Negative supercoil at gene boundaries modulates gene topology

Abstract

Transcription challenges the integrity of replicating chromosomes by generating topological stress and conflicts with forks1,2. The DNA topoisomerases Top1 and Top2 and the HMGB family protein Hmo1 assist DNA replication and transcription3,4,5,6. Here we describe the topological architecture of genes in Saccharomyces cerevisiae during the G1 and S phases of the cell cycle. We found under-wound DNA at gene boundaries and over-wound DNA within coding regions. This arrangement does not depend on Pol II or S phase. Top2 and Hmo1 preserve negative supercoil at gene boundaries, while Top1 acts at coding regions. Transcription generates RNA–DNA hybrids within coding regions, independently of fork orientation. During S phase, Hmo1 protects under-wound DNA from Top2, while Top2 confines Pol II and Top1 at coding units, counteracting transcription leakage and aberrant hybrids at gene boundaries. Negative supercoil at gene boundaries prevents supercoil diffusion and nucleosome repositioning at coding regions. DNA looping occurs at Top2 clusters. We propose that Hmo1 locks gene boundaries in a cruciform conformation and, with Top2, modulates the architecture of genes that retain the memory of the topological arrangements even when transcription is repressed.

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Fig. 1: Topological context of Pol II genes.
Fig. 2: Top2 and Hmo1 contribute to gene topology.
Fig. 3: Negative supercoil disruption causes disarray in nucleosome occupancy.
Fig. 4: Top2 mediates chromatin loop formation.

Data availability

All raw and processed data are available at the Gene Expression Omnibus (GEO) under the following accession numbers: GSE114410 (bTMP, RNA–DNA hybrids, Top1 protein ChIP-on-chip and RPB3 protein ChIP-on-chip); GSE114444 (RNA-seq, H3 ChIP-seq and ChIA-PET); GSE1625847 (Top2 protein ChIP-chip, Hmo1 protein ChIP-chip and RPB3 protein ChIP-chip).

Code availability

All the custom-made scripts used for this study are available in the GitHub repository at https://github.com/adhilmd/TopologyCustomAnalysis.

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Acknowledgements

We thank J. Roca for sharing TopA-expressing plasmids, and M. Bianchi, G. Liberi and all our laboratory members for discussions. We thank Cogentech and C. Valli, M. Riboni and S. Minardi for microarray and DNA sequencing. Research was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), the European Union, MIUR, Worldwide Cancer Research, and Telethon-Italy to M.F. Y.J.A. is supported by the European Community’s Seventh Framework Programme under grant agreement no. 246549 – Train 2009. N.G. is funded by the UK Medical Research Council (MR/J00913X/1; MC_UU_00007/13).

Author information

Y.J.A. and M.F. designed the experiments, interpreted results and prepared the manuscript. Y.J.A. and M.A. performed the experiments. M.A. performed statistical and computational analysis. R.C. provided technical input and N.G. provided bTMP and technical input for supercoil analysis.

Correspondence to Yathish Jagadheesh Achar or Marco Foiani.

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Peer review information Nature thanks Duncan Clarke, Anne Grove and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 DNA supercoil accumulation across the genome in wild-type conditions.

a, Schematic representation of bTMP binding to DNA in a supercoil-dependent manner. Based on normalized bTMP (IP/input) values, the genomic region is categorized into negative, positive and stable regions. b, Genome browser profile for bTMP binding on chromosome III. On the basis of peak intensities, bTMP peaks were called for negative (red), stable (blue) and positive (green) regions. Right, expanded view of chromosome III from 15 to 25 kb. For the bTMP profile, positive value peaks (normalized IP/input) that were above the threshold (+1.5) were designated as ‘negative supercoil’ (−σ), and negative value peaks (normalized IP/input) that were below the threshold (−1.5) were designated as ‘positive supercoil’ (+σ). Peaks in between the thresholds (from −1.5 to +1.5) were considered stable regions. c, Pie charts showing the coverage of negative, positive and stable regions based on bTMP-ChIP values plotted as percentage coverage for whole genome, intergenic regions, protein-coding regions and nucleosome-occupied regions. d, Gene correlation plot for negative supercoil (percentage) accumulation for wild-type cells in G1 and S phase (n = 6,706 genes; two-sided paired t-test, P < 2 × 10−16; Pearson correlation r = 0.86). e, Venn diagram comparison of G1 and S phase for bTMP binding with respect to peak number and base coverage. f, Genome browser view of the ASF2 locus on chromosome IV, showing bTMP peaks and accumulation of Pol II (Rpb3-ChIP) in G1 and S phase. g, Gal genes in chromosome II (from 270 to 285 kb), depicting bTMP and Pol II (Rpb3-ChIP) binding profiles under glucose and galactose conditions in S phase. h, Expanded view of chromosome III from 90 to 96 kb, containing a highly active gene (LEU2) close to tRNA (tL(CAA)C) and two moderately expressed genes (NFS1 and DCC1). Positive and negative supercoil and accumulation of Pol II (Rpb3-ChIP), Top1, Top2 and Hmo1 are shown.

