Structure/Function Analysis of Xenopus Cryptochromes 1 and 2 Reveals Differential Nuclear Localization Mechanisms and Functional Domains Important for Interaction with and Repression of CLOCK-BMAL1

Department of Biology, University of Virginia, Charlottesville, VA 22904-4328, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/2007; 27(6):2120-9. DOI: 10.1128/MCB.01638-06
Source: PubMed


Circadian rhythms control the temporal arrangement of molecular, physiological, and behavioral processes within an organism and also synchronize these processes with the external environment. A cell autonomous molecular oscillator, consisting of interlocking transcriptional/translational feedback loops, drives the approximately 24-hour duration of these rhythms. The cryptochrome protein (CRY) plays a central part in the negative feedback loop of the molecular clock by translocating to the nucleus and repressing CLOCK and BMAL1, two transcription factors that comprise the positive elements in this cycle. In order to gain insight into the inner workings of this feedback loop, we investigated the structure/function relationships of Xenopus laevis CRY1 (xCRY1) and xCRY2 in cultured cells. The C-terminal tails of both xCRY1 and xCRY2 are sufficient for their nuclear localization but achieve it by different mechanisms. Through the generation and characterization of xCRY/photolyase chimeras, we found that the second half of the photolyase homology region (PHR) of CRY is important for repression through facilitating interaction with BMAL1. Characterization of these functional domains in CRYs will help us to better understand the mechanism of the known roles of CRYs and to elucidate new intricacies of the molecular clock.

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Available from: Carla Beth Green, Jan 06, 2014
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    • "However, some animal-type cryptochromes from fungi and bacteria were detected in the CPD photolyase class I subfamily. Previous studies on the fusion proteins of 6–4 DNA photolyase suggest that evolutionary changes in the protein functionally separated the cryptochromes, but the core domain of photolyases were not significantly altered (25, 26). This mixed clade indicates that animal-type cryptochromes, including the 6–4 photolyases of fungi, bacteria and algae, have a higher sequence similarity to CPD photolyase class I compared with higher plants or vertebrates. "
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    ABSTRACT: Cryptochromes are flavoproteins that play a central role in the circadian oscillations of all living organisms except archaea. Cryptochromes are clustered into three subfamilies: plant-type cryptochromes, animal-type cryptochromes and cryptochrome-DASH proteins. These subfamilies are composed of photolyase/cryptochrome superfamily with 6–4 photolyase and cyclobutane pyrimidine dimer photolyase. Cryptochromes have conserved domain architectures with two distinct domains, an N-terminal photolyase-related domain and a C-terminal domain. Although the molecular function and domain architecture of cryptochromes are conserved, their molecular mechanisms differ between plants and animals. Thus, cryptochromes are one of the best candidates for comparative and evolutionary studies. Here, we have developed a Web-based platform for comparative and evolutionary studies of cryptochromes, dbCRY ( A pipeline built upon the consensus domain profile was applied to 1438 genomes and identified 1309 genes. To support comparative and evolutionary genomics studies, the Web interface provides diverse functions such as (i) browsing by species, (ii) protein domain analysis, (iii) multiple sequence alignment, (iv) homology search and (v) extended analysis opportunities through the implementation of ‘Favorite Browser’ powered by the Comparative Fungal Genomics Platform 2.0 (CFGP 2.0; dbCRY would serve as a standardized and systematic solution for cryptochrome genomics studies.Database URL:
    Database The Journal of Biological Databases and Curation 01/2014; 2014:bau037. DOI:10.1093/database/bau037 · 3.37 Impact Factor
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    • "Detailed structure/function analysis of the C-terminal region of mammalian CRY1 allowed us to identify a putative coiled-coil domain at the beginning of the C-terminal extension as a potential PER and BMAL1 binding site [35]. Deletion of the complete C-terminal extension (aa 471-606 of mouse CRY1) abolished the CLOCK/BMAL1 transcription inhibitory potential of CRY1, Similarly, Green and co-workers have demonstrated that the C-terminal extension of Xenopus laevis CRY proteins is crucial for transcription repression [36]. Interestingly, specific deletion of either the coiled-coil domain (aa 471-493) or the downstream tail region (aa 494-606) of mammalian CRY1 failed to eliminate its ability to inhibit CLOCK/BMAL1-mediated transcription [35], likely because these mutant proteins can still bind to CLOCK via a yet unidentified region of the core domain. "
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    ABSTRACT: Despite the sequence and structural conservation between cryptochromes and photolyases, members of the cryptochrome/photolyase (flavo)protein family, their functions are divergent. Whereas photolyases are DNA repair enzymes that use visible light to lesion-specifically remove UV-induced DNA damage, cryptochromes act as photoreceptors and circadian clock proteins. To address the functional diversity of cryptochromes and photolyases, we investigated the effect of ectopically expressed Arabidopsis thaliana (6-4)PP photolyase and Potorous tridactylus CPD-photolyase (close and distant relatives of mammalian cryptochromes, respectively), on the performance of the mammalian cryptochromes in the mammalian circadian clock. Using photolyase transgenic mice, we show that Potorous CPD-photolyase affects the clock by shortening the period of behavioral rhythms. Furthermore, constitutively expressed CPD-photolyase is shown to reduce the amplitude of circadian oscillations in cultured cells and to inhibit CLOCK/BMAL1 driven transcription by interacting with CLOCK. Importantly, we show that Potorous CPD-photolyase can restore the molecular oscillator in the liver of (clock-deficient) Cry1/Cry2 double knockout mice. These data demonstrate that a photolyase can act as a true cryptochrome. These findings shed new light on the importance of the core structure of mammalian cryptochromes in relation to its function in the circadian clock and contribute to our further understanding of the evolution of the cryptochrome/photolyase protein family.
    PLoS ONE 08/2011; 6(8):e23447. DOI:10.1371/journal.pone.0023447 · 3.23 Impact Factor
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    ABSTRACT: Members of the photolyase/cryptochrome family are flavoproteins that share an extraordinary conserved core structure (photolyase homology region, PHR), but the presence of a carboxy-terminal extension is limited to the cryptochromes. Photolyases are DNA-repair enzymes that remove UV-light-induced lesions. Cryptochromes of plants and Drosophila act as circadian photoreceptors, involved in light entrainment of the biological clock. Using knockout mouse models, mammalian cryptochromes (mCRY1 and mCRY2) were identified as essential components of the clock machinery. Within the mammalian transcription-translation feedback loop generating rhythmic gene expression, mCRYs potently inhibit the transcription activity of the CLOCK/BMAL1 heterodimer and protect mPER2 from 26S-protesome-mediated degradation. By analyzing a set of mutant mCRY1 proteins and photolyase/mCRY1 chimeric proteins, we found that the carboxyl terminus has a determinant role in mCRY1 function by harboring distinguished domains involved in nuclear import and interactions with other clock proteins. Moreover, the carboxyl terminus must cross-talk with the PHR to establish full transcription repression capacity in mCRY1. We propose that the presence of the carboxyl terminus in cryptochromes, which varies in sequence composition among mammalian, Drosophila, and plant CRYs, is critical for their different functions and possibly contributed to shape the different architecture and biochemistry of the clock machineries in these organisms.
    Cold Spring Harbor Symposia on Quantitative Biology 02/2007; 72(1):133-9. DOI:10.1101/sqb.2007.72.066
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