Prorok P et al. (2019), Involvement of G-quadruplex regions in mammalia...

XB-ART-56148

Nat Commun 2019 Jul 22;101:3274. doi: 10.1038/s41467-019-11104-0.

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Involvement of G-quadruplex regions in mammalian replication origin activity.

Prorok P , Artufel M , Aze A , Coulombe P , Peiffer I , Lacroix L , Guédin A , Mergny JL , Damaschke J , Schepers A , Cayrou C , Teulade-Fichou MP , Ballester B , Méchali M .

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Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation.

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Species referenced: Xenopus laevis
Genes referenced: actb h2bc21 ids kidins220 rai1

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	Fig. 1. Creation of an ectopic DNA replication origin. a Isothermal differential spectra (IDS; left panel) and circular dichroism spectra (CD; right panel) of potential OGRE/G4 sequences fond in the vicinity of replication origins. All tested sequences form G4 structures, as indicated by the strong negative peak around 295 nm and the two positive peaks around 240 and 273 nm (for IDS), and the strong positive peak around 260 nm (CD). The sequences are provided in Supplementary Table 1. b Ori1 that contains an OGRE/G4 element 240 bp upstream of the DNA replication initiation site (IS) was inserted by Cas9-stimulated homologous recombination into an origin-free region on chromosome 11 in NIH 3T3 mouse cells. The insertion of the 1907 bp fragment (marked in violet) occurred thanks to the two 500 bp homology arms (orange and green) present on the insertion template. The position of the primers (P) (sequences in Supplementary Table 2) used for the analysis of origin activity is also shown. c Ori1 activity in parental (control in black) and recombinant NIH 3T3 cells (in grey). As expected, a two-fold increase in DNA replication activity was detected in recombinant cells compared with parental cells, whereas the external origin Ori2 exhibited the same replication activity in both cell lines. Note that SNS activity was also detected at the 5′ and 3′ junctions of the insertion site, but not in the corresponding control regions. The background control regions Bcgd1 and 2 are located in origin-free regions; results are the mean ± SD of 3 independent experiments; p values were obtained using the two-tailed Student’s t test; p ≤ 0.05, p > 0.05. d Analysis of DNA replication, using the DpnI digestion method and colony counting (“Methods” section), in an EBV episomal plasmid transfected in HEK293 cells that express EBNA1. DNA replication activity was assayed using EBV episomal plasmids that carry or not (Delta) the OriP origin, or a 500 bp fragment of Ori2 containing an OGRE/G4 element in the sense or antisense orientation. Results are the mean ± SD of 3–7 independent experiments; p values were obtained using the two-tailed Student’s t test; p ≤ 0.05; ns not significant, p > 0.05
	Fig. 2. OGRE/G4 deletion strongly decreases the DNA replication activity of an endogenous origin. a The OGRE/G4 sequence of an endogenous origin (Ori1) was deleted and the deletion was confirmed using a restriction site close to the targeted sequence (see “Methods” section). b G4 formation propensity profiling of the Ori1 sequence targeted for deletion. The Ori1 sequence is located on chromosome 11 and presents a strong peak in the G4-Hunter score profile (red line). Such peak is not present upon OGRE/G4 deletion (alleles 1 and 2, blue and green dotted lines, respectively), and no point above 1 or below −1 is observed. This argues against the probability of G4 formation at this mutated locus. The striped lines on the top indicate the extent of the deletion in allele 1 (red and blue) and allele 2 (green and blue). c Nascent strand enrichment of Ori1 in parental NIH 3T3 cell line (black) and in mutant clones with the deletion (grey). Replication activity was strongly decreased after deletion of the OGRE/G4 sequence, whereas the activity of the external origin (Ori 2) did not vary. The background control regions Bcgd1 and 2 are located in origin-free regions. Results are the mean ± SD of 3 independent experiments; p values were obtained using the two-tailed Student’s t test; p ≤ 0.05; p ≤ 0.01; *p ≤ 0.001, p > 0.05. d Deletion of the OGRE/G4 did not affect the transcription level of the Rai1 gene, associated with Ori1. As a control, the housekeeping genes Actb and Gapdh were used. Results are the mean ± SD of 3 independent experiments; p values were obtained using the two-tailed Student’s t test; p > 0.05. Primer sequences are in Supplementary Table 2. e DNA replication activity was assessed as in Fig. 1c with the EBV origin, or with the 500 bp OGRE/G4 element of Ori2, or after scrambling or deletion of the same OGRE/G4 sequence. Results are the mean ± SD of 4–5 independent experiments; p values were obtained using the two-tailed Student’s t test; p ≤ 0.05; **p ≤ 0.01; p > 0.05. Note that data presented for episome containing delta oriP and OGRE/G4-containing origin were performed independently from results presented in the Fig. 1; ns not significant: p > 0.05