Extended Data Fig. 2 Negative and positive supercoil distribution in DNA topoisomerase mutants.

a, Pol II-coding genes were grouped into three categories; high, medium and low expression based on FPKM values from RNA-seq data (n = 6,706 genes; low = medium = high = 2,235 genes). b, Positive supercoil distribution in high-, medium- and low-expression genes. c, Base coverage percentage of accumulation of Top2 and Top1 accumulation at different intergenic spaces (<250 bp = 1,729 gene pairs, 251–500 bp = 2,224 gene pairs and >500 bp = 2,010 gene pairs) with respect to gene pairs grouped according to orientation. d, Accumulation of positive supercoil in wild-type and top2-1 cells in S phase. Pol II genes are plotted against average gene density on the y-axis. e, Meta-gene plot for negative supercoil in G1 synchronized wild-type and top2-1 cells. f, Accumulation of positive supercoil in wild-type and top1∆ cells in S phase. g, Accumulation of positive supercoil in wild-type and top1∆top2-1 cells in S phase. h, Genome browser profile of chromosome III from 90 to 96 kb, showing comparative bTMP binding in wild-type cells and topoisomerase mutants. i, Genome browser profile of chromosome III from 90 to 96 kb, showing Pol II (Rpb3-ChIP) and Top1 protein accumulation in wild-type cells and top2-1 mutants.

Extended Data Fig. 3 Negative and positive supercoil accumulation in hmo1 mutants.

a, Positive supercoil accumulation in wild-type and hmo1∆ cells in S phase. Pol II genes are plotted against average gene density on the y-axis. b, Positive supercoil accumulation in wild-type and hmo1∆top2-1 cells in S phase. c, Comparison of the accumulation of Top1 protein (Top1–10× Flag) in wild-type, hmo1∆ and hmo1∆top2-1 cells. d, Genome browser profile of chromosome III from 90 to 96 kb, showing comparative bTMP binding in wild-type, hmo1∆ and hmo1∆top2-1 cells in S phase. e, Genome browser profile of chromosome III from 90 to 96 kb, showing accumulation of Top1 in wild-type, hmo1∆, top2-1 and hmo1∆top2-1 mutants.

Extended Data Fig. 4 RNA–DNA hybrid accumulation in wild-type and mutant cells.

a, Meta-gene profiles for RNA–DNA hybrid comparison in wild-type cells (at 28 °C and 37 °C). b, Accumulation of RNA–DNA hybrids in different expression classes. Pol II genes were grouped into three categories: high-, medium- and low-expression genes based on FPKM values from RNA-seq (Extended Data Fig. 2a). c, Meta-gene profiles for RNA–DNA hybrids in S phase in wild-type, rnh1∆, rrm3∆ and sen1cl cells (a conditional lethal strain GAL::URL-HA-Sen1, which shows lethality in glucose). d, Meta-gene profiles for RNA–DNA hybrids in G1 synchronized wild-type and top2-1 cells. e, Meta-gene profile for RNA–DNA hybrid comparison in wild-type cells and top1∆top2-1 double-mutant cells. f, Meta-gene profiles for RNA–DNA hybrid comparison in wild-type, hmo1∆, and hmo1∆top2-1 cells. g, Gene density plot comparison of RNA–DNA hybrids in wild-type, hmo1∆ and hmo1∆top2-1 cells. h, Density plot showing the base coverage of RNA–DNA hybrids in genes with head-on or co-directional orientation with respect to replication fork. Genes within 1 kb (top, n = 347 genes) or 2 kb (bottom, n = 539 genes) of the replication origins were considered. i, Base coverage percentage of accumulation of RNA–DNA hybrids at different intergenic spaces (<250 bp = 1,729 gene pairs, 251–500 bp = 2,224 gene pairs and >500 bp = 2,010 gene pairs) with respect to gene pairs grouped according to orientation.

Extended Data Fig. 5 Ablation of negative supercoil at boundaries causes topological alterations and nucleosome repositioning.

a, Genome browser profile of chromosome III from 90 to 96 kb, showing comparative bTMP binding in wild-type[Control], wild type[TopA], top1∆top2-1 [Control] and top1∆top2-1 [TopA] cells after 120 min at restrictive temperature. b, Genome browser profile of chromosome V showing Hmo1 accumulation in wild-type, top1∆top2-1 [Control], and top1∆top2-1 [TopA] cells after 120 min at restrictive temperature. c, Absolute nucleosome count was derived from histone H3 ChIP-seq analysis of wild-type [Control], wild type [TopA], top1∆top2-1 [Control] and top1∆top2-1 [TopA] cells after 120 min at restrictive temperature. d, Nucleosome positions (150-bp intervals) in wild-type cells and top1∆top2-1 double mutants harbouring either control or TopA-expressing plasmids mapped with respect to TSS against Pol II gene percentage on the y-axis.

