Night-shift work is associated with peripheral clock gene dysregulation and circadian transcriptional network remodeling in female healthcare workers

Background: Night-shift work disrupts the circadian–melatonin axis through chronic light-at-night exposure and has been associated with metabolic, inflammatory, cardiovascular, and cancer-related disorders, but peripheral molecular signatures of occupational chronodisruption remain incompletely characterized. Female healthcare workers (HCWs), frequently exposed to prolonged night-shift schedules, represent a relevant study population. In a previous computational study, we identified a deregulated circadian gene panel (CLOCK, TEF, PER1, and PER3) suggestive of transcriptional remodeling in exposed workers. Here, we experimentally validated this signature and investigated the non-canonical circadian transcription factor YY1. Methods: Ninety-three female HCWs (47 day-shift, DSW; 46 night-shift workers, NSW) participated in the study. Peripheral blood expression of CLOCK, TEF, PER1, PER3, and YY1 was quantified by RT-qPCR. Associations with shift-work status and exposure metrics were evaluated using multivariable regression models adjusted for demographic, reproductive, lifestyle, and sleep-related covariates. Correlation, principal component, and clustering analyses were used to characterize transcriptional organization. Results: Compared with DSW, NSW exhibited significantly lower expression of CLOCK (p = 0.0362), TEF (p < 0.0001), and PER1 (p = 0.0003), whereas PER3 expression was unchanged (p = 0.7978). TEF and CLOCK expression progressively declined with longer duration and higher cumulative night-shift exposure. TEF showed excellent in-sample discrimination between groups (AUC = 0.925), while a four-gene model achieved an AUC of 0.940. YY1 expression was reduced in NSW (p = 0.0019), decreased across long-duration and high-exposure categories, and further improved multigene classification performance (AUC = 0.976). Network analyses revealed disrupted coordination among CLOCK, TEF, and PER1, whereas PER3 maintained stable expression despite reduced integration within the transcriptional network. Conclusions: Night-shift work exposure is associated with coordinated downregulation of CLOCK, TEF, and PER1, altered transcriptional network organization, and reduced YY1 expression. While PER3 expression is preserved, its network connectivity is selectively weakened in exposed workers. These findings identify peripheral transcriptional signatures that may provide a basis for future biomarker development and exposure characterization and support further investigation of chronobiology-driven preventive strategies within a precision medicine framework.