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Topoisomerase-modulated genome-wide DNA supercoiling domains colocalize with nuclear compartments and regulate human gene expression

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

DNA supercoiling is a biophysical feature of the double helix with a pivotal role in biological processes. However, understanding of DNA supercoiling in the chromatin remains limited. Here, we developed azide-trimethylpsoralen sequencing (ATMP-seq), a DNA supercoiling assay offering quantitative accuracy while minimizing genomic bias and background noise. Using ATMP-seq, we directly visualized transcription-dependent negative and positive twin-supercoiled domains around genes and mapped kilobase-resolution DNA supercoiling throughout the human genome. Remarkably, we discovered megabase-scale supercoiling domains (SDs) across all chromosomes that are modulated mainly by topoisomerases I and IIβ. Transcription activities, but not the consequent supercoiling accumulation in the local region, contribute to SD formation, indicating the long-range propagation of transcription-generated supercoiling. Genome-wide SDs colocalize with A/B compartments in both human and Drosophila cells but are distinct from topologically associating domains (TADs), with negative supercoiling accumulation at TAD boundaries. Furthermore, genome-wide DNA supercoiling varies between cell states and types and regulates human gene expression, underscoring the importance of supercoiling dynamics in chromatin regulation and function.

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Fig. 1: ATMP-seq enables quantitative genome-wide mapping of DNA supercoiling.
Fig. 2: Transcription-dependent negative and positive twin-supercoiled domains are locally present around genes.
Fig. 3: DNA SDs modulated by TOP1 and TOP2B are present throughout the human genome.
Fig. 4: Transcription activities, but not the consequent supercoiling accumulation in the local region, contribute to the formation of SDs.
Fig. 5: Genome-wide SDs colocalize with A/B compartments but are distinct from TADs.
Fig. 6: Negative DNA supercoiling is accumulated at the boundaries of TADs.
Fig. 7: Widespread dynamics and variations of genome-wide DNA supercoiling regulate human gene expression.

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Data availability

All genomics data produced for this study were deposited to the Sequence Read Archive under accession number PRJNA1003820. All other data needed to evaluate the conclusions in this paper are available in the article and the Supplementary Information. Source data are provided with this paper.

Code availability

R scripts and Python scripts for data processing and visualization are available on GitHub (https://github.com/qyao456/ATMP-seq.git).

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Acknowledgements

We thank Y. Cao (National Institutes of Health (NIH)) for the code of correlation analysis, J. Rowley (University of Nebraska) for providing the high-resolution A/B compartments data, L. Baranello (Karolinska Institute) for the HCT116-TOP1/TOP2A-mAID cell lines, R. Casellas (MD Anderson Cancer Center) for the HCT116-RAD21-mAID cell line, M. T. Kanemaki (National Institute of Genetics, Japan) for the HCT116-CTCF-mAID2 cell line and E. Lei (NIH) for the Drosophila S2 cell line. We thank D. Levens, M. Lichten, A. Kelly, S. Grewal, Y. Dalal and J. Barrowman for helpful discussions, comments and scientific editing. We also thank M. Wong, E. Conner and S. Shema of the CCR Genomics Core for NextSeq sequencing and J. Shetty and Y. Zhao of the National Cancer Institute (NCI) Sequencing Facility for NovaSeq sequencing. Cell sorting and flow cytometry analyses were performed at the NCI LGI Flow Cytometry Core supported by funds from the Center for Cancer Research. This work used the Biowulf computing cluster at NIH. This work was supported by the Intramural Research Program of the NIH NCI (1ZIABC011884).

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Author notes

  1. These authors contributed equally: Qian Yao, Linying Zhu.

Authors and Affiliations

  1. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Qian Yao, Linying Zhu, Zhen Shi & Chongyi Chen

  2. Laboratory of Genome Integrity, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Subhadra Banerjee

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Contributions

C.C. and Q.Y. conceptualized and designed the study. Q.Y. developed the assay and performed most of the experiments and bioinformatic data analysis. L.Z. performed the in vitro nucleosome assembly, ATMP fluorescence measurement and TT-seq and constructed the HCT116-TOP2B-mAID2 cell line. Z.S. helped with performing the assays. S.B. conducted the flow sorting enrichment of G1 cells. C.C. and Q.Y. wrote the paper.

