We show that, in the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2(Brdm1) mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with a dominant role of PERIOD2 in the positive regulation of the Bmal1 loop. In vitro analysis of CRYPTOCHROME inhibition of CLOCK: BMAL1-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the PERIOD and TIMELESS proteins. PERIOD2 is a positive regulator of the Bmal1 loop, and CRYPTOCHROMES are the negative regulators of the Period and Cryptochrome cycles.

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

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...

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Background: Breastfeeding is considered the most optimal mode of feeding for neonates and mothers. Human milk changes over the course of lactation in order to perfectly suit the infant’s nutritional and immunological needs. Its composition also varies throughout the day. Circadian fluctuations in some bioactive components are suggested to transfer chronobiological information from mother to child to assist the development of the biological clock. This review aims to give a complete overview of studies examining human milk components found to exhibit circadian variation in their concentration. Methods: We included studies assessing the concentration of a specific human milk component more than once in 24 h. Study characteristics, including gestational age, lactational stage, sampling strategy, analytical method, and outcome were extracted. Methodological quality was graded using a modified Newcastle-Ottawa Scale (NOS). Results: A total of 83 reports assessing the circadian variation in the concentration of 71 human milk components were included. Heterogeneity among studies was high. The methodological quality varied widely. Significant circadian variation is found in tryptophan, fats, triacylglycerol, cholesterol, iron, melatonin, cortisol, and cortisone. This may play a role in the child’s growth and development in terms of the biological clock.