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The landscape of promoter-centred RNA–DNA interactions in rice

Nature Plants volume 8, pages 157–170 (2022)Cite this article

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Abstract

Chromatin-associated RNAs play key roles in various biological processes. However, both their repository and conjugation genomic loci and potential functions remain largely unclear. Here, we develop an effective method for mapping of chromatin-associated RNA–DNA interactions, followed by paired-end-tag sequencing (ChRD–PET) in rice. We present a comprehensive interaction map between RNAs and H3K4me3-marked regions based on H3K4me3 ChRD–PET data, showing three types of RNA–DNA interactions—local, proximal and distal. We further characterize the origin and composition of the RNA strand in R-loop RNA–DNA hybrids and identify that extensive cis and trans RNAs, including trans-non-coding RNAs, are prevalently involved in the R-loop. Integrative analysis of rice epigenome and three-dimensional genome data suggests that both coding and non-coding RNAs engage extensively in the formation of chromatin loops and chromatin-interacting domains. In summary, ChRD–PET is an efficient method for studying the features of RNA–chromatin interactions, and the resulting datasets constitute a valuable resource for the study of RNAs and their biological functions.

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Fig. 1: ChRD–PET data for comprehensive mapping of RNA–DNA interactions in rice.
Fig. 2: Characterization of H3K4me3 ChRD–PET RNA–DNA interactions.
Fig. 3: Global view of cRNAs and ncRNAs around H3K4me3-marked DNAs.
Fig. 4: RNA tags of ChRD–PET data may reflect the RNA strand of R-loops.
Fig. 5: Epigenetic features of H3K4me3 ChRD–PET interactions.
Fig. 6: Features of chromatin associated-RNAs in 3D genome architecture.

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Data availability

The ChRD–PET, ATAC–seq, ssDRIP–seq and total RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession no. GSE163119. The ChIP–seq, ChIA–PET, strand-specific RNA-seq and Whole Genome Bisulfite Sequencing (WGBS) data used in this study were published previously48 and are available at NCBI GEO under accession nos. GSE131202 and GSE142570. Source data are provided with this paper.

Code availability

Custom scripts for data analysis are available at https://github.com/fengchuiguo1994/ChRDPETpipeline.

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Acknowledgements

We thank G. Cao (Huazhong Agricultural University) and colleagues J. Dai and Z. Zhang for their help in performing the RNA FISH experiment. This work was funded by the National Natural Science Foundation of China (nos. 32070612 to X.L., 31771402 and 31970590 to G.L. and 31701115 to Y.Z.) and Fundamental Research Funds for the Central Universities, and was partly supported by open funds from the National Key Laboratory of Crop Genetic Improvement (no. K201906).

Author information

Author notes

  1. These authors contributed equally: Qin Xiao, Xingyu Huang.

Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China

    Qin Xiao, Xingyu Huang, Yan Zhang, Yongqing Yang, Qing Zhang, Guoliang Li & Xingwang Li

  2. Hubei Key Laboratory of Agricultural Bioinformatics and Hubei Engineering Technology Research Center of Agricultural Big Data, 3D Genomics Research Center, Huazhong Agricultural University, Wuhan, China

    Xingyu Huang, Yan Zhang & Guoliang Li

  3. Tsinghua-Peking Joint Center for Life Sciences, Center for Plant Biology, Tsinghua University, Beijing, China

    Wei Xu & Qianwen Sun

  4. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China

    Zhe Hu

  5. College of Life Science, Xinyang Normal University, Xinyang, China

    Feng Xing

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  1. Qin Xiao
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Contributions

X.L. and G.L. conceived the project. Q.X. generated data with assistance from Y.Z., W.X., Y.Y., Q.Z., Z.H. and Q.S. X.H. performed data analysis with help from F.X. X.L., G.L., Q.X. and X.H. interpreted data and wrote the manuscript.

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Correspondence to Xingwang Li.

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Nature Plants thanks Chang Liu, Julia Chekanova and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ChRD-PET data analysis pipeline and reproducibility.

a, Main steps of ChRD-PET experiment are shown in the boxes. DNA fragment analysis for relevant steps is included on the right of the main steps. b, Analysis pipeline for ChRD-PET data. c, Bar charts showing the percentage of sequence reads with no linker (grey), one linker (purple), and more than one linker (green). Reads with one linker entered the downstream analysis. d, Bar charts showing the percentage of DNA and RNA reads that are unmapped (grey), uniquely mapped and mapped quality >= 20 (purple), uniquely mapped and mapped quality < 20 (green), and multi-mapped (light grey) in RNA-DNA interaction PETs. e, Scatter plots of DNA coverage signals between ChRD-PET replicates, with spearman correlation coefficient r = 0.990. Bin size: 10kb. f, Scatter plots of RNA coverage signals between ChRD-PET replicates, with spearman correlation coefficients r = 0.891. Bin size: the transcribed region of a gene. g, Scatter plot showing two replicates of ChRD-PET data. 2D contact map with Pearson correlation coefficient: r = 0.963. Bin size: 10kb. h, Scatter plot showing two replicates of ChRD-PET data. Loop frequency with Pearson correlation coefficient r = 0.987.