	Fig. 3. Changes in the replication origin repertoire upon G4 stabilization by PhenDC3. a PhenDC3 formula. b Volcano plot of origins affected by incubation of mouse ES cells with PhenDC3. After identification of the bound sites in all SNS-seq samples, differential binding analysis was performed. For each origin, the corrected p values (false discovery rates, −log10(FDR)) and the log2 fold change (FC) of control and PhenDC3-treated samples were plotted. The horizontal and vertical lines correspond to the thresholds for detecting differential origins. On the basis of the FC and peak reproducibility, origins were classified in five different classes, according to PhenDC3 effect (suppressed, reduced, reinforced, new, and insensitive), as described in “Methods” section. c Examples of the activity of origins in the indicated classes after incubation with PhenDC3 or in control cells. The corresponding genomic region is indicated and the origin color is according to the corresponding class in the Volcano plot. d Heatmap showing the read densities in origins affected by G4 stabilization (PhenDC3-treated vs. Control). The heatmap indicates the signal strength (number of reads) and density around each origin and was performed on 7 kb regions on each side of origins, as previously described4. The intensity (brown) is proportional to the read counts per 100 bp bins. Origins were sorted on the basis of the FC in signal strength. e Activity of origins (reads number) in each class in control (−) and PhenDC3-treated (+) cells
	Fig. 4. Nature of the OGRE/G4 in the different origin classes. a De novo motif found as the most representative in the new origin class using the RSAT suite60. For motifs found in the other classes see Supplementary Fig. 5A. b Fraction of OGRE/G4 sequences in function of the distance from the IS. The OGRE/G4 motif forms a relatively sharp peak upstream the IS at an average distance of 250 bp in all origin classes, but for the suppressed class. The CL95% is shown in pink. The fraction of OGRE/G4 sequences in shuffled regions and their CL 95% is shown in yellow and light yellow, respectively. c Isothermal differential spectra (IDS; upper panel) and circular dichroism spectra (CD; lower panel) of potential OGRE/G4 sequences associated with the new class of replication origins. All tested sequences form G4 structures, as indicated by the strong negative peak around 295 nm and the two positive peaks around 240 and 273 nm (for IDS, top panel), and the strong positive peak around 260 nm by CD (bottom panel). The IDS suggest that the tested sequences form predominantly G4 in parallel conformation. The possibility to adopt alternative folds, such as anti-parallel G4 structures, for some sequences is indicated by a minor peak around 295 nm. The sequences are provided in Supplementary Table 1. d Association of origins with OGRE/G4 motifs in the different classes. Insensitive, new, enforced and reduced origins are mainly G4-associated, but not suppressed origins. e FRET competition assays in which stabilization (∆T1/2, in °C) of the human telomeric quadruplex F21T by 0.5 µM PhenDC3 was analyzed in the absence (black bars), or in the presence of G-rich sequences from insensitive (upper panel) and new (lower panel) origins (3 or 10 µM strand concentration; dark green and light green bars, respectively), of positive (22Ag, 1XAV, both forming G4 structures), and negative (ds26 and dT30 are double- and single-stranded controls, respectively) control sequences. The means were obtained in independent expreiments ± SD. Efficient competition by quadruplex-forming oligonucleotides is evidenced by a sharp drop in stabilization. The origin oligonucleotide sequences are provided in Supplementary Table 1
