Kumar, PraveenPraveenKumarSingh, UmashankarUmashankarSingh2025-12-242025-12-242025-112692-820510.64898/2025.12.10.693595http://repository.iitgn.ac.in/handle/IITG2025/33726The formation of G-quadruplexes is fundamentally linked to the DNA sequence, particularly regions rich in guanine. Unlike other DNA features, evolution has not favored specific agents to stabilize G-quadruplex structures; their formation depends strictly on inherent sequence properties. Vertebrate promoters are notable for their consistently high GC-content and pronounced G/C strand asymmetry. The evolutionary acquisition of CGGBP1 in vertebrates has functionally constrained methylation-driven cytosine-to-thymine transition mutations at GC-rich transcription factor binding sites within promoters, thereby preserving GC-content in these regulatory elements across amniote genomes. CGGBP1 target sequences are associated with GC-rich DNA and display G/C asymmetry, while also modulating G-quadruplex formation. This underscores the importance of investigating G4-forming potential retention across vertebrates through CGGBP1’s role in protecting GC-content. In this study, we reveal the extensive potential for G-quadruplex formation in the promoters and genomes of over 100 vertebrate species. The evolutionary conservation of these GC-rich regions arises from selective mechanisms that protect both the G-rich strand, capable of forming the G4 structures, and the unused C-rich strand by suppressing C-to-T transitions. We demonstrate that G4-forming regions under nuclear constraints are governed by this restrictive process, mediated by CGGBP1. Furthermore, sequences most influenced by CGGBP1 levels also conform to this protective evolutionary strategy. Our data suggest that CGGBP1 acts not only to prevent excessive secondary structure formation but also to sustain genome-wide GC-richness by regulating methylation-associated C-to-T transitions in these sequences. Collectively, these findings underscore the evolutionary emergence of CGGBP1 as a guardian of genomic GC-richness.en-USCGGBP1meC-T transitionGenomeG-quadruplexEvolutionVertebratesE-Print0