Abstract
BACKGROUND: Corticotropin-releasing hormone (CRH), the major secretagogue of the hypothalamic-pituitary-adrenal (HPA) axis, is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions. N6-methyladenosine (m(6)A) RNA methylation is involved in the regulation of circadian rhythm, yet it remains unknown whether CRH expression and m(6)A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress. RESULTS: Chronic exposure to corticosterone (CORT) eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken. The circadian rhythms of clock genes in hippocampus, hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens. The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened, together with mRNA of other feeding-related neuropeptides. Interestingly, hypothalamic m(6)A level oscillates in an opposite pattern to CRH mRNA, with lowest m(6)A level after midnight (ZT18) corresponding to the peak of CRH mRNA before dawn (ZT22). CORT diminished the circadian rhythm of m(6)A methylation with significantly increased level at night. Further site-specific m(6)A analysis on 3'UTR of CRH mRNA indicates that higher m(6)A on 3'UTR of CRH mRNA coincides with lower CRH mRNA at night (ZT18 and ZT22). CONCLUSIONS: Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus, leading to dysfunction of HPA axis in the chicken. RNA m(6)A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.