A genome-scale mapping of 1,183 DNA-binding proteins around a consensus of promoter G-quadruplexes (Duan, Zheng, Hao and Tan, bioRxiv 2025) showed that nearly every protein is enriched, depleted, or in transition among three zones - the G-quadruplex itself, its proximal side, and its distal side - but the mapping is correlative. The open problem is to (i) determine the molecular mechanism that generates these spatial occupancy patterns - in particular whether promoter G-quadruplexes act as roadblocks or speed bumps that impede one-dimensional protein diffusion along DNA, how this couples to direct binding and inter-protein competition, and how it is modulated by the dynamic folding and unfolding of G-quadruplexes in cells - and (ii) establish whether particular occupancy patterns causally regulate transcription of individual genes.

Background

Guanine-rich single DNA strands can fold into G-quadruplexes (G4s), four-stranded structures stacked from planar G-tetrads (rings of four hydrogen-bonded guanine bases) that are stabilized by cations such as potassium. Sequence motifs capable of forming G4s (potential G-quadruplex sequences, PQSs) are strongly enriched in the promoters of human genes, near transcription start sites (TSSs, the genomic points where RNA synthesis begins), making G4s candidate structure-based regulatory elements that operate in addition to the classical sequence-based recognition of DNA by transcription factors (TFs, sequence-specific DNA-binding proteins). A second relevant strand of prior knowledge is target search: a DNA-binding protein lands at a random position on DNA and then performs facilitated one-dimensional diffusion (sliding) along the DNA until it finds its target site (Halford and Marko 2004); any stable non-duplex obstacle in the path can, in principle, slow, pause, or stop such translocation. Single-molecule and biochemical studies have documented this roadblock effect for helicases (nucleic-acid-unwinding enzymes such as BLM) colliding with G4s (Liu et al. 2010), and G4s are known to impede RNA polymerase transit (Robinson et al. 2021). What has been missing is genome-scale information on how the whole repertoire of DNA-binding proteins distributes around G4s. Duan, Zheng, Hao and Tan (bioRxiv 2025, DOI 10.1101/2025.01.01.630896) closed that descriptive gap: using ChIP-seq data (chromatin immunoprecipitation followed by sequencing, which maps where a given protein binds DNA) for 1,183 proteins from the ENCODE project consortium (Encyclopedia of DNA Elements, a public consortium that has produced genome-scale maps of protein-DNA binding in human cells), aligned around a consensus set of experimentally detected G4-forming promoter regions (G4(+), assembled from mapping data obtained with G4P, an engineered small artificial protein that binds folded G4s in living cells and thereby marks their genomic positions (Zheng et al. 2020), in several human cell lines), they found that the G4(+) region itself, its immediate proximal side, and its distal side act as three primary binding zones, with proteins adopting five recurring spatial states - enrichment at G4(+), depletion at G4(+), enrichment at the proximal side, depletion at the proximal side, and enrichment at the distal side - that vary across cell and tissue types. Histones (the protein spools of chromatin) were excluded from the proximal side, and their binding to G4(+) was switched on or off by lysine acetylation or methylation (post-translational histone modifications), respectively. The authors interpret the patterns as arising from protein competition for shared zones and propose - but do not test - that G4s act as physical 'roadblocks' or 'speed bumps' that slow, pause, or stop protein translocation, with two or more G4s on one DNA strand able to trap proteins between them, the whole process being modulated by G4 stability and by cellular G4-resolving activities (helicases and other enzymes that unfold G4 structures). The inference that 'the role a protein plays in transcription is defined by its spatial distribution controlled by G4 formation' therefore rests entirely on correlation. The mechanistic question (what actually generates the patterns) and the causal question (what the patterns do to transcription) are both open; reviews of G4-protein interaction methodology (Dai et al. 2023) and of single-molecule approaches to G4-protein interactions (Cho and Myong 2026) confirm that the tools to attack them exist but have not been combined into such a test.

Problem Statement

The common objective is to explain, mechanistically and functionally, the spatial organization that promoter G-quadruplexes impose on DNA-binding proteins. The problem has two linked parts: (1) Mechanism: identify and quantitatively characterize the molecular process (or combination of processes) that generates the observed occupancy patterns around the consensus promoter G4-forming regions mapped by Duan et al. - the three zones (G4(+), its proximal side, its distal side) and the five recurring spatial states - discriminating at least between direct structure-based binding of proteins to G4s with graded affinities, roadblock or speed-bump impedance of one-dimensional protein diffusion along DNA by stable G4s, trapping of proteins between paired G4s, and exclusion by inter-protein competition; the account must specify how these effects are modulated by the thermodynamic stability and the dynamic folding and unfolding of G4s and by cellular G4-resolving activities, so that the three-zone/five-state patterns (including histone exclusion from the proximal side and the acetylation/methylation-dependent histone switch at G4(+)) follow from the proposed mechanism rather than being restated as observations. (2) Causality: establish whether particular G4-protein occupancy patterns regulate transcription of the corresponding individual genes, with controls that separate G4-structure effects from effects of the underlying DNA sequence. The roadblock/speed-bump hypothesis is to be tested against the competing mechanisms, and any proposed mechanism must account for the observed patterns and their transcriptional consequences. Three further directions - the protein interactome of short-lived G4 sites, the interplay of DNA:RNA hybrid G4s with protein recruitment, and the interpretation of the unclassified minority of proteins and samples - are adjacent open questions outside this problem's completion boundary.

