supplement 1) were also done on both the MMR deficient and MMR proficient samples. We found that MMR status does not have any observable impact on the U-DNA pattern of the non-treated samples, while in case of drug-treated ones, some drug-dependent differences were detected. We have described and analyzed these new data at different sections of the Results, as well as in the figure legends (all changes are tracked in the revised manuscript). To discuss these results, the following texts are inserted to the Discussion of the revised manuscript: “We chose the HCT116 cancer cell line that is deficient in mismatch repair…”; “It has to be noted that MMR proficiency leads to a major decrease in the correlation with early replication timing…”; “This is further supported by the fact that in MMR proficient drug-treated samples higher U-DNA content…”; “Moreover, the MMR status has markedly different influence on the resulting U-DNA pattern in case of the two drugs (cf. Figure 3-4).”; “Similarly, we also detected slightly altered cell cycle distribution patterns in case of the two drug treatments, which were differently influenced by the MMR status…”.

Regarding the use of UGI, we have to point out that UGI is a well characterized specific, small (10 kDa) proteinaceous UNG-inhibitor (PMID: 7671300). UGI expression in HEK293 cells led elevated genomic uracil content, while did not affect the cytotoxicity, the cell cycle arrest, the γH2AX signaling upon RTX or 5FdUR treatments (PMID: 17942376). We refer to these data in the Introduction of the revised manuscript (“It has already been shown that UGI expression does not affect…”). In agreement with the literature, we also detected (i) elevated U-DNA content upon drug treatments (cf. Figure 1—figure supplement 1B-D), (ii) no major physiological effect on the non-treated cells in respect to their cell cycling (cf. Figure 5A performed upon the request of the reviewer #3), and (iii) no difference in the genomic uracil distribution as compared to the non-treated wild type cells (cf. Figure 3, Figure 3—figure supplement 3, Figure 3—figure supplement 4, Figure 4, Figure 4—figure supplement 2, Figure 4—figure supplement 3). Based on these arguments, we feel that repeating all of the experiments (overall 28 sequencing, the corresponding dot blots, flow cytometry, super-resolution imaging) on UNG-KO cells would not add fundamental new insights to this present study, but may form the basis for further research.

2) Their uracil mapping software lacks a proper negative control. They calculate log2 ratio and plot this ratio across the genome for treated and untreated cells. The input DNA has not undergone the pull-down and hence the DNA pulled down using anti-FLAG antibodies will always show a different genomic distribution than the input DNA. The U-DNA sequencing results from treated cells should be normalized with respect to U-DNA sequencing reads from the pull-down of untreated samples, not the input DNA.

The reviewer suggested to use the non-treated IP samples as controls for the treated ones. In a way, we agree, and we basically did this, when we compared the log2 curves (cf. Figure 3, Figure 3—figure supplement 3), assessed their colocalization with other factors or genetic features (Figure 4, Figure 4—figure supplement 2, Figure 4—figure supplement 3), and then compared the results of the drug-treated and non-treated samples in many respects. However, for the calculation of log2 ratio and for the peak calling, we decided to use the corresponding sonicated input samples as controls. We claim that this choice was reasonable because of the following arguments.

First, sonicated genomic DNA samples provide a more balanced coverage of the reference genome, but it is still not without some fluctuation. Such fluctuation might be slightly different in case of non-treated and drug-treated cells, and therefore, it is the best to compare each IP sample (enriched in U-DNA) to their corresponding input (sonicated) ones. As expected, we did not experience highly enriched sharp peaks, therefore, such fluctuations might have a distorting impact and need to be avoided.

Second, using input as a background control for peak calling or log2 ratio enrichment calculation meets the current ENCODE standards for ChIP-seq. Currently, corresponding input is required, while previous standard suggested either input or Ig control experiment (https://www.encodeproject.org/chipseq/histone/#restrictions). Accordingly, we have inserted the following statement to the Supplementary file 1: “…as it is also recommended by the current ENCODE standard…”.