Extended Data Fig. 6 Top2 mediates chromatin–chromatin interactions.

a, Heat map showing PET clusters in all of the chromosomes at 10-kb resolution. Inset, enlarged view of chromosome II. b, Table showing the number of paired-end reads, reads with linker on both ends, unique mapped PETs and inter-ligation clusters obtained after Top2 ChIA-PET analysis. c, Density map showing PET density at different interaction lengths. Inlet shows enlarged view of the region from 1,000 to 10,000 bp that was considered for analysis. d, Bar plot showing sizewise distribution of chromatin loops. e, Pie chart showing number of interactions matching with previous Top2-binding sites, number of interactions containing Top2 sites on either single or both sides of the interaction, number of genes within and outside the Top2-mediated chromatin loops and number of loops containing a single gene or multiple genes. Other categories include genes partially overlapping with the loops and all other elements. f, Genome browser view of ChIA-PET interactions along with Top2- and Hmo1-binding sites in chromosomes III and XVI.

Extended Data Fig. 7 A model for Hmo1, Top2 and Top1-mediated topological dynamics at Pol II genes.

a, Schematic representation of cruciform DNA structures arising at negatively supercoiled DNA formed by two branches in a B-DNA duplex structure (red and black) and two branches in a non-B-DNA duplex conformation (red or black). Cruciform DNA structures could form at gene boundaries and be stabilized by Hmo1. Gene looping is described in the blue area. b, Schematic representation of gene loop structures in S phase. Top2 associates with gene boundaries to harmonize topological transactions when transcribed genes are approached by incoming replication forks. We note that the topological dynamics described in the twin topological domain model7 are not represented in our scheme. c, In the absence of Hmo1, negative supercoil would lose the cruciform conformation and become a substrate for Top2. Top2 defects would delocalize Top1 at gene boundaries. The cruciform structures stabilized by Hmo1 would then become substrates for unscheduled Top1 activity that would convert them into aberrant intermediates such as single-stranded DNA, nicks and knots. In hmo1top2 double mutants, Top1 is not recruited at the gene boundaries, which remain in a negative supercoil context.

Extended Data Fig. 8 bTMP titration.

a, b, By using a biotin-labelled oligo as a reference point, we titrated in vivo binding of bTMP in yeast at different concentrations (from 0 to 800 ng). We diluted 4.16 pmol of biotin-labelled oligos into 200 μl of TE. The shown volumes of oligos were spotted onto a hybond membrane after equilibration with 1× maleic acid. Genomic DNA was isolated after UV (365 nM) cross-linking with the respective amount of bTMP and about 300 ng (a) or 1,500 ng (b) was spotted on the blot. The last sample was kept as a negative control for UV cross-linking. In Panel B, fivefold more oligo and genomic DNA was spotted compared to Panel A. Dot blot was developed with ExtrAvidin-Peroxidase antibody (Sigma-Aldrich, cat no: E2886). b, By measuring dot blot intensities, we estimated that 400 μg of bTMP was needed for 2 × 109 yeast cells.

Extended Data Fig. 9 bTMP binding normalization and dispersion profile for bTMP.

a, To avoid bias, normalization was performed to filter potential sequence-specific psoralen DNA binding23. To normalize the data, we first purified and sonicated genomic DNA and then performed the bTMP-ChIP procedure on purified DNA fragments. The correction for microarray readings was done by subtracting bTMP binding in vivo from bTMP binding on purified/sonicated genomic DNA as follows: (bTMP cells − IP/input) − (bTMP purified DNA − IP/input), which gives the normalized ratio of bTMP (bTMP − IP/input) binding. The meta ORF plot shows bTMP profiles with or without control DNA normalization of bTMP in wild-type S phase cells. b, c, Meta-gene plot showing the normalized mean bTMP ratio (b) and median bTMP (c) ratio in wild-type S phase cells. For both the plots, bTMP binding ratios for all protein-coding genes were plotted without smoothing, along with upper and lower confidence intervals (α = 0.05 or 95% limit). Dotted lines represent upper and lower confidence intervals (α = 0.05 or 95% limit). The confidence interval does not deviate significantly from the mean and median values.

Extended Data Table 1 List of S. cerevisiae strains

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Achar, Y.J., Adhil, M., Choudhary, R. et al. Negative supercoil at gene boundaries modulates gene topology. Nature 577, 701–705 (2020). https://doi.org/10.1038/s41586-020-1934-4

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