Source data

Extended Data Fig. 2 Characterization of H3 ChRD-PET and H3K4me3 ChRD-PET.

a, Venn diagram showing the peak overlap between H3K4me3 ChRD-PET DNA and H3K4me3 ChIP-seq. 24,028 ChIP-seq peaks overlapped with 23,686 ChRD-PET DNA peaks. p value was calculated using one-sided Fisher’s exact test. b, Length distribution of DNA and RNA reads in H3K4me3 ChRD-PET. c, d, Location analysis of uniquely mapped H3K4me3 ChRD-PET DNA reads (c) and RNA reads (d) compared with the expected genomic distribution. e, f, Location analysis of H3K4me3 ChRD-PET DNA peaks(e) and RNA peaks (f) compared with the expected genomic distribution. g, h, i, Simulated relationships between interaction cluster numbers and ChRD-PET sequence depths. The total sequencing data was first divided into 10 equal parts, a bin ð‘– ð‘¡ℎ was generated by merging the first ð‘– ð‘¡ℎ parts into the ð‘– ð‘¡ℎ bin, where ð‘– = 1, 2, 3 …, 10. the x-axis represents the bins of 1, 2, …,10 with increasing sequence depths. (g) Y-axis: DNA peak number (red), RNA peak number (blue) (h) Y-axis: the interaction cluster counts. (i) Y-axis: local interaction cluster number (purple), proximal interaction cluster number (grey) and distal interaction cluster number (green). j, Global view of H3K4me3 ChRD-PET interactions, along with epigenetic and transcriptional features. k, Global view of H3K4me3 ChRD-PET interactions, along with epigenetic and transcriptional features on chromosome 1. l, Pairwise correlation analysis (with gene expression: FPKM) between H3 ChRD-PET RNA, H3K4me3 ChRD-PET RNA and Total RNA-seq. m, Length distribution of H3 ChRD-PET DNA and RNA reads. n, Genomic distributions of uniquely mapped H3 ChRD-PET DNA reads (left) and RNA reads (right). o, Genome-wide contact heatmap at 1Mb resolution of H3 ChRD-PET. X-axis: H3 ChRD-PET RNA. Y-axis: H3 ChRD-PET DNA.

Extended Data Fig. 3 Features of RNA-DNA interactions in H3K4me3 ChRD-PET.

a, Three types of ChRD-PET interactions of exon RNA and intron RNA. b, Screenshot showing introns of MH05g0031500 (chr5: 1,278,000-1292000) participating in local interactions, along with the epigenomic and transcriptomic features. Top: gene (track 1), ChRD-PET RNA (red, track 2), ChRD-PET DNA (blue, track 3). Bottom: local interactions, ChRD-PET DNA (blue), ChRD-PET intron RNA (green), linker (black). c, Three types of ChRD-PET interactions of RNAs from single- and multi-exon gene. Y-axis: average number of interacting DNA on each RNA. d, Gene Ontology of hub RNAs (top 5% RNAs) in ChRD-PET. p value was calculated using one-sided Fisher’s exact test. p values are corrected for multiple comparison using the Yekutieli_FDR correction. e, Network showing the targets of SNAC1 RNA (red). ChRD-PET DNAs: yellow and blue, blue: DNAs showed in Fig. 2h. Gene Ontology of SNAC1’s targets are shown on the Bottom. p value was calculated using one-sided hypergeometric distribution test. p value is corrected for multiple comparison using the Benjamini-Hochberg correction. f, The motifs of ATAC-seq related to SNAC1 RNA targets. g, Three types of ChRD-PET interactions of transcription factor RNA and non-transcription factor RNA. Y-axis: average number of interacting DNA on each RNA.

Source data

Extended Data Fig. 4 Coding and non-coding RNA in H3K4me3 ChRD-PET.