Third, given that the U-DNA patterns of the drug-treated and non-treated samples are almost complementary to each other (cf. Figure 3A and Supplementary file 2), applying non-treated samples as controls would result in a signal increase artifact rather than better description of the true enrichment.

Fourth, using the non-treated IP sample as control in the peak calling or the log2 enrichment analysis would evoke technical problems about the scaling and the handling of non-covered regions. (1) If a region is not covered by reads in case of the non-treated IP sample, then it is hard to calculate the log2 ratio. The two usual solutions for such problem may be skipping databins of zero that would result in loosing data or introducing a pseudocount that might influence the results especially at the regions of lower enrichment. (2) It is not trivial how to compare a relatively big amount of DNA immunoprecipitated from the drug-treated samples to a much smaller amount of DNA originating from the non-treated cells (5:1, cf. Figure 1—figure supplement 2A), given that the deepness of the sequencing is approximately the same for both samples.

In addition to the discussion above, we also performed blank IP experiments (using empty beads without ∆UNG sensor) and sequencing to make sure that the U-DNA patterns are valid, and not due to a specific pull-down, even in case of the non-treated samples. Data are presented in Figure 1—figure supplement 2, and Supplementary file 1—table 1. The main text was also modified accordingly, see text segment: “Still, genome-wide sequencing data could be obtained from these negative control samples as well. We demonstrated that subtracting such control signals (for details see Supplementary file 1) will not affect the detected uracil distribution pattern regardless if the sample was drug-treated or not (Figure 1—figure supplement 2B-C). These control experiments provided confidence about the applicability and specificity of our U-DNA-IP method.”. Corresponding description was added to the Materials and methods (a paragraph with the title: “Controls of U-DNA-IP method” and “High-throughput DNA sequencing and data analysis”).

3) This method is unable to pinpoint uracils in the DNA that is pulled down. This is because, the uracils are in U:A pairs and hence they cannot be distinguished from normal T:A pairs after pull-down and sequencing. This is in contrast with most of the other available methods that can map uracils to a specific base pair. For this reason, this is a low resolution alternative to existing uracil mapping methods.

We agree that our method is a low-resolution technique, but addressing the case at issue, it is relevant, adequate and robust. In the given model system, under thymidylate synthesis inhibition potentially leading to a perturbation of dUTP/dTTP ratios, uracil incorporation into the genome during either replicative or repair DNA synthesis is a basically stochastic process. Under such circumstances, characteristic U-DNA patterns might be associated with uneven genome-wide distribution of the DNA synthesis foci (e.g. at replication forks or different repair foci) and/or actual repair activities at certain regions. We claim that our approach can reliably describe these patterns. We also feel that to pinpoint the individual uracil residues would not provide additional value to this description, but could be rather misleading, if we consider that from about 10 millions of cells (=20 millions of genomes), we read on average one per million only. This issue is covered in the Discussion of the revised manuscript (see text starting with: However, this aspect has lower impact, if we consider the basically stochastic nature of uracil…”).

We have reviewed the publications of other methods for determination of uracils in DNA and found that only the pre-digestion Excision-seq was explicitly stated to be capable to detect uracils with singlebase resolution. Excision-seq was used in the literature in samples with higher uracil content and smaller genome-size (PMID: 25015380). Although both UPD-seq (PMID: 31431505) and dU-seq (PMID: 29785056) use biotin-streptavidin system for the pull-down, and the biotin labeling could potentially allow site-specific detection, single-base resolution data have not been shown in either of these publications. In the revised manuscript, the first paragraph of the Discussion has been rephrased to provide a more elaborate comparison of the available methods.

The U-DNA-Seq method we present includes a refined analysis pipeline to ascertain its status as a useful alternative of the existing U-DNA sequencing methods. The use of the catalytically inactive UNG-based sensor renders it possible to complement the genome-wide distribution patterns with cellular localization studies. This advantage is also better underlined in the revised version, following the suggestion of the reviewer #3.

