Dimeric G-quadruplex motifs-induced NFRs determine strong replication origins in vertebrates

Replication of vertebrate genomes is tightly regulated to ensure accurate duplication, but our understanding of the interplay between genetic and epigenetic factors in this regulation remains incomplete. Here, we investigated the involvement of three elements enriched at gene promoters and replication origins: guanine-rich motifs potentially forming G-quadruplexes (pG4s), nucleosome-free regions (NFRs), and the histone variant H2A.Z, in the firing of origins of replication in vertebrates. We show that two pG4s on the same DNA strand (dimeric pG4s) are sufficient to induce assembly of an efficient minimal replication origin without inducing transcription. Dimeric pG4s in replication origins trigger formation of an NFR next to precisely positioned nucleosomes enriched in H2A.Z on this minimal origin and genome-wide. Thus, our data suggest a crucial role for dimeric pG4s in the organization and duplication of vertebrate genomes. It supports the hypothesis that a nucleosome close to an NFR is a shared signal for the formation of replication origins in eukaryotes. A Wt DT40 chicken cell line and clonal heterozygous or homozygous cell lines containing mutated version of the βA-globin fused to the IL2R gene and SV40 PolyA sequence, flanked with two USF binding sites inserted into a mid-late replicating region of chromosome one (chr1:71,981,290-73,532,377 bp, galGal5) were analyzed. Profiles of the histone variant H2AZ enrichments were obtained by chromatin immunoprecipitation DNA-sequencing (ChIP-seq). NFR and Nucleosome coverage profiles were obtained from MNAse-Seq and ATAC-seq experiments respectively. Isolated and clustered pG4 profiles were obtained from datas published in Zheng et al. 2020 under the accession number GSE133379 (supplementary files). We also considered G4s detected by ChIPSeq with a G4P protein (Zheng et al. 2020, GSE133379) for human 293T cells (GSM3907020,GSM3907021, GSM3907022, GSM3907023); human A549 cells (GSM3907012, GSM3907013, GSM3907014, GSM3907015); human H1975 cells (GSM3907016, GSM3907017, GSM3907018, GSM3907019); human HeLa-S3 cells (GSM3907024, GSM3907025), chicken DF-1 cells (GSM3907028, GSM3907029) and mouse 3T3 cells (GSM3907026, GSM3907027). Human ChIP-G4s were combined. Coverage plots represent the average number of genomic features in sliding windows of 20 bp, overlapping by 10 bp. Bed Graph files were generated.