RNAs anchoring replication complex control initiation and firing of DNA replication

Abstract

Coordinated initiation of DNA replication is essential to ensure efficient and timely DNA synthesis. Yet, molecular mechanism describing how replication initiation is coordinated in eukaryotic cells is not completely understood. Herein, we present data demonstrating a novel feature of RNAs transcribed in the proximity of actively replicating gene loci. We show that RNAs aNChoring ORC1 (ANCORs) to the histone variant H2A.Z are licensors of the DNA replication process. This ANCOR-H2A.Z interaction is essential for cells to initiate duplication of their genetic material. Widespread and locus-specific perturbations of these transcripts correlate with anomalous replication patterns and a notable loss of the H2A.Z replicative marker at the origin site. Collectively, we present a previously undescribed RNA-mediated mechanism that is associated with the generation of active replication origins in eukaryotic cells. Our findings delineate a strategy to modulate the origins of replication in human cells at a local and global level, with potentially broad biomedical implications.

Figure 1|. c-MYC G1/S phase RNAs interact with ORC1.

a. (Upper panel). Schematic of the c-MYC locus and respective primer sets indicated by numbers. The red bar indicates the c-MYC replication origin (region 9–10). (Bottom panel) ORC1 and H2A.Z enrichment at the c-MYC origin detected by chromatin immunoprecipitation (ChIP). The qPCR analysis was performed using distinct sets of primers designed as depicted in the schematic diagram provided above. The bars indicate the mean ± S.D of three independent experiments (n=3), Student’s t-test, **: p value 0.0085 (ORC1) and **: p value 0.0055 (H2A.Z); b Expression levels of RNAs originating within the promoter of c-MYC, G1 and later stages of the S phase: S1 (2hr), S2 (5hr). The bars represent the mean ± S.D of three independent experiments (n=3), Student’s t-test, *: p value <0.0001 (G1 vs WT), p value <0.0001 (S1 vs WT) and p value 0.00189 (S1 vs S2) c. RNA immunoprecipitation (RIP) qRT-PCR performed using two separate antibodies against human ORC1 (ab85830 and ab60). The association between c-MYC S-Phase RNAs anchoring ORC1 – ANCOR is shown d-f. In vitro RNA/DNA electrophoresis mobility shift assay (EMSA) and RNA pull-down assays. d. RNA secondary structures of the RNA probes corresponding to the c-MYC origin (RM9) and unrelated to the origin (MYC1, MYC2, and MYC3) calculated using RNAfold; e. RNA EMSA showing the preferential binding of ORC1 to RM9 (RNA) versus the unrelated MYC1 sequence and the homologous DNA sequence (dRM9); f. RNA pull down assay. Bound proteins were purified on streptavidin beads and immunoblotted with antibodies against ORC1 and H2A.Z. The experiment was repeated twice with similar results; g. Molecular dynamics simulations: frontal views of the representative structures of ORC1a, ORC1b and ORC1c states in complex with either dRM9 (DNA) or RM9 (RNA). RNA and DNA structures were derived using a RMSD-based clustering approach in the last half of simulation time. All molecules are color-coded as follows: orange: ORC1-BAH domain (residues 1–200); marine: ORC1 disordered region and AAA+ domain (residues 201–782); gray: orc1-WHD domain (residues 783–861); red and yellow: RM9 and dRM9, respectively; h. The figure shows a zoomed-in view of both the top and frontal perspectives, illustrating the preferred binding state of the ORC1-RM9 complex; i. Zoomed-in view showing the ORC1 amino-acid residues interacting with RM9.

Figure 2|. c-MYC ANCOR levels regulate DNA replication at the c-MYC locus.

a. Outline of the c-MYC locus is shown, indicating the position of the guide RNA-2 (gRNA-2) sequence, located 1.4 kilobases (kb) upstream of the c-MYC replication origin; b. Schematic representation of the doxycycline-responsive promoter (TET-ON) employed to clone gRNA-2 for the inducible CRISPR interference (CRISPRi) in K562 stably expressing dCas9-mCherry; c. Time-course analysis by quantitative reverse transcription-PCR (qRT-PCR) of scrambled control and gRNA-2 expression following a single 5 mM doxycycline addition; d. qRT-PCR analysis of c-MYC ANCOR and c-MYC expression over 6 days upon induction of gRNA-2 and scramble control with a single doxycycline addition. The mean ± S.D. of three independent experiments (n=3) is shown, Student’s t-test, ***: p value 0.0002 (c-MYC ANCOR) and **: p value 0.0021 (c-MYC mRNA); e. Quantification of nascent DNA (nasDNA) abundance at the c-MYC origin shows a significant decrease, 2.4- and 4.5-fold, at regions 7–8 and 9–10, respectively, encompassing the c-MYC origin in response to the suppression of c-MYC ANCOR. Measurements were taken at day 3 following induction of gRNA-2 and scramble control; f-g. CRISPR activator in HEK 293. f. gRNA-2 introduced in HEK 293 constitutively expressing VP64-dCas9 results in upregulation of c-MYC ANCOR and c-MYC mRNA. The mean ± S.D. of three independent experiments (n=3) is shown, Student’s t-test, *: p value 0.0161 (c-MYC ANCOR) and **: p value 0.0015 (c-MYC mRNA); g. Activation of c-MYC ANCOR was associated with an increase in newly synthesized DNA strands of 1.6- and 2-fold within regions 7–8 and 9–10, respectively, surrounding the c-MYC origin.