	Fig. 5. Transcription and epigenetic landscape in the different origin classes. a Venn diagrams showing the origin distribution between transcribed and intergenic regions in Control and PhenDC3-treated mES cells, and random distribution (Genome). Replication initiation sites are enriched in gene regions. b Genomic localization of the different classes of origins relative to transcription. Downregulated origins (suppressed and reduced) are mainly located at promoters. Random origins (dotted lines) are equally distributed in transcription-related regions. c Epigenetic marks associated with the different origin classes. All tested open chromatin marks were enriched around reduced and suppressed origins. New and reinforced origins were located mainly in highly methylated regions. d GSEA analysis of origins situated at promoters (TSS ± 2 kb, left panels) or in transcribed regions (TSS + 10 kb, right panels) for each class. A plot is drawn for each gene set. The x-axis of each plot represents differentially expressed genes ranked from upregulated (on the left) to downregulated (on the right). The enrichment score is indicated on the y-axis. The black horizontal bar indicates the genes present in the gene set. The highest enrichment score indicates the enrichment. If this score is on the left, the enrichment is higher for upregulated genes; if it is on the right, the enrichment is higher for the downregulated genes. Origins associated with the TSS follow the transcription changes upon G4 stabilization, whereas origins located in transcribed regions are insensitive to changes in transcription levels. At gene promoters, GSEA results show a significant association of downregulated genes with the suppressed origin class (after multi-testing correction using the Benjamini–Hochberg method, adjusted p < 0.03). The other origin classes show a similar trend, without reaching significance
	Fig. 6. At the activation step of DNA replication, OGRE/G4 elements compete for dsDNA but not for ssDNA replication. a Schematic representation of the replication kinetics of sperm nuclei in X. laevis low-speed egg extracts (LSE) in the presence of competing double-stranded (ds) oligonucleotides. LSEs were pre-incubated with competing oligonucleotides, sonicated salmon sperm DNA, or ultrapure H2O (mock) at 22 °C for 5 min. b Average DNA replication efficiency (mean + SD) of LSEs pre-incubated with competing oligonucleotides or controls (n = 6 for mock/salmon sperm DNA pre-incubated extracts, n = 3 for mock/random oligonucleotides). Total incubation time was 2 h. P values were obtained using the two-tailed Student’s t test; ****p < 0.0001, p > 0.05. c Replication kinetics of ssM13 complementary DNA strand synthesis in X. laevis high-speed egg extracts (HSE) pre-incubated with the indicated competing oligonucleotides or H2O (mock). d Competition by OGRE/G4 oligonucleotides does not affect pre-RC formation. Sperm nuclei were added to HSEs incubated with H2O (mock), sonicated salmon sperm DNA (S.Sperm), or the indicated oligonucleotides. Chromatin was isolated and immunoblotted with the indicated antibodies. Histone H2B level was used as loading control. e, f Competition by OGRE/G4 oligonucleotides affects DNA replication activation. Time-course analysis of replication initiation factor recruitment to chromatin after addition of sperm nuclei to LSEs pre-incubated with H2O (mock), sonicated salmon sperm DNA (S. sperm), or competing oligonucleotides. At the indicated time points, chromatin was isolated and immunoblotted with the indicated antibodies; ns not significant
	Fig. 7. G4 function in the DNA replication initiation. Based of the position of the OGRE/G4 that we already reported2 and the corresponding model3, we know that the OGRE/G4 element is always upstream of the initiation site itself, either on the + or the − DNA strand (left and right upper panels). In the corresponding lower panels, based on our present data, we confirm again the position of the G4 element relative to the initiation site. The position of the G4 might fit with the position of the preRC, but this does not obligatory imply it will recruit some factors to the pre-RC. First, G4 can adopt several types of structure which themselves might regulate the folding or the replication origin region. Second, G4 might play a role in the removal of the nucleosome positioned at the initiation site itself, a process necessary to load the replication machinery, and therefore regulate activation of DNA replication. Third, because G4 are themselves nucleosome-free regions, they could facilitate DNA helix opening at the initiation site

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