Abstract
Cryptochromes function in animal circadian regulation. Zebrafish are known to have six cryptochrome (cry) genes but their evolutionary relationships are not yet fully resolved. Here, comparative genomic analyses revealed that a local duplication of ancestral chordate Cry occurred likely before the first round of vertebrate genome duplication (VGD); following two successive rounds of VGD and subsequent gene losses, coelacanths retained cry1a, cry1b, cry2 and cry3; and following the third-round teleost genome duplication (TGD) and subsequent gene losses, zebrafish retained six cry genes, renamed as cry1aa (zcry1a in the old nomenclature), cry1ab (zcry1b), cry1ba (zcry2a), cry1bb (zcry2b), cry2 (zcry3) and cry3 (zcry4). Molecular evolutionary analyses suggested that zebrafish cry genes have evolved divergent functions, which is further supported by their distinct and rhythmic expression patterns as shown by both in situ hybridization and quantitative real-time PCR. Systematic cell transfection assays divided six Cry proteins into repressive Cry1aa, Cry1ab, Cry1ba and Cry1bb, and non-repressive Cry2 and Cry3. Cry2 is non-repressive because it lacks an effective protein-protein interaction domain although it does possess a nuclear localization signal (NLS) motif, whilst Cry3 lacks both an NLS motif and a protein-protein interaction domain. These findings provide a better understanding of evolution of zebrafish cry genes.
Highlights
Due to the third-round of genome duplication in vertebrate phylogeny, the teleost genome duplication (TGD), zebrafish harbors duplicates of most circadian clock genes[21,22,23]
We interrogated the six teleost fish genomes including fugu (Takifugu rubripes), tetraodon (Tetraodon nigroviridis), medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), cave fish Mexican tetra (Astyanax mexicanus) and zebrafish (Danio rerio), and several other animal genomes including of humans (Homo sapiens), chicken (Gallus gallus), zebra finches (Taeniopygia guttata), anoles (Anolis carolinensis), western clawed frogs (Xenopus tropicalis), coelacanths (Latimeria chalumnae), and fruit flies (Drosophila melanogaster), and uncovered a number of Cry genes (Supplementary Table S1)
Results showed that only Cry1bbD4, which we showed to enter the nucleus, repressed Clock1a:Bmal1b-mediated activation, while the other three truncated proteins Cry1bbD1, Cry1bbD2 and Cry1bbD3, which were located primarily in the cytoplasm, failed to repress Clock1a: Bmal1b-mediated activation (Fig. 7D)
Summary
Cryptochrome proteins belong to the DNA photolyase/cryptochrome family, which is classified into five subfamilies according to molecular phylogenetic analyses and functions: class I cyclobutane pyrimidine dimer photolyase, class II cyclobutane pyrimidine dimer photolyase, plant CRY, animal CRY including (6–4) photolyases (6-4PHR), and cryptochrome DASH1,2 All members of this family share an N-terminal phytolyase homology (PHR) domain that can bind to the flavin adenine dinucleotide (FAD) cofactor and a light-harvesting chromophore[1,2]. A previous study identified six zebrafish cry genes that were named as zcry1a, zcry1b, zcry2a, zcry2b, zcry[3] and zcry[429] Their evolutionary relationships and the mechanisms underlying their functional divergence, are not yet fully understood. Using an approach that integrates interrogation of animal genome sequences, and phylogenetic, splice site and conserved syntenic analyses, we determined that zebrafish have four cry[1] genes, cry1aa (zcry1a), cry1ab (zcry1b), cry1ba (zcry2a) and cry1bb (zcry2b); cry[2] (zcry3), which is the ortholog of mammalian Cry[2]; and cry[3] (zcry4), which is shared with amphibians, reptiles and birds but not mammals
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