Figure 3|. ORC1 engagement of H2A.Z by G1/S phase RNAs mark early replication origins.

a. Outline of the experimental setup used to identify nascent RNAs. Upon synchronization in the G1/S phase by double thymidine block, the growth medium was supplemented with the EU RNA analog at the indicated time points: S1 (2 hr) and S2 (5 hr). EU-labeled nascent RNA was collected at S1 and S2 by Click-iT conversion for RNA sequencing. b. Heatmap showing gene expression profiles of K562 at S1 (2 hr) and S2 (5 hr). Welch’s t-test: p-value = 0.02993 c. Workflow employed to predict early replication origins through the integration of S-phAse RNAs aNChoring ORC1 (ANCOR), detected by RNA immunoprecipitation with anti-ORC1 antibody followed by sequencing (RIP-seq) and H2A.Z-enriched sites captured by Chromatin Immunoprecipitation sequencing (ChIP-seq); d. Upper panel, Venn diagram showing early DNA replication origins as predicted by the integration of ANCOR and H2AZ-enriched loci. Lower panel, annotation of the predicted early DNA replication origins located within +/−100kb, +/−50kb, +/−20kb, +/−10kb +/−5kb and +/−2kb of the transcription start site (TSS). A significant proportion of the predicted early DNA replication origins are within close proximity to promoters and/or TSS of protein coding genes; e. Schematic showing K562 synchronization in G1/S phase cycle followed by incorporation of BrdU into nasDNA and pharmacological inhibition of transcription using DRB (100 mM). Samples were collected 3 hr after DRB treatment and every hour upon DRB reversal over the following 3 hr; f. Reversible downregulation of c-MYC ANCOR and c-MYC mRNA expression by qRT-PCR upon DRB treatment and the reversal. The mean ± S.D. was calculated for three independent experiments (n=3). Student’s t-test, ***: p value 0.0006 (c-MYC mRNA) and **: p value 0.0223 (c-MYC ANCOR); g. Flow cytometry analysis showing nonreversible inhibition of nas-DNA synthesis at all selected time points; h. DNA abundance within the c-MYC locus significantly decreases upon DRB treatment only within the region encompassing the origin (7–8 and 9–10). i. Heatmap showing analysis of the K562 Repli-Seq signal in G1/G1b, S1, S2, S3, S4, and G2 cell cycle fractions (GSE34399) centered at the ANCOR-H2A.Z predicted 46,360 early DNA replication origins in K562; l. Heatmap showing analysis of Repli-Seq signal centered at the replication peaks (GSE34399) in K562 cells.

Figure 4|. Impaired ORC1 engagement and loss of H2A.Z upon inhibition of ANCORs.

a. Heatmap showing a decrease in ORC1 enrichment by RIP-Seq at 46,360 predicted early DNA replication origins upon DRB treatment as compared to DMSO mock control in K562 cells. Welch’s t-test: p-value < 2.2e-16; b. Heatmap showing a decrease in H2A.Z enrichment by ChIP-Seq at 46,360 early DNA replication origins in response to DRB treatment as compared to DMSO mock control in K562 cells. Welch’s t-test: p-value = 4.473e-14; c. Box plot showing a global reduction in ORC1-RIP (upper panel) and H2A.Z-ChIP signals upon treatment with DRB in K562; d. Genomic snapshots of the c-MYC and FXN loci depicting the impact of pharmacological inhibition of transcription using DRB on ORC1 and H2A.Z enrichment at the respective origin sites. Transcriptional profile of the c-MYC and FXN loci for the S1 and S2 time points are presented. The rose gold bar indicates the well-documented c-MYC origin [4, 6], and the predicted origin by the ANCORs association for FXN; e. Heatmap showing the transcriptional profiles of K562 at S1 and S2 centered on ANCOR-predicted replication origins within +/−10kb of the transcription start site (TSS). The higher transcriptional activity in S1 supports the association of ANCOR-associated loci with early DNA replication origins.

Figure 5|. DNA Replication dynamics is perturbed by inhibition of ANCOR levels.

a. Depicted the experimental design for DNA fiber analysis. K562 cells synchronized at G1/S phase by double thymidine block were treated with DRB for 3hrs upon release into the S phase, and sequential additions of 5-iodo-2′-deoxyuridine (IdU) and 5-chloro-2′-deoxyuridine (CldU) were used to follow the progression of replication forks during transcriptional inhibition; b. Quantification of DNA fibers at the “origin initiation” and “origin termination” in K562 DRB-treated cells as compared to DMSO; c. Quantification of DNA fibers during the two consecutive pulses with IdU and CldU, respectively for K562 DRB-treated cells compared to DMSO; d. Single molecule analysis of replicated DNA (SMARD) performed on FXN locus. Origin initiation, DNA replication fork progression (3’ to 5’ and 5’ to 3’), and origin termination were monitored upon DRB treatment and DMSO control. Percentage of molecules with IdU incorporation is indicated in the column chart below each respective condition.

Figure 6|. Proposed model for DNA origin formation.

Depicted a schematic of ORC1 conformational changes that enable binding to RNA and formation of the RNP-complex. The RNA engagement of ORC1 to the origin site through H2A.Z enables licensing of early DNA replication origins (left box). Disruption of c-MYC ANCOR transcription by CRISPRi (dCAS9), affects ORC1 engagement at replication origin decreasing nasDNA synthesis (middle box). By contrast, induced c-MYC ANCOR transcription by CRISPRa (VP64), increases ORC1 engagement at replication origin increasing nasDNA synthesis (right box)