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. 2021 Jun 9;22(1):176.
doi: 10.1186/s13059-021-02390-3.

Transcription shapes DNA replication initiation to preserve genome integrity

Yang Liu #  1   2 , Chen Ai #  1 , Tingting Gan #  1   2 , Jinchun Wu  1   2 , Yongpeng Jiang  1   2 , Xuhao Liu  1 , Rusen Lu  1   2 , Ning Gao  2   3 , Qing Li  2   4 , Xiong Ji  1   2 , Jiazhi Hu  5   6
Affiliations

Transcription shapes DNA replication initiation to preserve genome integrity

Yang Liu et al. Genome Biol. .

Abstract

Background: Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.

Results: We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage.

Conclusion: RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

Keywords: DNA damage; DNA replication initiation; Genome instability; MCM redistribution; Transcription; Transcription-replication initiation collision.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
NAIL-seq identified DNA early replication initiation zones (ERIZs). a Schematic showing the ERIZs identified by NAIL-seq. Nascent DNA labeled with EdU is red, while that labeled with BrdU is green. The origin indicated by the gray arrows was identified by EdU/HU, but not by EdU and BrdU. The light blue boxes show the procedures for the indicated NAIL-seq libraries. CDKi is the CDK4/6 inhibitor, palbociclib. Rel., release; ERIZ, early replication initiation zone. b Early replication signals from NAIL-seq libraries in K562 cells. Bar plots show the distribution of EdU (red) or BrdU (green) signals in the indicated region. The early DNA replication regions identified by Repli-seq are shown in gray. For the dual-labeled samples, the G1-arrested K562 cells were released for 2.5 h to reach the G1/S transition and then sequentially labeled with EdU and BrdU, each for 15 min. The E-B panel shows the BrdU-subtracted EdU signals; red represents the EdU-dominant signal and green represents the BrdU-dominant signal. E-B peaks called by the RepFind pipeline are shown underneath. For EdU/HU, G1-arrested cells were released into medium supplied with 10 mM HU and 10 μM EdU for 12 h before harvesting. EdU/HU signals are shown in dark red with the identified ERIZs in blue underneath. The pink shadow box highlights a non-ERIZ region, while the cyan box highlights a typical region where EdU/HU displays a higher resolution than E-B. c The width distribution of NAIL-seq identified replication peaks and Repli-seq revealed early replicated regions in K562 cells. The Wilcoxon Rank-sum test was applied for statistical analysis. d Venn diagram showing the overlapping peaks of E-B and EdU/HU in K562 cells. The cyan circle represents the total number of E-B peaks and the orange circle represents the EdU/HU peaks. The EdU/HU peaks overlapping with E-B peaks are defined as ERIZs. e Replication timing analysis for the two categories of EdU/HU peaks in K562 cells. EdU/HU peaks are categorized as ERIZs (n = 2,265, red, overlapping with E-B peaks) and non-ERIZs (n = 891, black, independent of E-B peaks). The replication timing of each peak is defined by the mean of the wavelet-smoothed signals from six fractions of the ENCODE Repli-seq profile. f The peak width distribution of ERIZs in K562 and GM12878 cells. The total number (N) and the median width of ERIZs are shown in the legends
Fig. 2
Fig. 2
ERIZs are located in non-transcribed regions within active compartments. a Percentage of the width of ERIZs in the A compartments. Genome represents the percentage of A compartments in the hg19 genome in the indicated cells. b The relationship between the peaks of predictors and ERIZ appearance in GM12878 and K562 cells (see “Methods” for details). c Distribution of ERIZs in the context of active transcription in GM12878 cells. “+” indicates the forward strand and “–” indicates the reverse strand. The positions and transcribed direction of genes are shown at the bottom of the panel with the gene names. Forward-transcribed genes are marked in orange and reverse-transcribed genes are marked in blue. d Heatmaps of ERIZ-associated early replication initiation (from EdU/HU, red) and active transcription (blue) in GM12878 and K562 cells. The ERIZ-occupied non-transcribed regions (non-TRs) are ranked by width and centered on the midpoint flanked by two transcribed genes in each cell line. The non-TRs in A compartments, 20–100 kb in width, are displayed. Each line represents a non-TR
Fig. 3
Fig. 3
Transcription relocates early replication in K562 cells and mESCs. a, c Heatmaps of early replication signals (from EdU/HU) around ERIZ-associated non-TRs in K562 cells treated with the indicated concentrations of α-amanitin (a) or DRB (c). Cells were treated as shown in Additional file 1: Figure S4a. Non-TR, non-transcribed region. TR, transcribed region. Legends are depicted as described in Fig. 2d. b, d Box-plots showing the log2 ratio of read density between early replication in the ERIZ-associated non-TRs and that in the flanked transcribed regions in the A compartments of K562 cells treated with the indicated concentrations of α-amanitin (b) or DRB (d). The Wilcoxon rank-sum test was applied for statistical analysis. e Heatmaps of early replication signals (from EdU/HU) around the ERIZ-associated non-TRs in mESCs with or without RNA polymerase II. Cells were treated as illustrated in Additional file 1: Figure S4h. Endogenous RNA polymerase II tagged with mAID is marked as Pol II. Non-transcribed regions (non-TRs) are ranked by width and centered on the midpoint flanked by two transcribed genes in mESCs. The non-TRs within A compartments, 20–100 kb in width, are displayed. Each line represents a non-TR. f The log2 ratio of read density between early replication in ERIZ-associated non-TRs and that in the flanked transcribed regions within the A compartments of mESCs. WT, wild-type. The Wilcoxon rank-sum test was employed for statistical analysis. g Distribution pattern of early replication initiation with or without RNA polymerase II in mESCs
