DNA G-quadruplexes (G4s) in the CpG islands of the mouse Dele and Cdc6 genes activate luciferase reporter transcription, and guanine mutations that disrupt the G4 abolish this activation, yet the mechanism is unknown (Bay et al. 2017). It remains unresolved which transcription factors or regulatory proteins bind these G4s, whether activation proceeds by recruiting activator proteins, changing local DNA topology, or modulating promoter–enhancer communication, and why the G4-stabilizing ligand L1H1-7OTD inhibits reporter activation only when co-transfected with the plasmid. Within their full CpG islands the two G4s act oppositely — Dele G4 mutation lowers expression while Cdc6 G4 mutation raises it — suggesting context-dependent dual regulation, and all evidence still comes from episomal plasmids: endogenous G4 formation at the native loci and effects on endogenous gene expression have never been measured. Since 2021, genome-wide studies established that endogenous G4s act as transcription-factor binding hubs and can causally drive long-range enhancer interactions, and single-locus G4 causality has been demonstrated by CRISPR editing in yeast — but no study has returned to the Dele and Cdc6 loci. The problem is to dissect the mechanism endogenously at these loci: identify the regulatory proteins, establish the activation mode, define the cis-regulatory architecture of the Dele CpG island, determine when the Cdc6 G4 activates versus represses, and explain the ligand asymmetry.

Background

A G-quadruplex (G4) is a four-stranded DNA secondary structure formed by G-rich sequences, in which four guanines hydrogen-bond into a planar G-tetrad and two or more stacked tetrads are stabilized by a central cation such as potassium. G4-forming motifs are statistically enriched in gene regulatory regions, including promoters throughout the human genome (Huppert and Balasubramanian 2007). CpG islands (CGIs) are GC-rich genomic regions, typically located where transcription starts, in which the dinucleotide CpG is not depleted as it is elsewhere in the genome; a promoter is the DNA segment where RNA polymerase and transcription factors (TFs) assemble, an enhancer is a regulatory element that boosts promoter activity, and an insulator is a boundary element that can block enhancer–promoter communication. Most mechanistic work on promoter G4s concerns oncogene promoters such as c-MYC, where small-molecule stabilization of the G4 represses transcription (Siddiqui-Jain et al. 2002) and where specific proteins such as nucleolin were shown to bind the promoter G4 (Gonzalez et al. 2009); whether and how G4s can positively activate transcription remained far less clear (Bochman et al. 2012). Starting from a mouse CGI DNA microarray screen with a fluorescent G4 ligand that identified 1998 putative G4-forming sequences (Iida et al. 2013), Bay et al. (2017) confirmed G4 formation for ten sequences by circular dichroism (CD) spectroscopy, which reports G4 folding through characteristic ultraviolet absorbance signatures, and by dimethyl-sulfate footprinting, and then tested each sequence in luciferase reporter assays, where a cloned test sequence drives a firefly luciferase gene so that its regulatory activity is read out as enzyme activity. The Dele (death ligand signal enhancer) and Cdc6 (cell-division-cycle 6) G4 DNAs activated luciferase expression both upstream of a minimal promoter and in a promoterless construct, and guanine mutations disrupting the G-tetrads abolished the activation, indicating that the G4 structure itself carries enhancer- and promoter-like activity. Paradoxically, the telomestatin-derived macrocyclic heptaoxazole ligand L1H1-7OTD, which binds the top G-tetrad by aromatic π-stacking and stabilizes the G4 in CD melting experiments, inhibited the activation — but only when co-transfected with the plasmid, not when added to the medium a day after transfection. In full CGI constructs the two G4s behaved oppositely: mutating the Dele G4 lowered expression while mutating the Cdc6 G4 raised it. Because the Dele CGI hosts two divergently transcribed genes, Dele and 1700086O06Rik, the authors proposed (but did not demonstrate) that the CGI contains a Dele promoter with enhancer activity, a 1700086O06Rik promoter, and an insulator. The study concluded that these G4s are formed under physiological conditions in the cell and regulate transcription, yet every observation came from episomal reporter plasmids: endogenous G4 formation at the native loci and effects on endogenous gene expression were never measured, and the molecular mechanism of the activation was left open.

Problem Statement

Determine the molecular mechanism by which the G-quadruplexes in the Dele and Cdc6 CpG islands regulate transcription, at the native loci rather than on episomal reporter plasmids. Concretely, establish jointly: (1) which transcription factors or regulatory proteins bind the Dele and Cdc6 G4 structures in cells, and whether the activation these G4s produce arises from recruitment of activator proteins, from changes in local DNA topology, or from modulation of promoter–enhancer communication within the CpG island; (2) whether these G4s form in native chromatin at the endogenous Dele and Cdc6 loci under physiological conditions, and how specifically perturbing them changes endogenous expression of Dele, 1700086O06Rik and Cdc6, with controls that separate G4 structure from the underlying double-stranded sequence; (3) the cis-regulatory architecture of the Dele CpG island — the arrangement of the Dele promoter/enhancer, the 1700086O06Rik promoter, and the proposed insulator — and the conditions under which the Cdc6 G4 switches between its activating role (G4 sequence alone in a reporter) and its repressive role (G4 mutated within its full CpG island); and (4) the explanation for the ligand asymmetry — why co-transfected L1H1-7OTD inhibits reporter activation while post-transfection addition has no effect — distinguishing limited cellular or nuclear access of the ligand from ligand-occluded binding of transcription factors at the G4, given that the ligand stabilizes rather than disrupts the fold. These facets are one mechanistic question: an answer identifies the regulatory proteins acting at these G4s, shows at the endogenous loci how the G4 structure produces the observed transcriptional outcomes, defines the cis-regulatory architecture of the Dele CpG island including the conditions under which the Cdc6 G4 switches between activation and repression, and explains the ligand asymmetry.

References

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