Deletion of TOP2B promoter using CRISPR-Cas9 induces senescence in HEK 293T cells

DNA topoisomerase II beta (TOP2B) is crucial for the regulation of DNA topology, transcription, and genome stability. While its function has been extensively studied in post-mitotic cells such as neurons and cardiomyocytes, its role in proliferative cells, especially in the context of cellular senescence and aging, remains inadequately understood. Emerging evidence suggests that TOP2B expression declines with age and is implicated in several age-associated disorders, yet the molecular basis of its involvement in senescence remains poorly understood. In this investigation, we have specifically disrupted the promoter region of TOP2B in HEK 293T cells using CRISPR-Cas9 genome editing. The targeted region, spanning −533 to −481, which encompasses binding motifs for NF–Y and SP1, also accounts for a major proportion of TOP2B promoter activity. Knockout of this regulatory segment resulted in a marked reduction of TOP2B expression. The knockout cells exhibited classical features of senescence, including elevated ROS levels, increased senescence-associated β-galactosidase (SA-β-Gal) activity, and upregulation of key markers such as p53, p21, and p16.To examine the paracrine effects of senescence, conditioned media from TOP2B-deficient cells were given to wild-type HEK 293T cells, which also developed senescence-like phenotypes. These findings suggest that Loss of TOP2B expression is associated with the induction of senescence-like phenotypes in dividing cells and can propagate senescent signaling to neighboring cells. This study highlights a previously underappreciated role of TOP2B in the regulation of cellular aging and opens new avenues for understanding its potential contribution to age-related pathologies in proliferative cell types. • TOP2B knockout in HEK 293T cells induces oxidative stress and senescence. • Loss of TOP2B reduces cell proliferation and mitochondrial membrane potential. • TOP2B deficiency upregulates p16, p21, and p53, indicating activation of canonical senescence pathways. • Conditioned media from TOP2B-deficient cells propagate senescence via paracrine signaling.