Fig. 4
Fig. 4
Transcription redistributes MCM in the G1 phase. a Western blotting showing detection of the indicated proteins in G1-arrested K562 cells in the absence or presence of 10 μg/mL α-amanitin. G1-arrested K562 cells were treated with or without 10 μg/mL α-amanitin for 12 h before harvest. WCE, whole cell extract; Cyto-E, cytoplasmic extract; Chro-E, chromatin extract. b The distribution of ORC2 within the ERIZ-associated non-transcribed regions (non-TRs) in G1-arrested K562 cells in the absence or presence of 10 μg/mL α-amanitin. The non-TRs are the same as those shown in Fig. 3a. c Box-plot showing the log2 ratio of the read density of ORC2 between non-TRs and flanked TRs in the A compartments of K562 cells treated with 10 μg/mL α-amanitin. The Wilcoxon rank-sum test was applied for statistical analysis (p = 0.31). d The distribution of MCM5 in G1-arrested K562 cells in the absence or presence of 10 μg/mL α-amanitin. The data from ChIP-ed samples over the input samples are defined as fold change data, as in the ENCODE project and a previous report [35]. The fold change data were normalized to z-scores for heatmaps. The displayed regions are the same as in b. e Box-plot showing the log2 ratio of MCM5 signal enrichment in non-TRs and TRs in the A compartments of K562 cells treated with 10 μg/mL α-amanitin. The Wilcoxon rank-sum test was applied for statistical analysis. f, g, and h The distribution of early replication initiation from EdU/HU (f), ORC2 (g), and MCM5 (h) in ERIZ-adjacent active gene bodies with or without 10 μg/mL α-amanitin treatment. TSS, transcription start site; TTS, transcription termination site. ERIZ-flanked transcribed regions larger than 50 kb are ranked by width (with the smallest genes on top). For display, all transcribed regions were scaled to the same width and aligned at both TSS and TTS. Each line represents an individual transcribed gene
Fig. 5
Fig. 5
Replication initiation occurs at the transcription barrier. a Schematic of dCas9-gRNA-mediated transcription blockade. Four gRNAs were designed to bind the non-template strand at the fourth exon of CMIP. The orange arrow indicates the transcribed region and the red stop sign illustrates the dCas9-induced transcription blockade. b, d The mean of EdU/HU normalized read density (line) with the standard error (shadow) of three biological replicates near the dCas9/gRNA binding sites on CMIP in early S phase (b) or G1-phase (d) K562 cells treated with CMIP (red) or scrambled (cyan) gRNAs. gRNA-CMIP and gRNA-scramble indicate that cells were treated with dCas9/gRNA targeting CMIP or a control region, respectively. The regions between two dashed lines around 0 highlight the four binding sites of CMIP gRNAs. The 10-kb window of dCas9 binding sites is tiled by 1-kb bins (sliding by 200 bp). The read count per kilobase (RPK) was calculated within each 1-kb bin and normalized by the RPK of the B compartments from each biological replicate, defining the RPK ratio (see “Methods” for details). c, e The levels of EdU/HU read density upstream (− 2 k) or downstream (2 k) of the gRNA-CMIP binding sites in early S phase (c) or G1 phase (e) K562 cells. The read density was calculated in 2-kb bins. Student’s t-test, p = 0.025; *, p < 0.05; n.s., no significance. f ChIP-qPCR showing the distribution of MCM5 surrounding dCas9-binding sites in CMIP. A biological replicate of MCM5 ChIP-qPCR and the other two biological replicates are shown in Additional file 1: Figure S6i. t-test, mean ± SD. “Up” or “down” indicates the upstream or downstream non-transcribed regions of CMIP, respectively. The other five positions are the same as those shown in b and d. g Schematic of the transcription bulldozing model. Left: in the G1 phase, MCM double hexamers (MCM-DHs) loaded by ORC in transcribed regions are driven along gene bodies by RNA polymerase II to downstream non-transcribed regions. Accumulated MCM-DHs in non-transcribed regions initiate DNA replication in the early S phase. Right: when transcription is inhibited, MCM-DHs accumulate around the ORC binding site and initiate DNA replication at gene bodies
Fig. 6
Fig. 6
Relocated early replication initiation induces DNA damage. a Schematic showing DRB treatment of G1 or early-S phase K562 cells; a detailed description is given in the “Methods” section. The blue arrows show the samples that are presented in b and c. b Detection of nuclei-resident γ-H2AX foci in G1-arrested or early S phase K562 cells following transcription re-initiation via withdrawal of DRB. c Intensity of nuclei-resident γ-H2AX foci following transcription re-initiation via withdrawal of DRB in G1-arrested or early S phase K562 cells. Representative images are shown in b. t-test; ***, p < 0.001. d Detection of γ-H2AX foci induced by DRB withdrawal with or without RNase H1 (GFP-RNH1) overexpression. The white triangle indicates early S phase cells with GFP or GFP-RNH1 overexpression at 3.5 h after DRB withdrawal. Of note, the antibody against γ-H2AX is different from the one in b. e The number of nuclei-resident γ-H2AX foci with or without RNase H1 overexpression in the G1 or early S phase before or after withdrawal of DRB. t-test; n.s., no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001
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