Time-Resolved Analysis Reveals FEN1 as a Crucial Factor of Circadian Transcriptome Dynamics and Core Cellular Homeostasis in HEK 293T cell

Circadian rhythms orchestrate genome-wide transcriptional oscillations to coordinate cellular homeostasis. While the core clock machinery has been extensively characterized, its integration with other critical biological processes, such as the DNA damage response and cell cycle control, remains incompletely understood. Here, we identify the DNA replication and repair nuclease FEN1 as a critical modulator of circadian transcriptome dynamics in human cells. Time-series RNA-seq across six circadian time points in synchronized HEK293T cells revealed that FEN1 depletion induces widespread reprogramming of rhythmic gene expression, with over 30% of oscillatory transcripts exhibiting loss or gain of rhythmicity, phase shifts, or amplitude changes. Notably, reprogrammed genes were enriched in cell cycle, DNA damage response, and cellular senescence pathways. Systematic phase set enrichment analysis uncovered coordinated delays and inversions in G1/S checkpoint regulators and metabolic genes, disrupting temporal coherence. Despite retention of rhythmicity in core clock genes, amplitude attenuation and phase advances were observed, indicating compromised oscillatory stability. Functional validation demonstrated reduced S phase entry, G1 accumulation, and accelerated senescence in FEN1 knockdown cells, in line with transcriptomic predictions. qPCR analysis confirmed altered expression of representative target genes. These findings uncover an unrecognized role for FEN1 in sustaining circadian transcriptional architecture and reveal its broader impact on cell cycle control and aging programs.