Functional Evolution of the Photolyase/Cryptochrome Protein Family: Importance of the C Terminus of Mammalian CRY1 for Circadian Core Oscillator Performance

Abstract

Cryptochromes (CRYs) are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. Here, we show that the C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drive nuclear localization via two independent mechanisms and shift the equilibrium of shuttling mammalian CRY1 (mCRY1)/ mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquirement of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family.Circadian rhythms in physiology, metabolism, and behavior are generated by a genetically determined clock with an intrinsic periodicity of approximately 24 h. In mammals, the master clock resides in the neurons of the suprachiasmatic nucleus (SCN) in the ventral hypothalamus. To keep pace with the light-dark cycle, the SCN clock is daily entrained by light perceived via the retina and transmitted to the SCN via the retinohypothalamic tract (27, 31). Subsequently, this master clock synchronizes peripheral oscillators via neuronal and humoral signaling (1,19,24,46). Peripheral oscillators are thought to optimize organ performance by adjusting metabolic and physiological functions to the requirement at specific times of the day. SCN neurons, peripheral tissues, and in vitro-cultured fibroblasts generate circadian rhythms by means of a self-sustaining molecular oscillator that drives gene expression through interconnected positive and negative transcription/ translation feedback loops (28, 47). In the positive limb of the circadian oscillator, transcription of the Period (mPer1, mPer2, and mPer3), Cryptochrome1 (mCry1), and Rev-Erb␣ clock genes is activated by a heterodimer of the two helix-loop-helix /PAS domain transcription factors CLOCK and BMAL1, which act via CACGTG E-box enhancer elements (3,8,26,28,29,47). The mammalian CRY1 (mCRY1) and mCRY2 proteins are central components in the negative limb of this circuit, as they strongly inhibit CLOCK/BMAL1-mediated transcription and, as a consequence, shutdown their own expression (9,15,22). Cyclic expression of Bmal1 was found to occur through transcriptional activation by the orphan nuclear receptor ROR␣ (36) and inhibition by 26).Immunohistochemical analysis of the SCN has revealed synchronous circadian patterns of abundance and nuclear localization of mCRY and mammalian PER (mPER) proteins (6, 15). Moreover, as shown for mPER2, nuclear accumulation do...

Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes

“…This switch likely mediates CRY function through the structurally-identified constriction, formed by the phosphate-binding and PKL protrusion motifs. Besides these motifs, the CRY C terminus may assist in CLOCK/BMAL1 repression through FAD-regulated intermolecular interactions, which may explain its ability to impart repressive function to a noncircadian PHR (54). The combined results presented here reveal that the substrate recognition site, specific for (6-4) photoproducts, the cofactor binding sites, and the Trp electron-transfer pathway show differences from those of bacterial PHR/CRY, consistent with a distinct DNA repair mechanism.…”

The intricate dance of post-translational modifications in the rhythm of life

“…When the CRY2 NLS sequence is mutated, it can no longer facilitate the nuclear translocation of PER2 in cell culture 47 . In addition, PER2 nuclear localization is enhanced by CRY1 and is dependent on CRY1 binding 48,49 . However, nuclear entry of PER1 and PER2 is also enhanced by dimerization with PER3 via its cytoplasmic localization domain (CLD, amino acids 330-389 for mPER3) and its NLS (amino acids 726-734) 50 .…”

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

“…VOLUME 288 • NUMBER 49 • DECEMBER 6, 2013 portion of the CRY1 protein has been unclear, with some evidence that it is involved in nuclear localization or BMAL binding (7,8,68) and other evidence that it is dispensable for core clock function (6). However, even in the latter case, some role for the C terminus in period control was noted (6).…”

“…Although the mechanism by which CRYs repress CLOCK/BMAL1 activity is not well understood, the PHR domain appears to be sufficient for this function as long as it includes a coiled coil domain near the junction between the PHR and the tail (5-7). The role of the C-terminal tails is more mysterious, although the CRY1 C terminus may have a role in nuclear localization and may facilitate BMAL1 binding (7,8), and phosphorylation of the CRY2 C terminus destabilizes the protein (9,10).…”