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Correspondence to Chongyi Chen.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Julien Mozziconacci and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Drawbacks of previous psoralen-based supercoiling assays (A-F) and the performance of ATMP-seq in supercoiling measurement (G-J).

(A) Supercoiling-sensitivity of the TMP-DNA reaction. Crosslinking probability was reported based on experiments using an 8-kb plasmid (two biological replicates). (B) Top panel: Human genome coverage of non-crosslinked gDNA fragments remaining after three rounds of denaturation and exonuclease digestion, at 100-kb resolution, leading to genomic bias. Bottom panel: Human genome coverage of gDNA, suggesting the genomic bias of remaining non-crosslinked gDNA fragments in the top panel cannot be explained by gDNA sequencing data. (C) Schematic of TMP reverse-crosslinking, leading to three types of products including reverse-crosslinked (orange), remain-crosslinked (green) and DNA-damaged (blue). (D) Background noise revealed by analyzing the products of TMP reverse-crosslinking by 254-nm UV illumination for 1-5 min. (E) Background noise revealed by analyzing the products of TMP reverse-crosslinking by various conditions of alkaline treatment. Condition 1: 175 mM NaOH and 1.5 mM NaCl, 65 °C incubation for 3 h. Condition 2: 100 mM NaOH, 75 °C incubation for 30 min. Condition 3: 175 mM Na3PO4 and 1.5 mM NaCl, 65 °C incubation for 3 h. Condition 4: 100 mM Na3PO4, 70 °C incubation for 30 min. (F) Background noise revealed by analyzing the products of TMP reverse-crosslinking by 95% formamide and 10 mM EDTA, 90 °C incubation for 10-20 min. (G) ATMP monoadduct density upon reaction with supercoiling-relaxed DNA wrapped by nucleosomes with various densities. Nucleosome density was reported as the percentage of DNA bases wrapped by the nucleosome (two biological replicates). (H) Workflow of ATMP-seq’s sample and control to normalize DNA sequence bias. (I) Technical reproducibility of the ATMP-seq assay at 100-kb resolution, when sequenced by 60 million Illumina reads. (J) Correlation between ATMP-seq’s technical duplicates at 100-kb resolution, when sequenced by 60 million Illumina reads.

Source data

Extended Data Fig. 2 Elimination of nucleosome bias in the ATMP-seq assay.

(A) Distribution of ATMP-bound naked gDNA fragments or nucleosome-bound chromatin fragments in an example region at 100-kb resolution. (B) ATMP-seq readout normalized by naked gDNA (ATMP/gDNA) or nucleosome-bound chromatin (ATMP/Chrom), at various spatial resolutions. (C) Correlation between ATMP/gDNA and ATMP/Chrom at various spatial resolutions. (D) ATMP-seq readout (ATMP/gDNA) with different ATMP monoadduct densities (1 monoadduct per 20 kb, 15 kb, or 12 kb).

Extended Data Fig. 3 Overall relaxed supercoiling status across the human genome under perturbation and artifactual supercoiling distribution around genes reported by previous supercoiling assays.

(A) Schematic of ATMP-DBCO-647 fluorescence intensity measurement by FACS analysis. (B) Distribution of the ATMP-DBCO-647 fluorescence intensity measured by FACS in individual G1-phase GM12878 cells selected by DAPI staining. “Negative control” shows the autofluorescence background without the addition of ATMP or DBCO-647, “No ATMP control” shows the nonspecific DBCO-647 fluorescence background without the addition of ATMP. “Untreated control” shows the overall DNA supercoiling level under normal condition. “TPL”, “CPT”, and “ICRF” show the overall DNA supercoiling level upon transcription inhibition by triptolide (TPL), topoisomerase I inhibition by camptothecin (CPT), and topoisomerase II inhibition by ICRF-193 (ICRF), respectively. (C) Supercoiling level around all genes of Drosophila melanogaster genome on average, and at the TSS and TES with 3-kb extension on both sides, analyzed from the data of TMP-seq4. (D) Supercoiling level around all genes of Human genome on average, and at the TSS and TES with 3-kb extension on both sides, analyzed from the data of TMP-based method48. (E) Supercoiling level around all genes of Saccharomyces cerevisiae genome on average, and at the TSS and TES with 1-kb extension on both sides, analyzed from the data of biotinylated TMP (bTMP)-based pulldown method50.

Extended Data Fig. 4 Negative and positive twin-supercoiled domains around genes.