References

  1. Duan R-f, Zheng J-p, Hao Y-h, Tan Z (2025) G-Quadruplex-Protein Interactome at Human Gene Promoters. bioRxiv preprint 2025-01-02, DOI 10.1101/2025.01.01.630896, https://doi.org/10.1101/2025.01.01.630896
  2. Zheng K-w, Zhang J-y, He Y-d, Gong J-y, Wen C-j, Chen J-n, Hao Y-h, Zhao Y, Tan Z (2020) Detection of genomic G-quadruplexes in living cells using a small artificial protein. Nucleic Acids Research 48:11706-11720, DOI 10.1093/nar/gkaa841, https://doi.org/10.1093/nar/gkaa841
  3. Ren C-x, Duan R-f, Wang J, Hao Y-h, Tan Z (2024) Dominant and genome-wide formation of DNA:RNA hybrid G-quadruplexes in living yeast cells. Proceedings of the National Academy of Sciences USA 121:e2401099121, DOI 10.1073/pnas.2401099121, https://doi.org/10.1073/pnas.2401099121
  4. ENCODE Project Consortium (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489:57-74, DOI 10.1038/nature11247, https://doi.org/10.1038/nature11247
  5. Halford SE, Marko JF (2004) How do site-specific DNA-binding proteins find their targets? Nucleic Acids Research 32:3040-3052, DOI 10.1093/nar/gkh624, https://doi.org/10.1093/nar/gkh624
  6. Liu J-q, Chen C-y, Xue Y, Hao Y-h, Tan Z (2010) G-quadruplex hinders translocation of BLM helicase on DNA: a real-time fluorescence spectroscopic unwinding study and comparison with duplex substrates. Journal of the American Chemical Society 132:10521-10527, DOI 10.1021/ja1038165, https://doi.org/10.1021/ja1038165
  7. Robinson J, Raguseo F, Nuccio SP, Liano D, Di Antonio M (2021) DNA G-quadruplex structures: more than simple roadblocks to transcription? Nucleic Acids Research 49:8419-8431, DOI 10.1093/nar/gkab609, https://doi.org/10.1093/nar/gkab609
  8. Dai Y, Teng X, Zhang Q, Hou H, Li J (2023) Advances and challenges in identifying and characterizing G-quadruplex-protein interactions. Trends in Biochemical Sciences 48:894-909, DOI 10.1016/j.tibs.2023.06.007, https://doi.org/10.1016/j.tibs.2023.06.007
  9. Cho S, Myong S (2026) Single-molecule approaches to study G-quadruplex, R-loop, and protein interactions. Current Opinion in Structural Biology 98:103255, DOI 10.1016/j.sbi.2026.103255, https://doi.org/10.1016/j.sbi.2026.103255
  10. Marzano S, Pinto G, Di Porzio A, Amato J, Randazzo A, Amoresano A, Pagano B (2025) Identifying G-quadruplex-interacting proteins in cancer-related gene promoters. Communications Chemistry 8:64, DOI 10.1038/s42004-025-01462-w, https://doi.org/10.1038/s42004-025-01462-w
  11. Drápalová K, Dobrovolná M, Kledus F, Mergny J-L, Brázda V (2026) G-quadruplexes as regulatory platforms for human transcription factors: insights from ChIP-seq data. BMC Genomics, DOI 10.1186/s12864-026-13217-5, https://doi.org/10.1186/s12864-026-13217-5
  12. Cabrero-Martín A, Ruiz-Valdepeñas Montiel V, Velázquez-Gutiérrez J, Montero-Calle A, Garranzo-Asensio M, Reviejo AJ, Pingarrón JM, Barderas R, Campuzano S (2026) Decoding atypical G-quadruplexes and their interactome in liquid biopsies via integrated electroanalytical-proteomic technologies. Angewandte Chemie International Edition, article e3768019, DOI 10.1002/anie.3768019, https://doi.org/10.1002/anie.3768019
  13. Wang X, Qin G, Yang J, Zhao C, Ren J, Qu X (2025) A subcellular selective APEX2-based proximity labeling used for identifying mitochondrial G-quadruplex DNA binding proteins. Nucleic Acids Research 53:gkae1259, DOI 10.1093/nar/gkae1259, https://doi.org/10.1093/nar/gkae1259
  14. Mo J, Chen Z, Cui M, Fang X, Li R, Qin S, Zhou Z, Sun M, Wu J (2026) In situ proximity labeling of proteins associated with circRNA and RNA G-quadruplexes. Nature Chemical Biology 22(6):874-885, DOI 10.1038/s41589-025-01993-2, https://doi.org/10.1038/s41589-025-01993-2