Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüp-pel-like factor 9 (Klf9), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain-and loss-offunction experiments showed strong antiproliferative effects of Klf9. Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner.glucocorticoids | skin cancer B iological rhythms regulate cellular and physiological processes ranging from milliseconds to years. A well-studied timing system is the circadian (∼24-h) clockwork that allows organisms to anticipate diurnal variations in environmental conditions such as light, food availability, oxidative stress, pathogen exposure, or temperature. In mammals, the central circadian pacemaker resides in the suprachiasmatic nucleus (SCN), located in the anterior hypothalamus. Oscillations of SCN neurons are cell-autonomous, self-sustained, and synchronized to external time cues (Zeitgebers) such as light (1). The SCN, in turn, synchronizes peripheral clocks by systemic time cues such as neuronal input, hormonal signaling (e.g., cortisol), body temperature, and possibly many others (2). Interestingly, most cells in peripheral tissues also possess cell-autonomous clockworks with a similar molecular makeup to SCN neurons. These peripheral clocks are thought to generate or amplify daytime-dependent physiological and metabolic functions in a tissue-specific manner by circadian regulation of clock-controlled genes (3, 4).On a molecular level, circadian oscillation is generated by interlocked transcriptional-translational feedback loops. The transcription factor dimer CLOCK/BMAL1 drives expression of target genes such as Periods (Per1-3) and Cryptochromes (Cry1-2) by binding to E-box elements in their promoters. The negative feedback is formed by PER/CRY protein complexes that shuttle back into the nucleus, where they block CLOCK/BMAL1-mediated transactivation, thereby inhibiting their own transcr...
In mammals, circadian rhythms are generated by delayed negative feedback, in which period (PER1-PER3) and cryptochrome (CRY1, CRY2) proteins gradually accumulate in the nucleus to suppress the transcription of their own genes. Although the importance of nuclear import and export signals for the subcellular localization of clock proteins is well established, little is known about the dynamics of these processes as well as their importance for the generation of circadian rhythms. We show by pharmacological perturbations of oscillating cells that nuclear import and export are of crucial importance for the circadian period. Live-cell fluorescence microscopy revealed that nuclear import of the key circadian protein PER2 is fast and further accelerated by CRY1. Moreover, PER2 nuclear import is crucially dependent on a specific nuclearreceptor-binding motif in PER2 that also mediates nuclear immobility. Nuclear export, however, is relatively slow, supporting a model of PER2 nuclear accumulation by rapid import, slow export and substantial nuclear degradation.
A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular period variation in circadian clock networks (clonal period heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity.
The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in their variability across subjects. We also identified small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin and presents a novel methodology to quantify sources of variability in human circadian rhythms.
Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms.
Circadian clocks are molecular timekeeping mechanisms that allow organisms to anticipate daily changes in their environment. The fundamental cellular basis of these clocks is delayed negative feedback gene regulation with PERIOD and CRYPTOCHROME containing protein complexes as main inhibitory elements. For a correct circadian period, it is essential that such clock protein complexes accumulate in the nucleus in a precisely timed manner, a mechanism that is poorly understood. We performed a systematic RNAi-mediated screen in human cells and identified 15 genes associated with the nucleo-cytoplasmic translocation machinery, whose expression is important for circadian clock dynamics. Among them was Transportin 1 (TNPO1), a non-classical nuclear import carrier, whose knockdown and knockout led to short circadian periods. TNPO1 was found in endogenous clock protein complexes and particularly binds to PER1 regulating its (but not PER2’s) nuclear localization. While PER1 is also transported to the nucleus by the classical, Importin β-mediated pathway, TNPO1 depletion slowed down PER1 nuclear import rate as revealed by fluorescence recovery after photobleaching (FRAP) experiments. In addition, we found that TNPO1-mediated nuclear import may constitute a novel input pathway of how cellular redox state signals to the clock, since redox stress increases binding of TNPO1 to PER1 and decreases its nuclear localization. Together, our RNAi screen knocking down import carriers (but also export carriers) results in short and long circadian periods indicating that the regulatory pathways that control the timing of clock protein subcellular localization are far more complex than previously assumed. TNPO1 is one of the novel players essential for normal circadian periods and potentially for redox regulation of the clock.
In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila. Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the “kamikaze” properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.
11A ubiquitous feature of circadian clocks across life forms is its organization as a network of 12 coupled cellular oscillators. Individual cellular oscillators of the network often exhibit a 13 considerable degree of heterogeneity in their intrinsic periods. While the interaction of coupling 14 and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, 15 our knowledge of mechanisms governing network heterogeneity remains elusive. In this study, 16 we aimed to explore the principles that underlie inter-cellular period variation in circadian clock 17 networks (clonal period-heterogeneity). To this end, we employed a laboratory selection 18 approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging 19 from 22 h to 28 h. We report that while a single parent clone can produce progeny clones with a 20 wide distribution of circadian periods, heterogeneity is not entirely stochastically driven but has 21 a strong heritable component. By quantifying the expression of 20 circadian clock and clock-22 associated genes across our panel, we found that inheritance of different expression patterns in 23 at least three clock genes might govern clonal period-heterogeneity in circadian clock networks. 24 Furthermore, we provide preliminary evidence suggesting that epigenetic variation might 25 underlie such gene expression variation. 26 27 Evans et al., 2018). In this study, we aimed to explore the possible mechanisms underlying clonal-55 heterogeneity of circadian period in human circadian oscillator cells. 56 We hypothesised that clonal period-heterogeneity in mammalian cells is due to a) stochastic 57 variation (Geva-Zatorsky et al., 2006; Chang et al., 2008; Brock, Chang and Huang, 2009; Frank 58 and Rosner, 2012) and/or b) heritable variation (Dubnau and Losick, 2006; Gordon et al., 2009, 59 2013). Since the term 'stochastic' is used in the context of both non-heritable (external noise and 60 gene expression noise) as well as heritable gene expression variation (epigenetic stochasticity), 61 for the rest of this manuscript we define 'stochasticity' as any non-heritable variation (both 62 internal and external). To test the two hypothesis outlined above, we employed a laboratory 63 selection approach and derived a panel of 25 clonal cell lines (from a common ancestral/founding 64 culture) exhibiting a range of periods between 22h and 28h. We observed that the period-65 heterogeneity among progeny clones stemming from a single parent cell is not entirely stochastic 66 but has a substantial heritable component. We then measured expression of 20 clock and clock-67 associated genes in our panel and observed that variation in gene expression levels of at least 68 three clock genes (transcription factors) might underlie clonal period-heterogeneity. 69 Furthermore, we provide preliminary evidence that epigenetic variation might govern the 70 observed clonal-variation in gene expression. 71 72 73 74 75 157 period while D...
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