Zebrafish Timeless Negatively Regulates The Circadian Clock Through Binding To Cry1aa

While Drosophila contains two timeless genes, dtim and dtim2, acting in clock/photoreception and chromosome integrity/photoreception, respectively, mammals contain only one Tim gene and it’s clock function is still in debate. The TIM protein possesses evolutionarily conserved noncircadian functions critical for survival, and also is involved in period determination and DNA damage-dependent phase advancing of the circadian clock. Zebrafish timeless(tim) was identified based upon its sequence similarity to Drosophila’s essential circadian clock tim. The clock function of zebrafish timeless, however, is still unknown.Quantitative RT-PCR showed that timeless is rhytmically expressed in larvae, and down-regulated in bmal1 b and clock1 a mutants. We observed that zebrafish timeless promoter harbors one E’-Box element and seveal E like-Box elements. Luciferase reporter assays showed that the activities of timeless promoter is activated by Clock1 a and Bmal1 b but repressed by Cry1 ba in vitro. The ChIP assays showed that Bmal1 b binds to the E’-Box element of timeless promoter in vivo. These results indicated that timeless is controlled directly by the circadian clock.To examine functions of zebrafish timeless, here we used TALEN(Transcription activator-like effector Nuclease) technology to generate its mutants. The expression of core circadian genes bmal1 b, clock1 a, cry1 aa, per1 b and per3, and circadian clock controlled gene aanat2 are all up-regulated in timeless-/- compared with WT under DD condition, suggesting that Timeless acts as a repressor in zebrafish circadian clock.Under DD condition, the period of the locomoter activity of timeless-/- is lengthened half hour compared with WT. Furthermore, quantitative RT-PCR shows zebrafishTimeless appears to have tissue-specific regulatory roles in numerous peripheral organs.These results indicated that Timeless is essential for zebrafish circadian regulation.The cell transfection assay showed that the Timeless protein is distributed in both the cytoplasm and the nucleus. The Co-IP assays showed that Timeless through the LXXLL motif in the N-terminal binds to Cry1 aa, but not Per1 b, Per2 or Cry1 ba. The luciferase assays showed that timeless is able to strengthen the repressive function of Cry1 aa, suggesting that Timeless regulates the zebrafish circadian clock negatively through binding to Cry1 aaHigh-throughput deep sequencing-based transcriptome analsysis of timeless mutant larvae revealed that several genes involved in development and cell division cycle spice1, smac1 and tshz1 are all downregulated but an early response gene egr2a is up-regualted, suggesting that Timeless may contribute to regulation of developmemt and cell division cell in zebrafish. Taken together, these findings help define zebrafish Timeless’ circadian function as well as its role in development, and provide insights into how Timeless functions have evolved in the vertebrate circadian system.