(A) Supercoiling level around genes with low, medium, and high expression levels (Supplementary Table 3), relative to the supercoiling level in the gene body. (B) Supercoiling level around 6 categories of genes with different transcription levels (Supplementary Table 4), relative to the supercoiling level in the gene body. Dot plots are the quantitative relationship between the transcription level and the relative supercoiling level at TSS and TES, respectively. (C) Supercoiling level around 6 categories of genes with different lengths (Supplementary Table 5), relative to the supercoiling level in the gene body. Dot plots are the quantitative relationship between the gene length and the relative supercoiling level at TSS and TES, respectively. (D-E) Quantitative relationship between the relative supercoiling level and the transcription level (D) or the gene length (E), upon drug inhibition of transcription (TPL) or topoisomerase (CPT, ICRF).

Extended Data Fig. 5 Supercoiling change upon topoisomerase depletion around genes that form gene pairs and the genome-wide map of DNA supercoiling.

(A) Supercoiling level change upon TOP2B depletion around convergent genes, relative to the supercoiling level in the gene body. Untreated for the control, 5-ph-IAA for AID depletion. (B) Supercoiling level change upon TOP2B depletion around divergent genes, relative to the supercoiling level in the gene body. Untreated for the control, 5-ph-IAA for AID depletion. (C) Supercoiling level change upon TOP2A depletion around convergent genes, relative to the supercoiling level in the gene body. Auxinole (AUX) for the control, IAA AID depletion. (D) Supercoiling level change upon TOP2A depletion around divergent genes, relative to the supercoiling level in the gene body. Auxinole (AUX) for the control, IAA AID depletion. The semi-transparent shade of A-D means values ± SEM across the regions. (E) DNA-staining-based flow-sorting to enrich G1-phase cells. (F) Whole-genome mapping of DNA supercoiling level in G1-phase GM12878 human cells. (G) DNA supercoiling level in the rDNA region and the centromere repeats. (H) DNA supercoiling level in different types of repetitive regions in the human genome. Each dot represents a subfamily belonging to the specific type of repetitive elements.

Extended Data Fig. 6 Topoisomerase-modulated SDs across the human genome.

(A) SDs upon the inhibition of topoisomerase, in an example region at 100-kb resolution (technical replicate). (B) Fold of supercoiling level change upon topoisomerase inhibition within each genomic 100-kb bin. Dark and light color bars represent technical replicates. n = 22,361 (CPT, repeat 1), 22,190 (CPT, repeat 2), 19,613 (ICRF, repeat 1), 19,611 (ICRF, repeat 2). Mean values ± s.d. are marked in the violin plots using boxplot. (C) Distribution of DNA supercoiling level upon the inhibition of topoisomerase, within each genomic 100-kb bin (technical replicate). (D) Correlation between supercoiling level change upon topoisomerase inhibition and the original supercoiling level under normal condition, within each genomic 100-kb bin.

Extended Data Fig. 7 Genome-wide SDs and the genome-wide distribution of other chromatin features.

(A) Correlation between the distribution of chromatin accessibility (ATAC-seq) and DNA supercoiling level across the human genome at 100-kb resolution. SCC is spearman correlation coefficient. (B) Correlation between the distribution of various histone modifications (ChIP-seq of H3K27ac, H3K27me3, H3K4me3, H3K9me3) and DNA supercoiling level across the human genome at 100-kb resolution. SCC is spearman correlation coefficient. (C) Correlation between the distribution of CTCF binding sites (ChIP-seq of CTCF) and DNA supercoiling level across the human genome at 100-kb resolution. SCC is spearman correlation coefficient. (D) Percentage of genomic regions containing enhancers and promoters assigned to negative DNA supercoiling (red) and positive DNA supercoiling (green) at 500-bp resolution. (E-G) Correlation between DNA supercoiling level and the GC-content in GM12878 (E) and HCT116 (F) cells within each genomic 100-kb bin, and in Drosophila S2 (G) cells within each genomic 25-kb bin. r is Pearson correlation coefficient. (H-I) SDs and TADs in two example regions at 10-kb resolution. Top: Contract matrix to show TADs from the Hi-C data. Second: TAD insulation scores computed from the Hi-C data. Third: SDs from the ATMP-seq data. Bottom: CTCF binding sites from the ChIP-seq data.

