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Noctumin (NOC) is a circadian-regulated protein related to the yeast family of transcription factors involved in the cellular response to nutrient status. In mammals, NOC functions as a deadenylase but lacks a transcriptional activation domain. It is highly expressed in bone-marrow stromal cells (BMSCs), hepatocytes, and adipocytes. In BMSCS exposed to the PPARγ (peroxisome proliferator-activated receptor-γ) agonist rosiglitazone, Noc expression was enhanced 30-fold. Previously, we reported that N0C' mice had low body temperature, were protected from diet-induced obesity, and most importantly exhibited absence of Pparg circadian rhythmicity on a high-fat diet Consistent with its role in influencing BMSCs allocation, Noc-/- mice have reduced bone marrow adiposity and high bone mass. In that same vein, NOC overexpression enhances adipogenesis in 3T3-L1 cells but negatively regulates osteogenesis in MC3T3-E1 cells. NOC and a mutated form, which lacks deadenylase activity, bind to PPARγ and markedly enhance PPAR-γ transcriptional activity. Both WT and mutant NOC facilitate nuclear translocation of PPARγ. Importantly, NOC-mediated nuclear translocation of PPAR-γ is blocked by a short peptide fragment of NOC that inhibits its physical interaction with PPAR-γ. The inhibitory effect of this NOC-peptide was partially reversed by rosiglitazone, suggesting that effect of NOC on PPAR-γ nuclear translocation may be independent of ligand-mediated PPAR-γ activation. In sum, Noc plays a unique role in the regulation of mesenchymal stem-cell lineage allocation by modulating PPARγ activity through nuclear translocation. These data illustrate a unique mechanism whereby a nutrientresponsive gene influences BMSCs differentiation, adipogenesis, and ultimately body composition. [ABSTRACT FROM AUTHOR]

Background: Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. Results: cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. Conclusion: The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Cryptochromes (CRYs) are transcriptional repressors that are critical components of the circadian clock. Results: We have identified a phosphorylation site in the CRY1 tail that is negatively regulated by the DNA repair enzyme DNA-dependent protein kinase. Conclusion: Phosphorylation of CRY1 on Ser-588 increases its half-life and lengthens the circadian period. Significance: The C-terminal tail of CRY1 modulates period length. [ABSTRACT FROM AUTHOR]

Circadian clocks regulate numerous physiological processes that vary across the day-night (diurnal) cycle, but if and how the circadian clock regulates the adaptive immune system is mostly unclear. Interleukin-17-producing CD4+ T helper (TH17) cells are proinflammatory immune cells that protect against bacterial and fungal infections at mucosal surfaces. Their lineage specification is regulated by the orphan nuclear receptor RORgt. We show that the transcription factor NFIL3 suppresses TH17 cell development by directly binding and repressing the Rorgt promoter. NFIL3 links TH17 cell development to the circadian clock network through the transcription factor REV-ERBa. Accordingly, TH17 lineage specification varies diurnally and is altered in Rev-erba-/- mice. Light-cycle disruption elevated intestinal TH17 cell frequencies and increased susceptibility to inflammatory disease. Thus, lineage specification of a key immune cell is under direct circadian control. [ABSTRACT FROM AUTHOR]

The circadian clock in mammals is driven by an autoregulatory transcriptional feedback mechanism that takes approximately 24 hours to complete. A key component of this mechanism is a heterodimeric transcriptional activator consisting of two basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain protein subunits, CLOCK and BMAL1. Here, we report the crystal structure of a complex containing the mouse CLOCK:BMAL1 bHLH-PAS domains at 2.3 Å resolution. The structure reveals an unusual asymmetric heterodimer with the three domains in each of the two subunits—bHLH, PAS-A, and PAS-B--tightly intertwined and involved in dimerization interactions, resu|ting in three distinct protein interfaces. Mutations that perturb the observed heterodimer interfaces affect the stability and activity of the CLOCK:BMAL1 complex as well as the periodicity of the circadian oscillator. The structure of the CLOCK:BMAL1 complex is a starting point for understanding at an atomic level the mechanism driving the mammalian circadian dock. [ABSTRACT FROM AUTHOR]

The circadian clock drives rhythms in the levels of thousands of proteins in the mammalian cell, arising in part from rhythmic transcriptional regulation of the genes that encode them. However, recent evidence has shown that posttranscriptional processes also play a major role in generating the rhythmic protein makeup and ultimately the rhythmic physiology of the cell. Regulation of steps throughout the life of the messenger RNA (mRNA), ranging from initial mRNA processing and export from the nucleus to extensive control of translation and degradation in the cytosol have been shown to be important for producing the final rhythms in protein levels critical for proper circadian rhythmicity. These findings will be reviewed here., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

