Expressing the G-quadruplex-specific single-chain antibody HF1 in human cells (Fernando et al., 2009) changed the steady-state mRNA levels of 1,767 genes in a manner correlating with predicted G-quadruplex motifs near promoters and transcription end sites, establishing that quadruplex recognition has physiological consequences but leaving the mechanism, the direct-versus-indirect target distinction, the in-vivo structure and chromatin dependence of folding, and the causal role of endogenous unperturbed G-quadruplexes unresolved. Subsequent work mapped endogenous G-quadruplex structures genome-wide by antibody-based chromatin immunoprecipitation sequencing (ChIP-seq) and cleavage-under-targets-and-tagmentation (CUT&Tag) mapping, identified many cellular G-quadruplex-interacting proteins and G4-resolving helicases, and demonstrated structure-level causal regulation at individual loci by CRISPR abrogation (notably the MYC promoter G-quadruplex). What remains open is the genome-scale dissection: quantifying which genes are directly regulated by endogenous G-quadruplex formation, through which transcriptional steps and protein mediators, and under which chromatin-context rules, without relying on exogenous G-quadruplex binders whose effects are now known to be confounded.

Background

A G-quadruplex (G4) is a four-stranded nucleic acid secondary structure formed by guanine-rich sequences, in which four guanines hydrogen-bond into a planar tetrad and stacked tetrads are stabilized by monovalent cations such as potassium. Sequence motifs with quadruplex-forming potential (PQS, e.g. runs of three or more guanines repeated four times) are computationally predictable, and in the human genome they are statistically enriched in gene regulatory regions, particularly near transcription start sites (TSS, where transcription is initiated) and transcription end sites (TES, where termination occurs). Whether these predicted structures actually fold in cells and regulate transcription is a long-standing question in genome biology. Fernando, Sewitz, Darot, Tavaré, Huppert and Balasubramanian (2009) addressed it with a chemical-genomics experiment: they expressed HF1, a single-chain variable fragment (scFv) antibody selected to bind G4 DNA selectively over double-stranded DNA, in the human gastric cancer cell line HGC-27 and profiled mRNA by microarray. Of 18,105 protein-coding genes, 1,767 were differentially expressed (973 up, 794 down), and differentially expressed genes were significantly enriched for PQS in the 200 bp around the TSS and, with marked strand and position asymmetry, around the TES. The effects were strand-symmetric near promoters, which the authors took to suggest a DNA-level rather than RNA-level mechanism, and both up- and down-regulation occurred. Crucially, the study could not distinguish genes directly perturbed by local HF1-G4 binding from secondary effects of differentially expressed transcription factors, could not resolve whether G4 binding interferes with transcription factor binding, initiation, elongation, termination, or 3'-end processing, did not determine which predicted motifs actually fold in chromatin, and, as the authors stated explicitly, could not define the role of natural quadruplex-forming elements in the absence of the exogenous antibody binder. Later work transformed the empirical landscape. Hansel-Hertsch et al. (2016) introduced G4 ChIP-seq (chromatin immunoprecipitation followed by high-throughput sequencing), using the structure-specific BG4 antibody (a phage-display-derived single-chain antibody selected to bind G4 structures) on isolated chromatin (not expressed in situ), and mapped roughly 10,000 endogenous G4 structures in human cells, found predominantly in regulatory, nucleosome-depleted regions (nucleosomes are the basic DNA-histone packaging units; their positioning modulates DNA accessibility) and enriched at promoters of highly transcribed genes. Lyu et al. (2022) adapted CUT&Tag (cleavage under targets and tagmentation, an antibody- or nanobody-guided mapping method with lower background than ChIP-seq) to G4s and showed G4 formation at active promoters and enhancers in mouse embryonic stem cells, with G4 structures persisting independently of ongoing transcription. Resources such as the EndoQuad database (Qian et al., 2024) now compile experimentally validated endogenous G4 loci, and G4-interacting proteins are being identified in living cells (Lu et al., 2024), including G4-resolving DEAH-box helicases (a family of ATP-driven nucleic-acid unwinding enzymes) such as DHX36 and DHX9 (Karam et al., 2024; Gumina et al., 2024). On the causality front, Esain-Garcia et al. (2024) used CRISPR editing to abrogate folding of the endogenous MYC promoter G4 without deleting the region: loss of the structure (not the sequence) reduced MYC transcription through de novo nucleosome deposition and altered RNA polymerase II recruitment, and swapping in the KRAS G4 restored transcription, a direct single-locus demonstration that a G4 structure is a positive cis-regulatory element. Analogous targeted approaches followed (Qin et al., 2024; Obi et al., 2025; Roy et al., 2023 for synthetic insertions). At the same time, the interpretation of perturbation data has become more cautious: the clinically advanced G4-binding ligand CX-5461 was shown to be a potent collateral mutagen (Koh et al., 2024), so small-molecule-based evidence for G4 function carries G4-independent confounds, and a live 2025 controversy concerns whether G4-targeted CUT&Tag signal is inflated by chromatin accessibility (Thompson and Byrd, 2025, versus Melidis et al., 2025). Critical reviews (Johnson, 2020) emphasize that much of the evidence for endogenous G4 function remains associative. Thus the field has moved from correlation to single-locus causality, but a genome-scale causal and mechanistic account of endogenous G4 regulation of transcription is still missing.

Problem Statement

Establish, at genome scale in human cells and without reliance on exogenous G-quadruplex-binding perturbagens, the causal regulatory role of endogenous DNA G-quadruplexes in transcription. The single overarching objective is a causal-mechanistic account of endogenous G4-dependent transcriptional regulation, jointly supported by four linked results: (1) a direct-target map — the set of genes whose transcription is directly controlled by local endogenous G4 formation, with direct effects distinguished from indirect secondary cascades (for example via differentially expressed transcription factors), so that the direct fraction of G4-dependent expression changes is quantified; (2) a mechanistic partition — for the direct targets, which step(s) of the transcription cycle are affected (initiation, elongation, termination, or 3'-end processing) and which cellular proteins mediate the effect (for example G4-binding proteins, G4-resolving helicases, transcription factors, chromatin modifiers); (3) context rules — how chromatin context (for example nucleosome positioning, accessibility, histone modifications) and the endogenous folded conformation of each motif determine whether a sequence with quadruplex-forming potential actually forms a regulatory G4 in vivo and whether the regulatory outcome is activation or repression, including the relative contribution of DNA-level versus RNA G-quadruplex mechanisms at promoter-proximal sites; and (4) causal demonstration — evidence that removing and restoring endogenous G4 formation at the level of structure (not merely sequence) changes transcription in the predicted direction, generalizing beyond individual demonstration loci such as MYC. By genome scale is meant coverage of a substantial fraction of endogenous G4 loci across the genome in one or more human cell types, as opposed to single-locus or purely association-level studies; the deliverables are scientifically interdependent, since the direct-target map defines the genes over which the mechanistic partition and context rules are to be established, and the causal demonstration validates the structure-level interpretation of all three.

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