Fig 1: c-MYC G1/S phase RNAs interact with ORC1.a Upper panel: Schematic of the c-MYC locus, 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 c-MYC origin detected by ChIP - qPCR analysis. Bars indicate the mean ± SD of three independent biological replicates (n = 3), paired Student’s t test (two-tailed), **P value = 0.0085 (ORC1) and **P value = 0.0055 (H2A.Z). b RNAs originating within the promoter of c-MYC, measured for K562 synchronized in G1 and S phase: S1 (2 h), S2 (5 h). Bars show mean ± SD of three independent biological replicates (n = 3), unpaired Student’s t test (two-tailed), *P value < 0.0001 (G1 vs WT), P value < 0.0001 (S1 vs WT) and P value = 0.00189 (S1 vs S2). c RIP - qRT-PCR performed using two ORC1 antibodies (ab85830 and ab60). Bars indicate mean ± SD correspond to two technical replicates (n = 2). d–f In vitro RNA/DNA electrophoresis mobility shift assay and RNA pull-down assays. d RNA secondary structures of probes overlapping c-MYC origin (RM9) and unrelated origin coordinates (MYC1, MYC2, and MYC3) were calculated using RNAfold. e RNA EMSA showing the preferential binding of ORC1 to RM9. 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 three times with similar results; Protein marker (M, KDa): Precision Plus Protein™ (Bio-Rad, 1610374). g Molecular dynamics simulations: frontal views of the ORC1a, ORC1b, and ORC1c structures 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 the 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 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. Source data are provided as a Source Data file.
Fig 2: ORC1 engagement of H2A.Z by ANCORs controls DNA replication.a Schematic showing cell synchronization in the G1/S phase by double thymidine block. The growth medium was supplemented with EU at the indicated time points: S1 (2 h) and S2 (5 h). EU-labeled nascent RNA was bound to biotin by Click-it chemistry reaction, isolated by streptavidin beads, and sequenced. b Heatmap showing gene expression profiles of K562 at S1 and S2. Welch’s t test: P value = 0.02993. c Workflow employed to predict early replication origins through the integration of 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. Overlap between the two genomic interval sets was assessed by interval intersection, and the significance of the overlap was evaluated using a Fisher’s exact test (hypergeometric framework). Lower panel: Annotation of the predicted early DNA replication origins located within +/−100 kb, +/−50 kb, +/−20 kb, +/−10 kb, +/−5 kb, and +/−2 kb 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 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 h after DRB treatment and every hour upon DRB reversal over the following 3 h. f Reversible downregulation of c-MYC ANCOR and c-MYC mRNA expression by qRT-PCR upon DRB treatment. Bars indicate mean ± SD for three independent biological replicates (n = 3). A paired Student’s t test (two-tailed), ***P value = 0.0007 (c-MYC ANCOR) and ***P value = 0.0006 (c-MYC mRNA). g Flow cytometry analysis showing nonreversible inhibition of nasDNA synthesis at the selected time points. h DNA abundance within the c-MYC locus decreases upon DRB treatment within the region encompassing the origin (7–8 and 9–10). Bars indicate mean ± SD of two technical replicates (n = 2). Source data are provided as a Source Data file.
Fig 3: Impaired ORC1 engagement and loss of H2A.Z upon inhibition of ANCORs.a Heatmaps showing a decrease in ORC1 enrichment by RIP-seq (a) and H2A.Z enrichment by ChIP-seq (b) 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 < 2.2e‒16; Welch’s t test, P: 4.473e‒14. c Box plots representing normalized read counts calculated for (a, b) using bamliquidator. Differences in normalized read counts for ORC1-RIP (left panel, P < 2.2e‒16) and H2A.Z-ChIP (right panel, P: 4.473e‒14) at 46,360 predicted early DNA replication origins upon DRB treatment as compared to DMSO mock control in K562 cells were assessed by Welch two-sample t test, two-sided was calculated. Multiple-comparison adjustment: none. Box plots show the median (center line), 25th and 75th percentiles (box), and whiskers extending to the most extreme values within 1.5 × IQR; overlaid points represent individual observations. 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. The 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 (2 hr) and S2 (5 hr) centered on ANCOR-predicted replication origins within +/−10 kb of the transcription start site (TSS). The higher transcriptional activity in S1 supports the association of ANCOR-associated loci with early DNA replication origins. Significance of figures is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns, not significant.
