| The circadian clock regulates the life processes at physiological, biochemical, cellular and behavioral levels with a period of approximately 24 hours. Circadian dysrhythmias lead to insomnia, despair, and even cancer. The current molecular understanding of circadian regulation is that CLOCK and BMAL1 as a heterodimer bindto target genes’ E-box region and activate their expression. Then the negative regulatory factors(Per, Cry) form the PER-CRY heterodimer. They enter the nucleus interacted with the CLOCK: BMAL1 and released the transcriptional activation of it to complete a cycle of the feedback loop. E4bp4 as a basic leucine zipper(bZIP) transcription factor, was shown to inhibit the expression of the circadian clock genes through binding to the D-box in the promoter region, thereby playing a role in circadian regulation in chicken and mice. Derived from genome-wide duplication, there are six e4bp4 homologous genes in zebrafish the function of E4bp4 in the zebrafish circadian clock, however, is still unknown. In this study, we focus on studying the functions of e4bp4-2b(e4bp4-5).Quantitative real-time PCR showed that e4bp4-2b is rhythmically expressed in zebrafish larvae under LD(light/dark) and DD conditions, and the ISH indicated that e4bp4-2b was expressed primarily in the brain region. The e4bp4-2b promoter harbors several E-like boxes. Luciferase assays showed that the e4bp4-2b promoter activities are enhanced by co-transfected with Clock1 a and Bmal1 b but repressed by Cry1 aa. These result indicate that e4bp4-2b is regulated by the zebrafish circadian clock directly.The light treatment experiments revealed that e4bp4-2b expression is indeed induced by light. The e4bp4-2b promoter also harbors a D-box that may be important to the light response. Luciferase assay showeded that the e4bp4-2b promoter activities are enhanced by Tefa but repressed by E4bp4-2b itself. These results indicated that e4bp4-2b is a light-responded gene. However, how light induces e4bp4-2b expression through the D-box element would require further investigation.We also used TALEN to generate two e4bp4-2b mutant lines to examine its role in the zebrafish circadian clock. qRT-PCR showed up-regulation of a numbr of circadian clock genes, cry1 aa, per2, cloack1 a and bmal1 b in e4bp4-2b mutant zebrafish. Behavioral assays showed that e4bp4-2b mutant larvae display hyperactivity compared with wild types under LD condition. We also examined the roles of E4bp4-2b in regulation of cry1 aa. Ch IP assays showed that E4bp4-2b binds to the D-box in the cry1 aa promoter region. Luciferase assays showed that the cry1 aa promoter activities are repressed by E4bp4-2b. We also cloned the mutant form of E4bp4-2b cDNA for Luciferase assays showed that. The mutant form of e4bp4-2b cDNA lost the repression function. These findings indicated that E4BP4 is required for the zebrafish circadian clock, and particularly it represses cry1 aa expression.We also employed highthroughput sequencing technology to conduct transcriptome analysis of e4bp4-2b mutant larvae. o. Bioinformatic analysis of the sequencing results revealed more than 200 differentially expressed genes in e4bp4-2b mutant larvae. We selected four differentilly expressed genes including two up-regulated genes col28a1 and eva1 a, and two down-regulated genes capn2 and muc5 ac, revealed by the trancriptome analysis. Their expresssion patterns were reconfirmed by independent qRT-PCR. These studies revealed that E4bp4-2b may contribute to cancer and autophagy as well.In summay, this study showed that zebrafish e4bp4-2b is a circadian clock-controlled gene and also is requred for zebrafish circadian regulation. e4bp4-2b is also a lighted-responded gene. E4bp4-2b negatively regulates cry1 aa through D-box. In additon to its circadian functions, E4bp4-2b likely play roles in regulation of cancer and autophagy. These findings help ascertain E4bp4’s essential functions in zebrafish circadian regulation and set the stage for studing E4bp4’s functions in other life processes. |
PDF Full Text Request