a, Number of interactions of coding RNA, lncRNA, snRNA/snoRNA, and microRNA in ChRD-PET. Total RNAs: coding RNAs were from coding genes, while lncRNA, snRNA/snoRNA, and microRNA were predicted. RNAs in ChRD-PET interactions: RNAs from total RNAs and involved in ChRD-PET interactions. ChRD-PET interactions: ChRD-PET interactions related to each type of RNAs. b, c, Scatter plots of RNA reads detected by ChRD-PET in comparison with RNA-seq (b) and total RNA-seq (c) Bin size: the transcribed region of a gene. d, Histogram showing the number of DNA/coding RNA numbers in each coding RNA/DNA category. Bottom left: histogram showing the number of DNA regions interacting with different number of coding RNAs. For the second pillar, there are 5496 DNA peaks, and each peak interacting with 2 coding RNAs. Upper right: histogram showing the number of RNAs in each DNA peak category. For the second pillar, there are 4956 RNAs, and each RNA interacted with 2 DNA peaks. e, Cluster analysis of the pairwise correlation between the associated DNA regions of 8 types of snoRNA in ChRD-PET. Bin size: 25kb. f, Top: genomic loci, ChIP-seq, ChRD-PET and transcriptome information for lncRNA MSTRG.20623. Bottom: circlet plot of H3K4me3 ChRD-PET interactions related to lncRNA MSTRG.20623. g, Top: genomic loci, ChIP-seq, ChRD-PET and transcriptome information for microRNA osa-MIR2106. Bottom: circlet plot of H3K4me3 ChRD-PET interactions related to osa-MIR2106. h, Top: genomic loci, ChIP-seq, ChRD-PET and transcriptome information for the cluster of snoRNAs. Bottom: circlet plot of H3K4me3 ChRD-PET interactions related to the cluster of snoRNAs. i, j, k, l, m, Circlet plot of ChRD-PET interactions in which 5 types of snRNA were involved. U1(i), U2 (j), U4 (k), U5 (l), U6atac (m).

Source data

Extended Data Fig. 5 Intra-chromosome H3K4me3 ChRD-PET interactions.

a, b, c, d, RNAs engaged in intra-chromosome interactions on 12 chromosomes. Coding RNA (a), lncRNA (b), snRNA/snoRNA (c), microRNA (d).

Extended Data Fig. 6 Characterization of H3K4me3 peaks overlapping with R-loops.

a, Venn diagram showing the peak overlap between H3K4me3 ChIP-seq, H3K4me3 ChRD-PET DNA, and ssDRIP-seq. b, Density distribution of H3K4me3 ChIP-seq, H3K4me3 ChRD-PET DNA, and ssDRIP-seq at transcription start site. c, The percentage of S9.6 foci colocalized/non-colocalized with H3K4me3 foci in 29 rice nuclei. Averagely, 91% S9.6 foci were colocalized with H3K4me3 foci, 9% non-colocalized. Biological replicates, n=29. p value was calculated using two-sided Wilcoxon rank-sum test. Centre line represents the median, box extent ranges from 25th to 75th percentile, and whiskers extend at most to 1.5× the interquartile range (distance between the first and third quartiles). d, Number of ChRD-PET interactions for coding RNA, lncRNA, snRNA/snoRNA and, microRNA in the overlap and non-overlap portion of ChRD-PET DNA and ssDRIP-seq peaks. e, Histogram showing the number of R-loop derived RNA-DNA interactions related to ncRNAs in H3K4me3 ChRD-PET filtered by RNase H treated H3K4me3 ChRD-PET and 8-bp RNA-DNA complementary pairing test. f, Histogram showing the number of R-loops in different categories of RNA. g, Histogram showing the number of RNAs in different categories of R-loops.

Source data

Extended Data Fig. 7 Analysis of RNase H treated H3K4me3 ChRD-PET interactions.

a, Density distribution of RNase H treated H3K4me3 ChRD-PET DNAs around the H3K4me3 peak center. b, Density distribution of RNase H treated H3K4me3 ChRD-PET sense (red) and antisense (grey) RNA at the annotated genes region. c, Length distribution of RNase H treated H3K4me3 ChRD-PET DNA and RNA reads. d, Genomic distributions of uniquely mapped RNase H treated H3K4me3 ChRD-PET DNA (left) and RNA reads (right). e, Genome-wide contact heatmap at 1Mb resolution of RNase H treated H3K4me3 ChRD-PET. X-axis: RNase H treated H3K4me3 ChRD-PET RNA. Y-axis: RNase H treated H3K4me3 ChRD-PET DNA. f, Histogram showing the number fraction of R-loop derived RNA-DNA interactions and non R-loop derived RNA-DNA interactions in local, proximal, distal interactions. g, Screenshot showing osa-MIR2106 mediated R-loop formation in trans. Track 1: ChRD-PET RNA coverage signals. Arrows point out the trans location of RNA strand involved in R-loop. Track 2: ssDRIP-seq coverage signals and peak in the forward strand. Track 3: ssDRIP-seq coverage signals and peak in the reverse strand. Track 4: RNase H treated ssDRIP-seq. Track 5: ChRD-PET DNA coverage signals. Track 6: H3K4me3 ChIP-seq coverage signals and peak. Track 7: R-loop formation site. Track 8: annotation genes. Track 9: sequences and R-loop structure, RNA (red), DNA (blue).

