GR-mediated transcriptional regulation of m^(6)A metabolic genes contributes to dietinduced fatty liver in hens

Background:Glucocorticoid receptor(GR)mediated corticosterone-induced fatty liver syndrome(FLS)in the chicken by transactivation of Fat mass and obesity associated gene(FTO),leading to demethylation of N6-methyladenosine(m^(6)A)and post-transcriptional activation of lipogenic genes.Nutrition is considered the main cause of FLS in the modern poultry industry.Therefore,this study was aimed to investigate whether GR and m^(6)A modification are involved in high-energy and low protein(HELP)diet-induced FLS in laying hens,and if true,what specific m^(6)A sites of lipogenic genes are modified and how GR mediates m^(6)A-dependent lipogenic gene activation in HELP diet-induced FLS in the chicken.Results:Laying hens fed HELP diet exhibit excess(P<0.05)lipid accumulation and lipogenic genes activation in the liver,which is associated with significantly increased(P<0.05)GR expression that coincided with global m^(6)A demethylation.Concurrently,the m^(6)A demethylase FTO is upregulated(P<0.05),whereas the m^(6)A reader YTHDF2 is downregulated(P<0.05)in the liver of FLS chickens.Further analysis identifies site-specific demethylation(P<0.05)of m^(6)A in the mRNA of lipogenic genes,including FASN,SREBP1 and SCD.Moreover,GR binding to the promoter of FTO gene is highly enriched(P<0.05),while GR binding to the promoter of YTHDF2 gene is diminished(P<0.05).Conclusions:These results implicate a possible role of GR-mediated transcriptional regulation of m^(6)A metabolic genes on m^(6)A-depenent post-transcriptional activation of lipogenic genes and shed new light in the molecular mechanism of FLS etiology in the chicken.

Chronic corticosterone disrupts the circadian rhythm of CRH expression and m^(6)A RNA methylation in the chicken hypothalamus

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 lowestm^(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.