Circadian Clock-controlled Transcriptional Corepressors Groucho1 And Groucho2 Are Essential For The Zebrafish Circadian Clock

groucho was first discovered in Drosophila and was shown to have transcriptional co-repression function. In Neurospora crassa, the transcriptional activation of frq not only depends on WHITE-COLLAR complexes in vitro but also relys on transcriptional corepressors RCO-1. By evolutionary tree we found two RCO-1 homologous genes, namely gro1 and gro2 in zebrafish. But for the zebrafish, whether the transcriptional regulation of gro1 and gro2 gene could be activited by the core circadian clock and whether the gro1 and gro2 gene could participate in the core circadian clock feedback loop are unclear.Quantitative real-time(qRT-PCR) detected gro1 and gro2 are rhythmically expressed in larvae under both Light/dark(LD) and constant dark(DD) conditions, and up-regulated in the per1 b mutant fish but down-regulated in the bmal1 b mutant fish. Through luciferase reporter assay, we found that the promoter activities of gro1 and gro2 are enhanced by Clock1 a and Bmal1 b but repressed by Cry1 ab. Through ChIP assay, we also found that Bmal1 b could bind to E-Boxes in the promoter regions of gro1 and gro2, indicating that both gro1 and gro2 are controlled directly by the circadian clock in zebrafish. To further study the function of gro1 and gro2, we also employed CRISPR-Cas9 to generate a number of zebrafish mutant lines for gro1 and gro2. The mRNA expression of circadian clock genes has significantly changed. Under LD, gro1 mutant fish display less locomotor activity; gro2 mutant fish display more locomoter activity. Under DD, gro1 mutant fish display lower amplitude, lengthened period and delayed phase; conversely, gro2 mutant fish exhibit higher amplitude, shortened period and advanced phase; when knockdown the expression of both gro1 and gro2, the locomotor activity has also been affected, so these results suggested that both Gro1 and Gro2 are critical for maintaining zebrafish locomotor rhythmicity. By CoIP assay, we found that Gro1 and Gro2 protein could not directly bind to core clock protein of zebrafish, which means that Gro1 and Gro2 protein may affect the core clock protein in other manners, which influences the expression of core clock genes and the behavior of normal rhythm.Together, our results demonstrate that transcriptional corepressors gro1 and gro2 are direct targets of the circadian clock and also play essential roles in maintaining rhythmicity of zebrafish key circadian clock genes and locomotor activities. These findings for the molecular mechanism of zebrafish circadian clock study as well as other life clock process research have important significance.