Age-associated G-quadruplex accumulation and DDX5 loss shape chromatin landscapes in human astrocytes

Aging disrupts genome organization and transcriptional fidelity, but the role of non-canonical DNA structures in the aging process remains unclear. G-quadruplexes (G4s), stable guanine-rich DNA and RNA structures are established regulators of gene expression and genome integrity, yet their contribution to physiological aging is unknown. Using fluorescent imaging with primary human astrocytes derived from individuals spanning early to late adulthood (22-73 years) reveals an accumulation of G4s and a reduced nuclear expression of the G4-resolving helicase DDX5 in aging cells. To investigate how these changes relate to genome architecture, we performed ATAC-seq to profile chromatin accessibility and G4 CUT&Tag to profile the G4 landscape across all astrocyte cultures. Older cells exhibited global chromatin compaction and focal G4 enrichment, with gains occurring in both accessible and closed chromatin regions, indicating locus-specific and context-dependent regulation. To determine whether DDX5 modulates these features, we overexpressed DDX5 in young astrocytes and identified transcriptional targets involved in chromatin organization and genome maintenance. Acute DDX5 knockout caused focal G4 accumulation without widespread chromatin changes, indicating that DDX5 maintains and modulates G4 dynamics at defined genomic regions. Together, these findings reveal G4s as dynamic, age-sensitive features of the genome with potential roles in epigenetic regulation and establish DDX5 as a modulator of G4 dynamics and genome integrity during human brain aging.