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 (m6A) RNA methylation is involved in the regulation of circadian rhythm, yet it remains unknown whether CRH expression and m6A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress. 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 m6A level oscillates in an opposite pattern to CRH mRNA, with lowest m6A level after midnight (ZT18) corresponding to the peak of CRH mRNA before dawn (ZT22). CORT diminished the circadian rhythm of m6A methylation with significantly increased level at night. Further site-specific m6A analysis on 3’UTR of CRH mRNA indicates that higher m6A on 3’UTR of CRH mRNA coincides with lower CRH mRNA at night (ZT18 and ZT22). 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 m6A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.

中文翻译：

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慢性皮质酮破坏鸡下丘脑 CRH 表达和 m6A RNA 甲基化的昼夜节律

促肾上腺皮质激素释放激素 (CRH) 是下丘脑-垂体-肾上腺 (HPA) 轴的主要促分泌素，它与时钟基因错综复杂地交织在一起，以调节各种身体功能的昼夜节律。N6-甲基腺苷（m6A）RNA甲基化参与昼夜节律的调节，但尚不清楚CRH表达和m6A修饰是否与鸡下丘脑的时钟基因振荡，以及慢性压力下昼夜节律如何变化。长期暴露于皮质酮 (CORT) 消除了鸡血浆 CORT 和褪黑激素水平的昼夜模式。CORT处理的鸡海马、下丘脑和垂体生物钟基因的昼夜节律均受到不同程度的干扰。最显着的变化发生在下丘脑，其中 CRH mRNA 的昼夜波动变平，以及其他与进食相关的神经肽的 mRNA。有趣的是，下丘脑 m6A 水平与 CRH mRNA 的波动模式相反，午夜后 m6A 水平最低 (ZT18) 对应于黎明前 CRH mRNA 的峰值 (ZT22)。CORT 降低了 m6A 甲基化的昼夜节律，夜间水平显着增加。对 CRH mRNA 的 3'UTR 的进一步位点特异性 m6A 分析表明，CRH mRNA 的 3'UTR 上较高的 m6A 与夜间较低的 CRH mRNA 一致（ZT18 和 ZT22）。我们的研究结果表明，慢性应激破坏了下丘脑 CRH 表达的昼夜节律，导致鸡 HPA 轴功能障碍。