Core Circadian Clock Proteins Research Articles

The E3 Ligases Spsb1 and Spsb4 Regulate RevErbα Degradation and Circadian Period.

The time-dependent degradation of core circadian clock proteins is essential for the proper functioning of circadian timekeeping mechanisms that drive daily rhythms in gene expression and, ultimately, an organism's physiology. The ubiquitin proteasome system plays a critical role in regulating the stability of most proteins, including the core clock components. Our laboratory developed a cell-based functional screen to identify ubiquitin ligases that degrade any protein of interest and have started screening for those ligases that degrade circadian clock proteins. This screen identified Spsb4 as a putative novel E3 ligase for RevErbα. In this article, we further investigate the role of Spsb4 and its paralogs in RevErbα stability and circadian rhythmicity. Our results indicate that the paralogs Spsb1 and Spsb4, but not Spsb2 and Spsb3, can interact with and facilitate RevErbα ubiquitination and degradation and regulate circadian clock periodicity.

Unveiling the Impact of the Core Circadian Clock Protein FREQUENCY on Output in Neurospora crassa

Circadian rhythms are cellular oscillations that tune an organism's biology to the 24‐hour light/dark cycle on Earth. This cellular time‐keeping mechanism aids in the anticipation of environmental changes and increases organismal survival. These rhythms are maintained by a transcriptional/translational negative feedback loop, called the core circadian clock, that is evolutionarily conserved throughout most branches of life. In this core clock, positive arm proteins act to transcriptionally activate negative arm proteins. The negative arms proteins act back on the positive arm, in a time specific manner, to halt the negative arm's activation before degradation and re‐initiation, thus setting the period of the clock. Beyond this core clock, positive arm proteins also act to transcriptionally activate a variety of other genes that do not participate in the core clock mechanism in a time dependent manor, referred to as clock‐controlled genes (ccgs.) Most research into the mechanisms of circadian regulation on output has focused on transcriptional activation by the positive arm proteins. However, recently published work has demonstrated that there are many oscillating proteins for which the corresponding mRNA is not rhythmic. This has led to the theory that the regulation of circadian output is also accomplished via post‐translational mechanisms, which we hypothesize are imparted by the negative arm proteins.In the model organism Neurospora crassa, the negative arm comprises FREQUENCY (FRQ), Frequency‐Interacting RNA Helicase (FRH), and Casein Kinase 1 (CK‐1). Importantly, FRQ has been computationally and experimentally proven to be an intrinsically disordered protein (IDP). Aside from lacking a single, predetermined three‐dimensional structure, IDPs play key roles in cellular signaling and regulation due, in part, to the transient but highly specific and varied interacting partners they have. These attributes make FRQ a prime candidate to impart post‐translational circadian regulation. The aim of this study is to characterize protein complexes, centered around FRQ, that change in a time specific manner, to determine the impact these interactions could impart post‐translationally on circadian regulatory pathways. To this end, Neurospora samples where crosslinked and purified through co‐immunoprecipitation before protein identification through liquid chromatography mass spectrometry. Initial results suggest that FRQ plays a role in post‐translational regulation on metabolism, the cell cycle, and a variety of other functions.Support or Funding InformationThis work was funded through the support of the NIGMS/NIH R35GM128687 grant and the Rensselaer Polytechnic Institue Start Up Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy.

From an epidemiological standpoint, disruptions to circadian rhythms have been shown to contribute to the development of various disease pathologies, including breast cancer. However, it is unclear how altered circadian rhythms are related to malignant transformations at the molecular level. In this article, a series of isogenic breast cancer cells representing disease progression was used to investigate the expression patterns of core circadian clock proteins BMAL1 and PER2. Our model is indicative of 4 stages of breast cancer and includes the following cells: MCF10A (non-malignant), MCF10AT.Cl2 (pre-malignant), MCF10Ca1h (well-differentiated, malignant), and MCF10Ca1a (poorly differentiated, malignant). While studies of circadian rhythms in cancer typically use low-resolution reverse transcription polymerase chain reaction assays, we also employed luciferase reporters BMAL1:Luc and PER2:Luc in real-time luminometry experiments. We found that across all 4 cancer stages, PER2 showed relatively stable oscillations compared with BMAL1. Period estimation using both wavelet-based and damped-sine-fitting methods showed that the periods are distributed over a wide circadian range and there is no clear progression in mean period as cancer severity progresses. Additionally, we used the K-nearest neighbors algorithm to classify the recordings according to cancer line, and found that cancer stages were largely differentiated from one another. Taken together, our data support that there are circadian discrepancies between normal and malignant cells, but it is difficult and insufficient to singularly use period evaluations to differentiate them. Future studies should employ other progressive disease models to determine whether these findings are representative across cancer types or are specific to this series.

Cell-Autonomous Regulation of Astrocyte Activation by the Circadian Clock Protein BMAL1

Circadian clock dysfunction is a common symptom of aging and neurodegenerative diseases, though its impact on brain health is poorly understood. Astrocyte activation occurs in response to diverse insults and plays a critical role in brain health and disease. We report that the core circadian clock protein BMAL1 regulates astrogliosis in a synergistic manner via a cell-autonomous mechanism and a lesser non-cell-autonomous signal from neurons. Astrocyte-specific Bmal1 deletion induces astrocyte activation and inflammatory gene expression invitro and invivo, mediated in part by suppression of glutathione-S-transferase signaling. Functionally, loss of Bmal1 in astrocytes promotes neuronal death invitro. Our results demonstrate that the core clock protein BMAL1 regulates astrocyte activation and function invivo, elucidating a mechanism by which the circadian clock could influence many aspects of brain function and neurological disease.

The clock gene period differentially regulates sleep and memory in Drosophila.

