Abstract
G-quadruplexes (G4s) are four-stranded nucleic acid structures that are predominantly found at promoters, replication origins, and topologically associating domain (TAD) boundaries. In these regions, they play active roles in transcriptional regulation, DNA replication, and chromatin organization. However, their regulatory functions are inseparable from their capacity to destabilize the genome, which has direct implications in cancer biology and cancer immunotherapies. When G4 homeostasis is dysregulated, these structures become sources of genome instability. Using DHX36 knockout (KO) in the Jurkat T-lymphoblastoid cancer cell line, we showed that the loss of this helicase, which exhibits exceptionally high specificity and affinity for G4s, results in genome-wide enrichment of DNA double-strand breaks (DSBs) at G4 sites, confirming that G4 motifs are key mediators of instability in regulatory regions. The DNA breakage arising from G4 dysregulation has functional consequences. Loss of DHX36 results in accumulation of DNA in the cytoplasm, activation of the cGAS-STING innate immune signaling pathway, upregulation of NF-κB transcriptional programs, and secretion of proinflammatory cytokines. At the same time, loss of DHX36 drives aberrant R-loop accumulation associated with transcriptional activation, particularly of cytotoxic effector and innate immune genes, inducing a partial transition toward a cytotoxic-like state in CD4+ T cells. At the chromatin level, DHX36 KO alters CTCF occupancy, creating de novo CTCF binding sites enriched for G4s, R-loops, and DSBs. One-third of these de novo CTCF binding sites lack the canonical CTCF motif, suggesting that structural features associated with genomic stress may contribute to CTCF recruitment independently of canonical sequence recognition. These chromatin-level changes intersect with an intrinsic susceptibility to DNA breakage at CTCF-binding sites. Using an unbiased genome-wide approach, we demonstrated that DSBs are preferentially enriched at strong CTCF binding sites but not at weaker sites. Strong CTCF binding sites are enriched for energetically favorable alternative DNA secondary structures, including G4s, which function as recognition elements for topoisomerase II (TOP2) binding and cleavage. Reduction of the CTCF protein level amplifies this fragility, particularly at TAD boundaries, where G4s may also function as boundary insulators under limited CTCF conditions. Together, these results establish a mechanistic framework linking G4 dysregulation to rewiring of chromatin topology, genome instability, and the activation of immune signaling pathways, with implications for the therapeutic targeting of G4 biology in cancer.