4) The manuscript lacks a proper positive control. It may be useful for the authors to introduce uracils by other means such introduction of AID gene to introduce uracils in the Ig locus and then demonstrate that their method can detect such localization of uracils. In the absence of a positive control, it is hard to know whether their method works correctly. It is possible- for example- that the treatment of cells with RTX or 5FdUR arrests the cells in early S phase and that is why the uracil incorporation occurs in early replicating regions. In which case, the lack of uracil-enriched fragments from the late replicating heterochromatin regions is a somewhat trivial result.

We agree that a relevant positive control is valuable. Reviewer #2 suggested expression of AID to introduce uracils specifically into the Ig locus in order to detect such localization of uracils by our method. However, overexpression of AID without its proper regulation and targeting is expected to lead to deamination events outside the Ig loci also, as it was reported in case of tumorigenesis (PMID: 26845615). We therefore designed another positive control experiment. Namely, a uracil-containing spike-in sequence was added to genomic DNA fragments, then the U-DNA-IP was performed and the enrichment of the spike-in was followed by qPCR, as it has been already mentioned above in the reply to the Editor’s main point #2. Corresponding sentences are inserted to the Results (“To further confirm the capability of U-DNA-IP, uracil-containing spike-in DNA was combined with non-treated genomic DNA samples (cf. Materials and methods). In these samples U-DNA-IP led to 4.5 fold enrichment of the uracil-containing spike-in DNA compared to the uracil-free spike-in as determined by qPCR.”), and to the Materials and methods (a paragraph with the Title: “Controls of U-DNA-IP method”).

We note that the robustness and the reproducibility over biological replicates reported in our study also support the applicability of our method. Moreover, comparison of U-DNA-Seq results to the independent dU-seq data also confirmed the reliability of our method (cf. Discussion of the submitted manuscript (Re-analysis of the earlier published dU-seq data…) and Appendix 1).

The reviewer also mentioned the possibility “that the treatment of cells with RTX or 5FdUR arrests the cells in early S phase and that is why the uracil incorporation occurs in early replicating regions. In which case, the lack of uracil-enriched fragments from the late replicating heterochromatin regions is a somewhat trivial result.”. We agree that the S-phase arrest will influence the pattern of uracil incorporation, and now in the revised manuscript, it is presented and discussed more extensively. Clear correlation has already been shown between the U-DNA pattern and early replicating segments in the submitted manuscript (cf. Figure 4C-D). Further genome segmentation analysis, presented in the revised manuscript, also confirmed this (Figure 4—figure supplement 3). In addition, upon the request of the reviewer #3, cell cycle analysis was also performed (Figure 5) supporting the presence of a cell cycle arrest upon the applied drug treatment. The connection of the cell cycle arrest and the correlation between U-DNA pattern and early replicating segments is now presented in the revised manuscript (cf. Figure 4C-D and Figure 5). We propose that these results provide additional insights. U-DNA-Seq might give a molecular approach to the replication arrest, while flow cytometry provides a phenotypic description of cell cycle phases. Interestingly, drug dependent and also MMR status dependent differences were detected by both approaches, which are discussed in the Discussion of the revised manuscript (“Similarly, we also detected slightly altered cell cycle distribution patterns…”). It also has to be noted that correlation with the replication timing is not exclusive, and DNA synthesis coupled to either transcriptional activity or epigenetic remodeling might also have an impact on the resulting genomic U-DNA pattern in the drug-treated samples.

5) The authors dismiss the dU-seq (Shu et al., 2018) rather lightly. Shu and colleagues verified their findings by enrichment of centromeric regions showed by LC-MS/MS that centromeric uracils increased in UNG-knockout cells. Finally, they reintroduced UNG in the cells and showed that this reversed the uracil localization to centromeres. As noted above, the authors of the current manuscript have not done such careful controls in their experiments.

