Kubo Y et al. (2010), Cryptochrome genes are highly expressed in the ...

XB-ART-41196

PLoS One 2010 Feb 02;52:e9273. doi: 10.1371/journal.pone.0009273.

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Cryptochrome genes are highly expressed in the ovary of the African clawed frog, Xenopus tropicalis.

Kubo Y , Takeuchi T , Okano K , Okano T .

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Cryptochromes (CRYs) are flavoproteins sharing high homology with photolyases. Some of them have function(s) including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates. Although CRYs and the other circadian clock components have been extensively studied in the higher vertebrates such as mice, only a few model species have been studied in the lower vertebrates. In this study, we identified two CRYs, XtCRY1 and XtCRY2 in Xenopus tropicalis, an excellent experimental model species. Examination of tissue specificity of their mRNA expression by real-time PCR analysis revealed that both the XtCRYs showed extremely high mRNA expression levels in the ovary. The mRNA levels in the ovary were about 28-fold (XtCry1) and 48-fold (XtCry2) higher than levels in the next abundant tissues, the retina and kidney, respectively. For the functional analysis of the XtCRYs, we cloned circadian positive regulator XtCLOCK and XtBMAL1, and found circadian enhancer E-box in the upstream of XtPer1 gene. XtCLOCK and XtBMAL1 exhibited strong transactivation from the XtPer1 E-box element, and both the XtCRYs inhibited the XtCLOCK:XtBMAL1-mediated transactivation, thereby suggesting this element to drive the circadian transcription. These results revealed a conserved main feedback loop in the X. tropicalis circadian clockwork and imply a possible physiological importance of CRYs in the ovarian functions such as synthesis of steroid hormones and/or control of estrus cycles via the transcription regulation.

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Species referenced: Xenopus tropicalis
Genes referenced: adm ag1 bmal1 clock crygdl.43 dnai1 nos1 nos3 phr prl.2

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	Figure 4. Cellular localization of GFP-fused XtCRYs in HEK 293 cells. Each expression vector was transfected and observed using a fluorescence microscope (upper, GFP; middle, DAPI) or differential interference microscope (lower, DIC).
	Figure 1. Phylogenetic tree of CRY family proteins.XtCRY sequences and their related sequences obtained from the NCBI Entrez Protein database (accession nos. are shown in Table S1.) were analyzed in the conserved region of the CRY family proteins (corresponding to Arg10-Pro447 in XtCRY1) using the Neighbor-Joining method and CLUSTAL W. CRY DASH proteins (XlCRY DASH, zCRY DASH, and AtCRY DASH) were used as the outgroup (not shown). Bootstrap probabilities (p) are represented by closed circles on the nodes (p>98%) or values near the nodes. Abbreviations are defined as follows: h, human; m, mouse; c, chicken; Xl, Xenopus laevis; Xt, Xenopus tropicalis; z, zebrafish; Ag, Anopheles gambiae; Am, Apis mellifera; Ap, Antheraea pernyi; Dm, Drosophila melanogaster; Dp, Danaus plexippus; PHR, photolyase.
	Figure 2. Cry mRNA levels and their daily variations in X. tropicalis tissues estimated by quantitative RT-PCR.Each tissue (n = 4) was collected at ZT6 and ZT18. Each Cry mRNA level was calculated as a value relative to that of the Xtβ2M or XtHprt1 gene. Error bars represent ±SEM. (A) Daily changes in the Cry mRNA levels in eleven tissues. Messenger RNA levels are shown as a ratio to XtHprt1 mRNA levels, which showed relatively small changes between ZT6 and ZT18 in many tissues (except for the ovary). The Gusb gene was used as another internal control gene. p<0.05, * p<0.02, Student's t-test. (B,C) Tissue specificity of XtCry mRNA levels. Messenger RNA levels at ZT6 and ZT18 are averaged and shown as a ratio to Xt(2M mRNA levels, which showed relatively small changes among eleven tissues. B; XtCry1, C; XtCry2.
	Figure 3. Effects of XtCRY on CLOCK:BMAL1-mediated transcriptional activation from XtPer1 E-box elements.(A) Schematic diagrams of the XtPer1 promoter region in which there is one CACGTG E-box (depicted as a closed triangle) and a TATA-box (depicted as a closed box). (B) Twenty-five nanogram of a firefly luciferase reporter with XtPer1 E-box-SV40-luc reporter, 0.5 ng of Renilla luciferase reporter, pRL-CMV as an internal control (Promega), 125 ng of XtCLOCK expression vector, 12.5 ng of XtBMAL1 expression vector, and 0, 10, 20, or 50 ng of XtCRY expression vector were mixed. The total amount of plasmids was adjusted to 1 µg per well by adding pcDNA3.2/V5-DEST empty vector. All data presented are the means ±SD for three independent experiments. *p<0.0001, Student's t-test, p<0.05, Tukey-Kramer test, comparing the effect of 4, 5, 6 to 2; or 7, 8, 9 to 2.

References [+] :

Amano, Expression and functional analyses of circadian genes in mouse oocytes and preimplantation embryos: Cry1 is involved in the meiotic process independently of circadian clock regulation. 2009, Pubmed