Expression of corticotropin releasing hormone in olive flounder (Paralichthys olivaceus) and its transcriptional regulation by c-Fos and the methylation of promoter

Abstract

Corticotropin-releasing hormone (CRH) has been implicated in multiple physiological processes, such as circadian rhythms, food intake and anxiety-like behavior. In telelost fishes, CRH is predominantly expressed in the caudal neurosecretory system (CNSS), a much low level in hypothalamus and gonads, while undetectable levels in other tissues. However, the mechanisms governing this tissue-specific expression remain unknown. In this study, firstly, we investigated the expression pattern of CRH mRNA in different tissues of olive flounder (Paralichthys olivaceus). Secondly, we found that flounder exhibited low locomotor activity in the daytime, concomitant with the highest CRH mRNA expression in CNSS at noon. Thirdly, we examined whether epigenetic mechanisms through DNA methylation are involved in tissue-specific expression of CRH mRNA. Promoter methylation of the CRH gene was assessed through bisulphate sequencing methods. In the proximal promoter, almost no methylation was detected in the muscle, hypothamus and CNSS. However, different methylation was detected in the distal promoter of CRH. The methylation was 63% in CNSS, while that in hypothalamus and muscle was 85% and 96%, respectively. Lastly, bioinformatics analysis revealed that a conserved AP-1 binding site (5-TCACTGA-3) was located in the proximal promoter of CRH gene. In vivo experiment, lipopolysaccharide (LPS) intraperitoneally injection in the flounder up-regulated c-Fos and CRH mRNA in CNSS (P<0.01). In CNSS tissue culture experiment, forskolin treatment significantly induced the expression of c-Fos, c-Jun and CRH mRNA (P<0.01). Collectively, our data provide the evidence that distal promoter methylation and c-Fos signal pathway are involved in transcriptional regulation of CRH expression in flounder.