It is our intention to provide a useful comparison among the relevant methods. Towards this end, we present a well detailed assessment of the dU-seq method in the Discussion section, and also in Appendix 1. We do not question the validity and reliability of the LC-MS/MS data of the Shu et al. publication and to emphasize this, we added the following sentence into the Discussion section: It has to be noted that the authors also applied additional experimental approaches, like 3D-PCR for sensitive detection of U:G pairs, mass spectrometry on genomic DNA fraction enriched in centromeric DNA regions.”

However, there are serious concerns about the capability of dU-seq method to reveal centromeric localization (cf. Appendix 1). These are the following: (1) Short-reads were mapped to the highly repetitive centromeres using a routine mapping software without any extra care or quality control. (2) The data analysis pipeline is not published in sufficient clear details, major points are missing, e.g. whether only the uniquely mapped reads were used or not, whether blacklist and deduplication were applied or not, or which reference genome set was used. (3) Their peak calling was also not reported correctly, it is not fully described if they used controls, and which parameters they set in the MACS2. (4) Their probable approach was to subtract peaks of the control experiment from the peaks of pulled down samples, which is also questionable (cf. above). (5) Their results are shared on the GEO only partially, making a critical evaluation quite problematic.

Finally, we agree with the reviewer that our method has the novel potential for direct visualization of uracils in human genomic DNA. Here, we demonstrated that our technique allows (1) detection of genomic uracil in eukaryotic cells, (2) co-staining with other factors, and (3) measurement of co-localization for the predicted correlating factors from the U-DNA-Seq results. These are important steps towards its application in more sophisticated experimental setup also combined with time-resolved U-DNA-Seq and RNA-seq that might lead to deeper understanding of the molecular mechanisms, and especially the impact of uracil incorporation upon treatments with TS inhibitors. Our present results contribute novel information relevant to existing questions such as: (1) What kind of genomic uracil patterns can arise upon two widely used thymidylate synthase inhibitor drugs in a valid cancer cell line model? (2) Is there any correlation between the U-DNA patterns characteristic for these drug treatments and epigenetic factors or other genomic features? (3) Is there any connection between the known cell cycle arrest and the U-DNA pattern? (4) Is there any difference between the U-DNA patterns induced by treatments with the two drugs? (5) Is there any impact of the MMR status on the U-DNA pattern and does it correlate with mechanistic differences? Further studies based on our present results will reveal more mechanistic details.

Reviewer #3:

The authors develop a new approach to measure the location and density of incorporated uracils in the human genome. They identify an interesting pattern of uracil incorporation in the human genome under normal and thymidine-depleted (using TS inhibitors) conditions, and associate these patterns with specific chromatin features. They also generalize the tool to visualize uracil incorporation with fluorescence microscopy. Both of these are significant advances of use to the DNA repair community. Moreover, these data may eventually be used to probe molecular and cellular consequences caused by common chemotherapies.

1) This study would be improved by employing computational methods designed to identify patterns across multiple independent genomic signals. The one-by-one correlations the authors present provide some insight into general correlations between uracil density, replication timing, and histone modifications, but this ad hoc approach could miss important correlations across all these signals. The segway software (Hoffman, Noble, et al) was developed specifically for this purpose and could be applied to data they have in hand to understand broader genome-wide correlations.

We highly appreciate this suggestion and fully agree. In the revised version, we have performed the suggested Segway analysis on the U-DNA-Seq data also including those obtained from MMR proficient cells (latter experiments were performed according to the request of reviewer #2). We have used ChIP-seq data on HCT116 cells available in the ENCODE database, and also used data from our own H3K36me3 ChIP-seq experiment (performed according to the request of reviewer #1). The results shed light on the relations of the different correlating factors and features, suggesting that none of the histone markers alone can explain the detected U-DNA patterns, and that the correlation with the replication timing or the chromatin structure should be taken into account with higher impact. The resulted signal distribution is presented in Figure 4B and further calculations regarding the replication timing scores and AT content of these genomic segments are shown in Figure 4—figure supplement 3. Corresponding texts are inserted into the respective figure legends; the Materials and methods (“Genome segmentation analysis on our U-DNA-Seq data,…", and “Replication timing scores and AT content were calculated on the genomic segments…”); the Results (a complete section starting with “To understand broader genome-wide correlations, a genome segmentation approach was employed…“, and two sentences “The replication timing correlation and the AT content were also calculated…”, and “Furthermore, Segway analysis strengthened…”); and the Discussion (“Such combinatorial behavior was further demonstrated by the genome segmentation analysis…"). The applied commands/scripts are reported in the Supplementary file 3 with the subtitle “Genome segmentation analysis of U-DNA-Seq results and ChIP-seq data from the ENCODE using Segway genome segmentation algorithm.”. The title of Supplementary file 3 was changed accordingly. Similarly, Supplementary file 4 was completed with the calculation of replication timing and AT content.

