Functional Identification Of Cis-regulatory Elements Of Cold-Responsive Genes And Characterization Of Cold-inducible Promotor Region Of Ciarta Gene In Zebrafish

Fishes are poikilotherms whose body temperature changes as the ambient water temperature changes. The molecular networks regulating temperature responses in fishes are not clear so far. Cis-element is a DNA sequence in a gene which has the function in transcriptional regulation by interacting with the trans-factors. In a previous study, we found that two cis-regulatory elements, AGMAACCA and SAGTCAA are enriched in the set of cold-responsive genes in zebrafish. This study is to verify their cold responsive roles through in vivo studies.We respectively cloned the cis-elements and their mutant elements to drive EGFP expression in a Tol2 transposable element-based transgenic vector, and injected into zebrafish eggs through microinjection. We examined the expression level of EGFP mRNA to determine the regulatory ability of these cis-elements using quantitative real-time PCR. Under cold stress, the cis- element AGMAACCA work as an enhancer to inducing the expression of EGFP, whereas the cis-element SAGTCAA works as a repressor to repress the expression of EGFP. In the mutation assays, the regulatory ability in cold stress was abolished. Thus we identified two novel cis-regulatory elements, one is cold-inducible, and another cold-repressive from the zebrafish genome. The discovery opened a new way to understanding cold-responsive network in zebrafish.Previous study in the laboratory showed that expression of ciarta(circadian associated repressor of transcription a) gene, also known as c1orf51, was commonly induced in eight tissues of zebrafish under cold stress. Ciarta is known to associate with rhythmic expression, and participate in biological clock setting. To identify the core cold-regulatory regions of ciarta promoter, five fragments with different lengths(-1992 ~ +100 bp,-1354 ~ +100 bp,-881 ~ +100 bp,-634 ~ +100 bp and-247 ~ +100 bp) were cloned into pGL4.10, and transfected into zebrafish-derived ZF4 cell lines. The cold-regulatory ability of these 5 fragments were quantified by examining the changes in the luciferase activity in cold temperatures. Results showed that all of the five fragments could significantly induce the gene expression at cold temperature but with different capacities. Of the 5 fragments, one fragment(-881 ~ +100 bp) shows the highest cold-induced ability and increases the downstream gene’s expression up to 5.61±0.53 fold. Compared with the fragment(-634 ~ +100 bp) which has less inducing capability, the fragment(-881 ~ +100 bp) contains a coldinducible cis-element which is known to be a BCL6 binding site. These findings verify the cold-induced ability of BCL6 binding site and provide a foundation for efficiently inducing the expression of certain genes under cold stress in zebrafish.