Fig 4: Gene expression dynamics and ORC1/H2A.Z redistribution at early replication origins following DRB treatment.a, b Heatmaps showing gene expression profiles of K562 at S1 (2 h) and S2 (5 h) of genes with high-confidence predicted early DNA replication origins within +/− 10 kb from TSS (N = 6913). Genes were stratified into two subgroups based on log2(Fold Change): a increased expression at S1 (log2(Fold Change) S1 vs. S2 > 1; n = 3545). Welch two-sample t test, two-sided was employed, obtaining P: 9.739e‒05 and b decreased expression at S1 (log2(Fold Change) S1 vs. S2 < 1; n = 3368). Welch two-sample t test, two-sided was employed, obtaining P: 2.657e‒12. Expression levels (log2(FPKM) values) for each subgroup were plotted, illustrating transcriptional dynamics of early replication origin–associated genes. Heatmaps showing decreased ORC1 recruitment by RIP-seq (c) and H2A.Z enrichment by ChIP-seq (d) at high confidence 19,230 predicted DNA replication origins upon DRB treatment versus DMSO. Box plots representing normalized read counts calculated for (a, b) using bamliquidator. Differences in normalized read counts for ORC1-RIP (e), P: 0 and H2A.Z-ChIP (f), P: 0 at high confidence 19,230 predicted DNA replication origins upon DRB treatment versus DMSO mock control in K562 cells were assessed by Welch two-sample t test. Two-sided was calculated. Multiple-comparison adjustment: none. Box plots show the median (center line), 25th and 75th percentiles (box), and whiskers extending to the most extreme values within 1.5 × IQR; overlaid points represent individual observations. Significance of figures is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns, not significant.
Fig 5: Structural and functional characterization of ORC1-RM9 interactions and ORC1, RM9, H2A.Z in vitro reconstitution.a RM9 interacting with ORC1a residues highlighted in pink. ARG297, SER450, GLU575, THR593, and ARG846 predominantly mediate the interaction during the simulation. The molecules are colored as follows: white: disordered region (residues 201–479), marine: AAA+ domain (residues 481–782), red and green: RM9 with native ORC1 and RM9 with mutated proteins, respectively. The mutated residues are displayed in sticks and colored in magenta. b Schematic representation of the structural domains of ORC1 (top panel). Zoomed-in view highlighting the ORC1 amino acid residues in the lateral pocket in interaction with RM9 (bottom panel). c Light Shift RNA EMSA with biotinylated RM9 (20 pmol), showing preferential binding of ORC1 to the Intrinsically Disordered Region (IDR; residues 201–782, lane 1), with no detectable binding to the BAH domain (residues 1–200, lane 2) or the Winged-Helix Domain (WHD; residues 783–861, lane 3). A mutant ORC1 protein (R297E, S450Y, T593Y, R846E) was also tested (lane 5), showing no binding. This REMSA experiment was performed three times. d RNA EMSA of in vitro reconstituted complexes containing ANCOR probe (RM9), ORC1, and recombinant nucleosomes containing H2A.Z (Active Motif, Cat. No. 81072). Free RNA probe (RM9) was loaded in control lanes 1 and 5. The RM9 was incubated with nucleosome containing H2A.Z alone, or with ORC1 alone, lanes 2 and 3, respectively. A specific complex formation is observed when H2A.Z, ORC1, and RM9 are together (lanes 4 and 6). Addition of anti-H2A.Z (lane 7) and anti-ORC1(lane 8) increases or decreases the intensity of the signal, consistent with antibody-dependent stabilization or perturbation of ORC1-RM9-H2A.Z assembly. e Western blot analyses used to validate the quality of the recombinant proteins employed for the RNA EMSA assay. The protein marker (M, KDa) is indicated in the figure and corresponds to Precision Plus Protein™ (Bio-Rad, 1610374). The ORC1 and recombinant nucleosomes containing H2A.Z were tested both separately and in combination to eliminate the possibility of mutual interference. Source data are provided as a Source Data file.
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