2) More characterization of any DNA damage response in these cells would be helpful to understand whether human uracil incorporation patterns are a "neutral" pattern in the absence of DDR (e.g., similar to high levels of seemingly innocuous uracil incorporation found in dut ung E. coli and yeast) , or represent a stress-induced condition caused by uracil incorporation followed by some checkpoint activation. Some flow cytometry would be useful to see the effect of RTX on cycling of these cells, and H2AX phosphorylation could be measured in bulk by western and in the microscopy experiments upon treatment.

We agree that more characterization on the phenotype caused by the drug treatments could improve our understanding of the U-DNA pattern. Hence, based on the suggestion of the reviewer, we performed two types of flow cytometry experiments addressing the potential effect of drug treatments on the cell cycle progression as well as on the DNA-damage signaling under the same conditions that were used in the U-DNA-Seq and the colocalization dSTORM ICC experiments.

First, we addressed the cell cycle using propidium iodide and 5′-bromo-2′-deoxyuridine (BrdU) staining in all those HCT116 samples, for which we provided U-DNA-Seq data. Our results can be summarized as follows. (1) In non-treated cells, UGI expression did not cause any visible changes in the cell cycle progression. (2) Both RTX and 5FdUR treatment (in the presence of UGI) caused significant changes, resulting in arrest-like patterns, which are not identical in case of the two drugs, in good agreement with the detected differences of the corresponding U-DNA patterns. (3) MMR status has an influence on these patterns, which is in good agreement with the MMR status related changes in the correlations between U-DNA patterns and replication timing. These results are presented in Figure 5 of the revised manuscript. Corresponding texts are inserted into the figure legend; the Results (a paragraph that is started with As the uracil distribution pattern in drug-treated cells shows correlation…”); the Discussion (the following sentences: “We demonstrated that UGI expression did not cause any observable…”, “… or the less tight control on cell cycle arrest (cf. Figure 5) allowing more extended replicative synthesis.”, “Similarly, we also detected slightly altered cell cycle distribution patterns in case of the two drug treatments…”), and the Materials and methods (a section with the title “Cell cycle analysis and γH2AX staining”).

Second, regarding the potentially induced DNA damage response (DDR), we also performed flow cytometry measurements using γH2AX immunostaining. The results revealed an increased γH2AX level indicative for DDR in case of both drug treatments. These results are shown in the Figure 5—figure supplement 1. Corresponding texts are inserted into the figure legend; the Results (“As expected (cf. (Meyers et al., 2001)), 5FdUR and RTX treatment…”); the Discussion (“However, equally induced DNA damage response (reported by γH2AX)…”); and the Materials and methods (“Occurrence of DSBs was investigated by immunofluorescent staining of γH2AX…”).

3) Given previous indications that replication timing is somehow linked to uracil incorporation, more discussion is warranted to reconcile similarities and differences in this relationship in E. coli, yeast, and human uracil incorporation patterns relative to replication timing and associated changes in nucleotide pools.

To discuss the correlation between uracil incorporation and replication timing, we have inserted a new paragraph into the Discussion starting with “Our data showing that under normal conditions…”. Here, we show differences between the timing of genomic uracil incorporation in our drug-treated cells and in other biological systems previously published in the literature, and the possible reasons are also discussed.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Revisions:

The following minor revisions are strongly recommended for acceptance to publish in eLife.