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Extended Data Fig. 8 R-loop distribution and epigenetic features of ChRD-PET.

a, Density distribution of H3K4me3 ChRD-PET DNA at H3K4me3 peak center. Three categories of H3K4me3 ChRD-PET DNA related to R-loop were analysed: H3K4me3 anchors overlapping with hybrid RNA: DNAs, H3K4me3 anchors overlapping with R-loop and associating with proximal RNA, and H3K4me3 anchors not overlapping with R-loop. b, Density distribution of H3K4me3 ChIP-seq at H3K4me3 peaks center in different categories as same as classification in a. c, Conservation analysis of ChRD-PET RNA in different categories of interacting DNA with same classification as a. d, Methylation ratios of peaks with H3K4me3 modification in different categories as same as classification in a. Random: regions on the genome randomly generated by BEDTools. e, Methylation ratios of ChRD-PET RNA in different categories of interacting DNA with same classification as a. Random: regions on the genome randomly generated by BEDTools. f, Density distribution and heatmap of H3K4me3 ChIP-seq and ATAC-seq for three types of peaks classified in Fig. 5c. g, DNA methylation level of three types of peaks classified in Fig. 5c.

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Extended Data Fig. 9 Analysis of ChIA-PET and ChRD-PET at chromatin loop level.

a, Table for ChRD-PET DNA and RNA from peak or anchor and the number of ChRD-PET interactions. DNA peak: DNA with the H3K4me3 ChIP-seq peak (Dp). DNA anchor: DNA with the H3K4me3 ChIP-seq peak and in ChIA-PET interaction (Da). Peak RNA: RNA transcript from DNA peak (Rp). Anchor RNA: RNA transcript from DNA anchor (Ra). Rest RNA: RNA transcript from a region that is neither DNA peak nor DNA anchor (Rr). b, Histogram showing ChRD-PET interaction number for six kinds of PETs. c, Right: Venn diagram showing the overlap of three group RNAs: RNAs interacting with basal peaks, RNAs interacting with anchors, RNAs interacting with both basal peaks and anchors. Left: expression level of cognate genes of three group RNAs. RNA without interaction, n=36,040. p value was calculated using two-sided Wilcoxon rank-sum test. Centre line represents the median, box extent ranges from 25th to 75th percentile, and whiskers extend at most to 1.5× the interquartile range (distance between the first and third quartiles). d, Similar example to Fig. 6g showing the interactions of model 3. Six anchors and four RNAs were in the complex of ChIA-PET and ChRD-PET interactions. Among them, anchor 802 and anchor 804 in the ChIA-PET interactions loops and both interacting with 4 ChRD-PET RNAs of MH01g0150100, MH01g0150300, MH01g0150400 and MH01g0150700. e, Screenshot showing the details of d. f, Similar example to Fig. 6g showing the interactions of model 3. Three anchors and six RNAs were in the complex of ChIA-PET and ChRD-PET interactions. Among them, anchor 13131 and anchor 13133 in the ChIA-PET interactions loops and both interacting with 4 ChRD-PET RNAs of MH02g0618900, MH03g0317500, MH03g0317700 and MH03g0318400. In addition, non-coding RNA MH02gsn00210 (U1) and MSTRG.12745 interact with anchor 13133.

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Extended Data Fig. 10 Analysis of ChIA-PET and ChRD-PET at CID level.

a, Heatmap showing chromatin-enriched RNA across whole chromatin interaction domain (CID) in MH63. Y-axis: chromatin-enriched RNA. X-axis: MH63 chromatin interaction domain (CID). b, Number of ChRD-PET RNAs inside/outside the CID. c, Histogram showing the number of coding RNAs and ncRNAs related ChRD-PET interactions in or out of the CID. d, Density distribution of ChRD-PET DNA and ChIP-seq around CID boundary. e, Number of ChRD-PET interactions in A/B compartment. A/B compartment, which is a type of high-order 3D genome architecture unit composed of multiple interconnected CIDs (chromatin interacting domains). In the DNA-DNA interaction matrix heatmap, each chromosome is partitioned into mega base-scale gene-rich and gene-poor chromatin blocks corresponding to transcriptionally active “A” and inactive “B” compartments, respectively. AA: RNA from A compartment, DNA from A compartment. AB: DNA from A compartment, RNA from B compartment. BA: DNA from B compartment, RNA from A compartment. BB: RNA from B compartment, DNA from B compartment. f, Gene Ontology analysis of CID (614) hub RNAs.

Supplementary information

Reporting Summary

Supplementary Table 1

Contains 13 tables with peak results, ChRD–PET interaction results, GO enrichment analysis results and more.

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Xiao, Q., Huang, X., Zhang, Y. et al. The landscape of promoter-centred RNA–DNA interactions in rice. Nat. Plants 8, 157–170 (2022). https://doi.org/10.1038/s41477-021-01089-4

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