Abstract
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.
Highlights
The circadian clock uses properties of both self-sustained oscillation and sensitivity to environmental light for its resetting
The full-length XtClock sequence has already been identified in the Entrez Nucleotide database (NCBI), and in this study, we were able to determine full-length XtCRY1, XtCry2, XtBmal1 and Xtb2M sequences
The amino acid sequences of the X. tropicalis clock proteins were similar to those of Xenopus laevis, a closely related species of X. tropicalis, indicating that the main frame of circadian clockwork is conserved between the two Xenopus species
Summary
The circadian clock uses properties of both self-sustained oscillation and sensitivity to environmental light for its resetting. Several clock genes show circadian transcriptional oscillations that are served by positive and negative regulatory factors. CLOCK and BMAL are transcription factors constituting a positive regulatory complex that binds to the CACGTG-type E-box, a core circadian enhancer element [1]. Cryptochromes (CRYs) are unique molecules in that they retain the structure of blue-light photoreceptors, which are highly related to photolyases [2,3], and the vertebrate CRYs operate as negative factors inhibiting E-box-mediated circadian gene transcription [4]. There are a certain number of reports on clock molecules in zebrafish and Xenopus laevis [5,6], temporal and spatial expression and/or the function of clock molecules in other species, especially in the lower vertebrates, are far less investigated. Further investigation into the expression and circadian function of XtCRYs yielded findings of extremely high expression of cryptochrome mRNA in the ovary of this lower vertebrate
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