Stability Matters: Revealing Causal Roles of G-Quadruplexes (G4s) in Regulation of Chromatin and Transcription

<h4>Background</h4>G-quadruplexes (G4s) are non-canonical higher-order nucleic acid structures that form at guanine-rich motifs, with features spanning both secondary and tertiary structural levels. These dynamic structures play pivotal roles in diverse cellular processes. Endogenous G4s (eG4s) function through their dynamically formed structures, prompting the hypothesis that their thermostability, as a key structural property, may critically influence their functionality. This study investigates the relationship between G4 stability and other functional genomic signals within eG4 regions and examines its broader impact on chromatin organization and transcriptional regulation.<h4>Methods</h4>We developed a mapping strategy to associate in vitro-derived thermostability metrics and multi-omics functional signals with eG4 regions. A stability-centric analytical framework combining correlation analysis and causal inference using the Bayesian networks was applied to decipher causal relationships between G4 stability and the other related signals. We further analyzed the association between the stability of transcription start site (TSS)-proximal eG4s and the biological functions of their downstream genes.<h4>Results</h4>Our analyses demonstrate that G4 thermostability exerts causal effects on epigenetic states and transcription factor binding, thereby influencing chromatin and transcription regulation. We further show distinct network architectures for G4-binding versus non-binding transcription factors. Additionally, we find that TSS-proximal eG4s are enriched in genes involved in core proliferation and stress-response pathways, suggesting that eG4s may serve as regulatory elements facilitating rapid stress responses through genome-wide coordination.<h4>Conclusions</h4>These findings establish thermostability-though measured in vitro-as an intrinsic property that shapes eG4 functionality. Our study not only provides novel insights into the functional relevance of G4 thermostability but also introduces a generalizable framework for high-throughput G4 data interpretation, significantly advancing the functional decoding of eG4s across biological contexts.