CpG islands (CGI) are genomic regions associated with gene promoters and involved in gene expression regulation. Despite their high CpG content and unlike bulk genomic DNA methylation pattern, these regions are usually hypomethylated. So far, the mechanisms controlling the CGI methylation patterning remain unclear. G‐quadruplex (G4) structures can inhibit DNA methyltransferases 1 enzymatic activity, leading to CGI hypomethylation. Our aim was to analyse the association of G4 forming sequences (G4FS) and CGI methylation as well as to determine the intrinsic and extrinsic characteristics of G4FS that may modulate this phenomenon. Using methylation data from human embryonic stem cells (hESCs) and three hESC‐derived populations, we showed that hypomethylated CpGs located inside CGI (CGI/CpG) tend to be associated with highly stable G4FS (Minimum free energy ≤ −30 kcal·mol−1). The association of highly stable G4FS and hypomethylation tend to be stronger when these structures are located at shorter distances (~ < 150 bp) from GCI/CpGs, when G4FS and CpGs are located within open chromatin and G4FS are inside CGI. Moreover, this association is not strongly influenced by the CpG content of CGI. Conversely, highly methylated CGI/CpG tend to be associated with low stability G4FS. Although CpGs inside CGIs without a G4FS tend to be more methylated, high stability G4FS within CGI neighbourhood were associated with decreased methylation. In summary, our data indicate that G4FS may act as protective cis elements against CGI methylation, and this effect seems to be influenced by the G4FS folding potential, its presence within CGI, CpG distance from G4FS and chromatin accessibility.

Maturation of mRNA in humans involves modifying the 5′ and 3′ ends, splicing introns, and installing epitranscriptomic modifications that are essential for mRNA biogenesis. With respect to epitranscriptomic modifications, they are usually installed in specific consensus motifs, although not all sequences are modified suggesting a secondary structural component to site selection. Using bioinformatic analysis of published data, we identify in human mature-mRNA that potential RNA G-quadruplex (rG4) sequences colocalize with the epitranscriptomic modifications N 6 -methyladenosine (m 6 A), pseudouridine (Ψ), and inosine (I). Using the only available pre-mRNA data sets from the literature, we demonstrate colocalization of potential rG4s and m 6 A was greatest overall and occurred in introns near 5′ and 3′ splice sites. The loop lengths and sequence context of the m 6 A-bearing potential rG4s exhibited short loops most commonly comprised of single A nucleotides. This observation is consistent with a literature report of intronic m 6 A found in SAG (S = C or G) consensus motifs that are also recognized by splicing factors. The localization of m 6 A and potential rG4s in pre-mRNA at intron splice junctions suggests that these features could function together in alternative splicing. A similar analysis for potential rG4s around sites of Ψ installation or A-to-I editing in mRNA also found a colocalization; however, the frequency was less than that observed with m 6 A. These bioinformatic analyses guide a discussion of future experiments to understand how noncanonical rG4 structures may collaborate with epitranscriptomic modifications in the human cellular context to impact cellular phenotype.

Sequences of DNA typically adopt B-form duplexes in genomes, although noncanonical structures such as G-quadruplexes, imotifs, Z-DNA, and cruciform structures can occur. A challenge is to determine the functions of these various structures in cellular processes. We and others have hypothesized that G-rich G-quadruplex-forming sequences in human genome promoters serve to sense oxidative damage generated during oxidative stress impacting gene regulation. Herein, chemical tools and a cell-based assay were used to study the oxidation of guanine to 8-oxo-7,8-dihydroguanine (OG) in the context of a cruciformforming sequence in a gene promoter to determine the impact on transcription. We found that OG in the nontemplate strand in the loop of a cruciform-forming sequence could induce gene expression; conversely when OG was in the same sequence on the template strand, gene expression was inhibited. A model for the transcriptional changes observed is proposed in which OG focuses the DNA repair process on the promoter to impact expression. Our cellular and biophysical studies and literature sources support the idea that removal of OG from duplex DNA by OGG1 yields an abasic site (AP) that triggers a structural shift to the cruciform fold. The AP-bearing cruciform structure is presented to APE1, which functions as a conduit between DNA repair and gene regulation. The significance is enhanced by a bioinformatic study of all human gene promoters and transcription termination sites for inverted repeats (IRs). Comparison of the two regions showed that promoters have stable and G-rich IRs at a low frequency and termination sites have many AT-rich IRs with low stability.