1) The criticism of other methods are given the disproportionate significance in the discussion. Although the effort to highlight the advance and innovation of the technique used in the current manuscript over other previously published methods is completely valid and helpful, overly long and highly critical discussion of other methods diminishes the meaningful discussion of the findings in this paper and becomes unnecessary distraction. There is also a risk of misstating what the other methods do or point out deficiencies that may have simple explanations. A separate review paper comparing various methods would better suit such discussion. I recommend that the authors reorganize the Discussion section so that it starts by describing what this manuscript has accomplished and briefly summarize the pros and cons of all the methods towards the end.

We agree with the Editors’ recommendation, and accordingly we have restructured the Discussion. Comparison of the different methods was significantly shortened and moved from the beginning of the Discussion (see paragraph starting with "As we demonstrated here, the genome-wide uracil distribution patterns have relevance…"). For harmonization, a new first paragraph is inserted in the Discussion starting with "Here we focus on the alteration of U-DNA distribution pattern".

2) Clarification is required regarding the replication scores derived from Weddington et al. Is the distribution of scores correlated with timing (negative = early, positive = late?). It's unfortunate that ENCODE Repli-seq data is not available for the HCT116 cell line; this would have been a better comparison.

Thank you for pointing out the meaning of replication score was not defined. We have now inserted the following sentences with an extra reference into the figure legends of Figure 4—figure supplement 2: "Replication timing scores are derived from E/L Repli-seq experiments, where cycling cells are pulse-labeled with BrdU, then sorted to early and late S-phase fractions by flow cytometry, and BrdU labeled genomic DNA fragments are pulled down and subjected to NGS. Signal tracks are computed from the read coverages in early over late S-phase samples, therefore the higher score means earlier replication (Marchal et al., 2018). The scale goes from -2.5 to 5." Similar description is inserted to the figure legend of Figure 4—figure supplement 3: "Replication timing scores are derived from E/L Repli-seq experiments, the higher score means earlier replication (Marchal et al., 2018)."

Please add discussion or speculate on the possible mechanism regarding why uracil enrichment upon treatment both appear at similar replication timing, relative to the control samples (Figure 4—figure supplement 3)?

This is already discussed in details in the following sections:

"Uracil appearance via thymine replacing misincorporation implies prior DNA synthesis involved in either replication, or transcription-coupled DNA repair, or epigenetic reprogramming (e. g. erasing the methyl-cytosine epigenetic mark). Importantly, we found that uracil pattern showed the highest correlation with the features (early replication, active promoters and DNase hypersensitive sites, and CpG islands) linked exactly to these processes (cf. Figure 4C)."

With even more details starting with: "The antifolate or nucleotide-based thymidylate synthase inhibitors, such as 5-FU, RTX or 5FdUR are known to lead to cell cycle arrest…"

We also inserted a short reference: "also supported by Figure 4—figure supplement 3".

https://doi.org/10.7554/eLife.60498.sa2

Article and author information

Author details

  1. Hajnalka L Pálinkás

    1. Genome Metabolism Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
    2. Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
    3. Doctoral School of Multidisciplinary Medical Science, University of Szeged, Szeged, Hungary
    Contribution
    Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    palinkas.hajnalka@ttk.hu
    Competing interests
    No competing interests declared
  2. Angéla Békési

    1. Genome Metabolism Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
    2. Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2294-3002
  3. Gergely Róna

    1. Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
    2. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States
    3. Perlmutter Cancer Center, New York University School of Medicine, New York, United States
    4. Howard Hughes Medical Institute, New York University School of Medicine, New York, United States
    Contribution
    Conceptualization, Investigation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Lőrinc Pongor

    1. Cancer Biomarker Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
    2. Department of Bioinformatics and 2nd Department of Pediatrics, Semmelweis University, Budapest, Hungary
    Contribution
    Data curation, Software, Formal analysis
    Competing interests
    No competing interests declared
  5. Gábor Papp

    Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
    Contribution
    Data curation, Software, Formal analysis, Visualization
    Competing interests
    No competing interests declared
  6. Gergely Tihanyi

    1. Genome Metabolism Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
    2. Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3729-6709
  7. Eszter Holub

    Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  8. Ádám Póti

    Genome Stability Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
    Contribution
    Formal analysis, Visualization
    Competing interests
    No competing interests declared
  9. Carolina Gemma

    Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom
    Contribution
    Resources
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4890-9972
  10. Simak Ali

    Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom
    Contribution
    Resources
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1320-0816
  11. Michael J Morten

    Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  12. Eli Rothenberg

    Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1382-1380
  13. Michele Pagano

    1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States
    2. Perlmutter Cancer Center, New York University School of Medicine, New York, United States
    3. Howard Hughes Medical Institute, New York University School of Medicine, New York, United States
    Contribution
    Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3210-2442
  14. Dávid Szűts

    Genome Stability Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
    Contribution
    Supervision, Writing - review and editing
    Competing interests
    No competing interests declared
  15. Balázs Győrffy

    1. Cancer Biomarker Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
    2. Department of Bioinformatics and 2nd Department of Pediatrics, Semmelweis University, Budapest, Hungary
    Contribution
    Supervision, Writing - review and editing
    Competing interests
    No competing interests declared
  16. Beáta G Vértessy

    1. Genome Metabolism Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
    2. Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    vertessy@mail.bme.hu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1288-2982

Funding

National Research, Development and Innovation Office of Hungary (K119493)

  • Beáta G Vertessy

National Research, Development and Innovation Office of Hungary (NVKP_16-1-2016-0020)

  • Beáta G Vertessy

National Research, Development and Innovation Office of Hungary (2017-1.3.1-VKE-2017-00002)

  • Beáta G Vértessy

National Research, Development and Innovation Office of Hungary (2017-1.3.1-VKE-2017-00013)

  • Beáta G Vértessy

National Research, Development and Innovation Office of Hungary (VEKOP-2.3.2-16-2017-00013)

  • Beáta G Vértessy

National Research, Development and Innovation Office of Hungary (NKP-2018-1.2.1-NKP-2018-00005)

  • Beáta G Vértessy

National Research, Development and Innovation Office of Hungary (NVKP_16-1-2016-0037)

  • Balázs Győrffy

National Research, Development and Innovation Office of Hungary (2018-1.3.1-VKE-2018-00032)

  • Balázs Győrffy

National Research, Development and Innovation Office of Hungary (KH-129581)

  • Balázs Győrffy

Ministry of Human Capacities (BME FIKP-BIO)

  • Beáta G Vertessy

Cancer Research UK (C37/A18784)

  • Carolina Gemma

National Institutes of Health (R35-GM136250)

  • Michele Pagano

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We gratefully acknowledged the kind help of György Török and László Homolya in acquiring fluorescent images via STED microscopy. We also wish to say sincere thanks to György Várady in FACS sorting and flow cytometry experiments, to Gábor Tusnády for providing access to computational capacity, and to Balázs Ligeti for providing storage and computational capacity by the infrastructure of PPKE ITK. We acknowledge the ENCODE Consortium (ENCODE Project Consortium, 2012) and the ENCODE production laboratory(s) generating the particular dataset(s) as well as the contributors of the UCSC Table Browser (Karolchik et al., 2004; Kuhn et al., 2013) data. We also acknowledge the contributors of Ensembl (Zerbino et al., 2018), ReplicationDomain (Weddington et al., 2008), Cistrome Data Browser (Mei et al., 2017) for making their data publicly available.

Senior Editor

  1. Jessica K Tyler, Weill Cornell Medicine, United States

Reviewing Editor

  1. Nayun Kim, University of Texas Health Science Center at Houston, United States

Publication history

  1. Received: June 29, 2020
  2. Accepted: August 23, 2020
  3. Version of Record published: September 21, 2020 (version 1)
  4. Version of Record updated: September 28, 2020 (version 2)

Copyright

© 2020, Pálinkás et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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