Duan, R.-f. | Search bioRxiv

bioRxiv2025

G-Quadruplex-Protein Interactome at Human Gene Promoters

DNA-protein interactions at gene promoters play a critical role in gene expression. The promoters of human cells are highly enriched in guanine-rich sequences, which can form four-stranded G-quadruplex (G4) structures. G4s are emerging as a distinct class of structure-based regulatory elements in gene regulation, and their interaction with proteins is essential for the role G4s play. Currently, our understanding of G4-protein interaction is mainly on a case-by-case basis, without systematic information. In this work, we examined the spatial occupancy of 1,183 human DNA-binding proteins, including transcription factors, histones and their modifying enzymes, around the consensus G4-forming region, G4(+), using data from the ENCODE project. We found that the G4(+), its immediate proximal side, and its distal side serve as three primary protein binding sites. Nearly all proteins are either enriched or depleted at these sites, likely due to competition, or in a spatiotemporal transition between the sites, resulting in different degrees of variation or persistence within or across cell/tissue types. Notably, histones were excluded from the proximal side of G4(+), and their binding to G4(+) was turned on and off by acetylation and methylation, respectively. Furthermore, the distal side is preferentially enriched for H3K23me2 and H3K4me2. Our experiments also revealed corresponding patterns of G4-protein interaction. Taken together, our results suggest a general role for G4s in dynamically defining and coordinating chromatin architecture and DNA-protein interactions at gene promoters for transcriptional regulation, a task that is unlikely to be accomplished by sequence-based DNA recognition. O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/630896v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1eb0b6eorg.highwire.dtl.DTLVardef@3b7d63org.highwire.dtl.DTLVardef@1a4f4dborg.highwire.dtl.DTLVardef@c760f7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO A protein can be either enriched or suppressed at G-quadruplexes (G4s), at the proximal side of G4s, or enriched at the distal side of G4s in a 2 kb neighborhood. Regulatory roles of proteins may result from spatial transitions between the different binding regions. C_FIG

genomics↗

bioRxiv2023

Genome-Wide Formation of DNA:RNA Hybrid G-Quadruplexes in Live Yeast Cells

Guanine-rich nucleic acids form G-quadruplex (G4) structures that play a critical role in cellular processes. Previous studies have mostly focused on monomeric intramolecular G4s with four consecutive guanine tracts (G-tracts) from a single strand. However, this structural form has never been confirmed in eukaryotic cells. Here, we report the formation of hybrid G4s (hG4s), consisting of G-tracts from both DNA and RNA, in the genome of living yeast cells. Analysis of Okazaki fragment syntheses and G4-specific probing reveals that hG4s can efficiently form with as few as a single DNA guanine-guanine (GG) tract due to the participation of G-tracts from RNA. This finding increases the number of G4-forming sites in the yeast genome from 38 to 587,694, a more than 15,000-fold increase. Interestingly, hG4s still form and even dominate at genomic G4 sites that are theoretically capable of forming the monomeric intramolecular DNA G4s by themselves. Compared to DNA G4s (dG4s), hG4s exhibit a wider range of kinetics, higher prevalence, and greater structural diversity and stability. Most importantly, hG4 formation is tightly coupled to transcription through the involvement of RNA, allowing hG4s to function in a transcription-dependent manner. Overall, our study establishes hG4s as the overwhelmingly dominant G4 species in the yeast genome and emphasizes a renewal of the current perception of the structural form, formation mechanism, prevalence, and functional role of G4s in eukaryotic genomes. It also provides a sensitive and currently the only method for detecting the structural form of G4s in living cells. SignificanceThe identification of hybrid G-quadruplexes (hG4s) has disclosed a previously unrecognized structural form of G4s as the most common and abundant G4 species in the yeast genome. It reveals not only a dominant rule governing the formation of G4s in eukaryotic genomes, but also a unique genotype that allows G4-mediated transcriptional regulation to take feedback from the output as input, thus allowing the creation of feedback loops at the transcriptome scale.

biochemistry↗