Circadian regulation is a conserved phenomenon across the animal kingdom, and its disruption can have severe behavioral and physiological consequences. Core circadian clock proteins are likewise well conserved from Drosophila to humans. While the molecular clock interactions that regulate circadian rhythms have been extensively described, additional roles for clock genes during complex behaviors are less understood. Here, we show that mutations in the clock gene period result in differential time-of-day effects on acquisition and long-term memory of aversive olfactory conditioning. Sleep is also altered in period mutants: while its overall levels don't correlate with memory, sleep plasticity in different genotypes correlates with immediate performance after training. We further describe distinct anatomical bases for Period function by manipulating Period activity in restricted brain cells and testing the effects on specific aspects of memory and sleep. In the null mutant background, different features of sleep and memory are affected when we reintroduce a form of the period gene in glia, lateral neurons, and the fan-shaped body. Our results indicate that the role of the clock gene period may be separable in specific aspects of sleep or memory; further studies into the molecular mechanisms of these processes suggest independent neural circuits and molecular cascades that mediate connections between the distinct phenomena.

Just-So Stories and Origin Myths: Phosphorylation and Structural Disorder in Circadian Clock Proteins

Some longstanding dogmas in the circadian field warrant reexamination in light of recent studies focused on the role of post-translational modifications and intrinsic disorder in core circadian clock proteins of mice and fungi. Such dogmas include the role of turnover in circadian feedback loops and the origin myths describing evolutionary relatedness among circadian clocks. In this Essay, the authors recapitulate recent findings on circadian clock protein regulation by taking an unconventional approach in the form of a dialog between Wizard and Apprentice.

Disruption of normal circadian clock function in a mouse model of tauopathy

Disruption of normal circadian rhythm physiology is associated with neurodegenerative disease, which can lead to symptoms such as altered sleep cycles. In Alzheimer's disease (AD), circadian dysfunction has been attributed to β-amyloidosis. However, it is unclear whether tauopathy, another AD-associated neuropathology, can disrupt the circadian clock. We have evaluated the status of the circadian clock in a mouse model of tauopathy (Tg4510). Tg4510 mice display a long free-running period at an age when tauopathy is present, and show evidence of tauopathy in the suprachiasmatic nucleus (SCN) of the hypothalamus — the site of the master circadian clock. Additionally, cyclic expression of the core clock protein PER2 is disrupted in the hypothalamus of Tg4510 mice. Finally, disruption of the cyclic expression of PER2 and BMAL1, another core circadian clock protein, is evident in the Tg4510 hippocampus. These results demonstrate that tauopathy disrupts normal circadian clock function both at the behavioral and molecular levels, which may be attributed to the tauopathy-induced neuropathology in the SCN. Furthermore, these results establish the Tg4510 mouse line as a model to study how tauopathy disrupts normal circadian rhythm biology.

Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2

SummaryTranscriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5, 6, 7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock.

Modulation of glucocorticoid receptor induction properties by core circadian clock proteins

Glucocorticoid (GC) plays important roles in diverse physiological processes including metabolism and immune functions. While circadian control of GC synthesis and secretion is relatively well appreciated, circadian control of GC action within target tissues remains poorly understood. Here, we demonstrate that CLOCK/BMAL1, the core circadian clock components, reduces maximal GR transactivation (Amax) as well as efficacy (EC50) by a novel mechanism that requires binding to DNA and transactivation of target genes. Accordingly, we observe that PER1 and CRY1, the primary targets of CLOCK/BMAL1 action, reduce maximal GR transactivation while not affecting the efficacy. Moreover, we observe hyper-activations of GRE-dependent transcription in BMAL1- or PERs-deficient MEFs. In addition, endogenous GC target genes expression negatively correlates with the CLOCK/BMAL1 activity. Considering that GC sensitivity is widely implicated in human health and diseases, these results provide valuable insights into plethora of GC-related physiology and pathology.

Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina

Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue.

Circadian function in cancer: Regulating the DNA damage response

Proper circadian clock function is essential for the coordination of cellular functions within an organism in response to light and dark cycles. Although epidemiological studies have linked altered circadian rhythms to cancer susceptibility (1, 2), the molecular mechanisms tying the circadian clock to cancer are poorly understood. In PNAS, Lee and Sancar (3) demonstrate that loss of the core circadian clock proteins Cry1 and Cry2 can sensitize tumor cells to DNA damage-induced apoptosis. This effect is shown to be particularly relevant in cells that have lost p53 (TP53), a key tumor suppressor and mediator of chemosensitivity, which is mutated or deleted in the majority of human cancers. These findings imply unique roles for circadian rhythm in regulating the DNA damage response and point to a potential avenue for treating refractory p53-deficient cancers.

Circadian control of XPA and excision repair of cisplatin-DNA damage by cryptochrome and HERC2 ubiquitin ligase

Cisplatin is one of the most commonly used anticancer drugs. It kills cancer cells by damaging their DNA, and hence cellular DNA repair capacity is an important determinant of its efficacy. Here, we investigated the repair of cisplatin-induced DNA damage in mouse liver and testis tissue extracts prepared at regular intervals over the course of a day. We find that the XPA protein, which plays an essential role in repair of cisplatin damage by nucleotide excision repair, exhibits circadian oscillation in the liver but not in testis. Consequently, removal of cisplatin adducts in liver extracts, but not in testis extracts, exhibits a circadian pattern with zenith at approximately 5 pm and nadir at approximately 5 am. Furthermore, we find that the circadian oscillation of XPA is achieved both by regulation of transcription by the core circadian clock proteins including cryptochrome and by regulation at the posttranslational level by the HERC2 ubiquitin ligase. These findings may be used as a guide for timing of cisplatin chemotherapy.