Extended Data Fig. 8 Genome-wide SDs and A/B compartments.

(A) SDs and A/B compartments in an example region in HCT116 cells at 100-kb resolution. Top: Negative SDs (yellow) and positive SDs (grey). Bottom: A compartment (blue) and B compartment (grey). (B) Correlation between DNA supercoiling level and the A/B compartment score in HCT116 cells, within each genomic 100-kb bin. (C-D) Hi-C map in two example regions in HCT116 cells. Auxinole (AUX) for the control, IAA for AID depletion of TOP1. (E) Curve of contact frequency from the Hi-C data. Auxinole (AUX) for the control, IAA for AID depletion of TOP1. (F) A/B compartments in an example region in HCT116 cells at 100-kb resolution. Auxinole (AUX) for the control, IAA for AID depletion of TOP1.

Extended Data Fig. 9 Variations of genomic DNA supercoiling between cell states and types, and the role of DNA supercoiling in the regulation of gene expression.

(A) Percentage of dead cells in normal and quiescent cell population analyzed by flow cytometry, after cell staining with Live/Dead fixable near-IR dead cell stain kit. (B) Distribution of the DNA supercoiling level within each genomic 100-kb bin in GM12878 cells and HCT116 cells. (C) Distribution of DNA supercoiling level and the transcription activity (GRO-seq) in two example regions in GM12878 and HCT116 cells, at 500-bp resolution. Dotted lines mark the TSS of DEGs to show the corresponding supercoiling difference. Pink areas highlight the DNA supercoiling difference in genomic regions without transcription or DEGs. (D) DNA supercoiling in an example genomic region in GM12878 and HCT116 cells, at 100-kb and 10-kb resolutions. Pink areas highlight the difference. (E) Pearson correlation coefficients of the genome-wide DNA supercoiling between technical duplicates, between ATMP-seq normalized by naked gDNA and nucleosome-bound chromatin, if any nucleosome effect, and between GM12878 and HCT116 cells, at 100-kb, 10-kb, and 1-kb resolutions. (F-G) Biological duplicate dataset of nascent transcription activity of protein-encoding genes in negative SDs (negative, n = 2,487) or positive SDs (positive, n = 271), upon the AID depletion of TOP1 (F) or TOP2A (G) P values were calculated using nonparametric Wilcoxon two-sided tests. Auxinole (AUX) for the control, IAA for AID depletion. Boxplots show the median, first and third quartiles as a box, and the whiskers indicate the most extreme data point within 1.5 lengths of the box.

Extended Data Fig. 10 Making supercoiled and nucleosome-wrapped DNA templates (A-C), adjusting the TMP-DNA reaction (D-E), and verifying AID depletion of target protein (F-J).

(A) 1-D gel to measure the level of negative and positive DNA supercoiling. (B) 2-D chloroquine gel to measure the relaxed, negatively supercoiled, and positively supercoiled DNA templates. (C) Estimation of the nucleosome density on a plasmid DNA of different length, by quantifying the level of unconstrained DNA supercoiling after nucleosome removal. At least two biological duplicates were examined over at least two independent experiments for data presented in A-C and only one representative result was shown. (D) Adjusting the TMP-DNA reaction by tuning TMP concentration. Crosslinking probability was reported based on experiments using an 8-kb plasmid. (E) Adjusting the TMP-DNA reaction by tuning UV-A power. Crosslinking probability was reported based on experiments using an 8-kb plasmid. Data of D-E are representative of two biological replicates. At least two biological replicates were examined for data in A-E. (F-H) Verifying AID depletion of topoisomerase. For TOP1 (F) and TOP2A (G), the corresponding cell lines were incubated with 500 µM IAA for 2 h, while the control was 200 µM Auxinole 24 h incubation. For TOP2B (H), the corresponding cell line was incubated with 1 µM 5-ph-IAA for 4 h. (I-J) Verifying AID depletion of cohesin (I) and CTCF (J). For RAD21, the corresponding cell line was incubated with 500 µM IAA for 6 h, while the control was 200 µM Auxinole 24 h incubation. For CTCF, the corresponding cell line was incubated with 1 µM 5-ph-IAA for 4 h.

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Yao, Q., Zhu, L., Shi, Z. et al. Topoisomerase-modulated genome-wide DNA supercoiling domains colocalize with nuclear compartments and regulate human gene expression. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01377-5

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