Within the retina there is a circadian clock that controls the 24-h timing of processes such as hormone release, cell movement, and gene transcription. In an effort to better understand the molecular nature of this retinal clock, a differential display (DD) screen was performed to isolate a gene with high amplitude circadian rhythmicity in the Xenopus retina. A novel gene expressed in the early evening in photoreceptor cells was isolated and named nocturnin for night factor. This article outlines the steps we took to study a protein of unknown function, particularly highlighting the analyses one can perform when little more than the primary sequence of a gene is known. In addition, we describe the results of sequence analysis that assisted in predicting the function of nocturnin. We have shown that nocturnin acts as a deadenylase in vitro, removing the poly(A) tail from a mature messenger RNA in a process that either leads to degradation or translational silencing of a message. Although the role of nocturnin in the retina is unknown, future studies to identify target mRNAs that are deadenylated by nocturnin will assist in elucidating its physiological role in this tissue.

The frog (Xenopus laevis) retina has been an important model for the analysis of retinal circadian rhythms. In this paper, several isoforms of X. laevis casein kinase I (CKI) were analyzed to address whether they are involved in the phosphorylation and degradation of period protein (PER), as they are in the circadian oscillators of other species. cDNAs encoding two splice variants of CKI(delta) (a full-length form and deletion isoform, which is missing an exon that encodes a putative nuclear localization signal and two evolutionarily conserved protein kinase domains) were isolated and analyzed, together with a previously isolated CKI(epsilon) isoform. Both CKI(delta) and CKI(epsilon) were shown to be constitutively expressed in the photoreceptors of the retina, where a circadian clock has been localized. Both the full-length CKI(delta) and CKI(epsilon) were shown to have kinase activity in vitro, and the full-length CKI(delta) phosphorylated and degraded Drosophila PER when expressed in Drosophila S2 cells. The expression and biochemical characteristics of these CKIs are consistent with an evolutionarily conserved role for CKI in the Xenopus retinal clock. The CKI(delta) deletion isoform did not exhibit kinase activity and did not trigger degradation of PER. Subcellular localization of both CKI(delta) isoforms was cytoplasmic in several cell culture lines, but the full-length CKI(delta) , and not the deletion CKI(delta) isoform, was localized to both the nucleus and the cytoplasm in Drosophila S2 cells. These results indicate that the sequences missing in the deletion CKI(delta) isoform are important for the nuclear localization and kinase activity of the full-length isoform and that one or both of these features are necessary for degradation of Drosophila PER.

Xenopus laevis retina is an important experimental model system for the study of circadian oscillator mechanisms, as light input pathways, central oscillator mechanisms, and multiple output pathways are all contained within this tissue. These retinas continue to exhibit robust circadian rhythms even after being maintained in culture for many days. The usefulness of this system has been improved even further by the development of a technique for simple genetic manipulation of these animals, which is complemented by expanded genomics resources (Xenopus genome project, microarray, etc.). By taking advantage of the transgenic technique in Xenopus described in this article, many types of analysis can be done on the primary transgenic animals within a couple of weeks after transgenesis. The availability of many cell-type-specific promoters and well-characterized cell types within the Xenopus retina provides the advantage of cell-specific modification of clock function using this method; in other words, contributions of different cell types within the circadian system can be analyzed independently by "molecular dissociation" of these cells. This article describes both how this transgenic technique is useful and various considerations that should be taken into account when these types of experiments are planned and interpreted. Application of these new techniques to studies of clock function provide an opportunity to rapidly assess gene expression and?or function in the context of the intact retina.

Xenopus laevis retinas, like retinas from all vertebrate classes, have endogenous circadian clocks that control many aspects of normal retinal physiology occurring in cells throughout all layers of the retina. The localization of the clock(s) that controls these various rhythms remains unclear. One of the best studied rhythmic events is the nocturnal release of melatonin. Photoreceptor layers can synthesize rhythmic melatonin when these cells are in isolation. However, within the intact retina, melatonin is controlled in a complex way, indicating that signals from many parts of the retina may contribute to the production of melatonin rhythmicity. To test this hypothesis, we generated transgenic tadpoles that express different levels of a dominant negative Xenopus CLOCK specifically in the retinal photoreceptors. Eyes from these tadpoles continued to produce melatonin at normal levels, but with greatly disrupted rhythmicity, the severity of which correlated with the transgene expression level. These results demonstrate that although many things contribute to melatonin production in vivo, the circadian clock localized in the retinal photoreceptors is necessary for its rhythmicity. Furthermore, these data show that the control of the level of melatonin synthesis is separable from the control of its rhythmicity and may be controlled by different molecular machinery. This type of specific "molecular lesion" allows perturbation of the clock in intact tissues and is valuable for dissection of clock control of tissue-level processes in this and other complex systems.