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Precise protein-protein interactions contribute to 24-hour timekeeping in mammals
Full text linkNearly all walks of life, from single cell cyanobacteria to humans, have evolved an intimate connection to the light dark cycle and coordinate physiology and behavior to the solar day. This phenomenon is known as circadian rhythms and is an adaptation that organisms use to anticipate daily environmental changes. In mammals, disruption of circadian rhythms through environmental stimulus or genetic means leads to the onset of many diseases such as: diabetes, cardiovascular disease, premature aging and cancer. Nearly every cell in the human body has an endogenous molecular clock that controls integrated biochemical processes on a ~24-hour period. At the core of this molecular clock is the heterodimeric basic helix-loop-helix Per:Arnt:Sim (bHLH-PAS) transcription factor CLOCK:BMAL1 that together with its repressors, Period (PER) and Cryptochrome (CRY), forms an autoregulatory feedback loop that results in the rhythmic transcription of nearly 40% of the genome including essential genes in metabolism, hormone secretion and the cell cycle. While this model for transcription-translation feedback loop has been accepted for over two decades, it is still not well understood how CLOCK:BMAL1 activity is directly regulated to generate intrinsic 24-hour timing in mammals. Using a combination of biochemistry, structural and cell biology we have elucidated a mechanism by which CRY1 directly interacts with CLOCK:BMAL1 to form a critical repressive circadian complex in circadian rhythms. We have also discovered an uncharacterized PAS domain containing protein in humans, PAS Domain containing protein 1 (PASD1), that inhibits CLOCK:BMAL1 activity and suppresses circadian cycling in cancer cells, providing a molecular link from oncogenesis to circadian disruption.Chapter 2 describes our work to elucidate how CRY1 directly inhibits CLOCK:BMAL1 activity. CRYs close the tightly regulated transcriptional feedback loop to control circadian rhythms, however, the mechanistic underpinnings of how CRYs interact with and repress CLOCK:BMAL1 have remained elusive. Previous studies in our lab showed that tuning affinity of CRY1 for the transactivation domain (TAD) of BMAL1 controls circadian period by competing with the coactivator CBP/p300. CRY1 also binds to CLOCK, although it was not yet understood how multivalent interactions with CLOCK:BMAL1 contribute to CRY1 function. I have shown that CRY1 directly binds the CLOCK:BMAL1 PAS-AB core and this interaction is driven by a single domain in this multi-domain protein complex, CLOCK PAS-B. Furthermore, I have worked with experts in small angle x-ray scattering analysis to generate a model of the CRY1:CLOCK:BMAL1 complex that situates CRY1 atop the CLOCK:BMAL bHLH PAS-AB domains in the solution envelope providing the first low resolution description of the CRY1:CLOCK:BMAL1 complex. These studies have paved the way for circadian-directed therapeutics and have provided a basis for comparative analysis between the CRY1 and CRY2 proteins as described in Chapter 5. Further studies on the assembly of circadian repressive complexes, including CRY and PERIOD proteins, is described in Chapter 6. In Chapter 4 we report the discovery of a previously uncharacterized repressor of circadian rhythms, PAS domain containing protein 1 (PASD1). Upon joining Dr. Partch’s lab I chose to work on the “long-shot project” – an interesting but undeveloped project that was based entirely off Dr. Partch’s initial discovery of an uncharacterized gene that bears significant similarity to a clock protein, potentially possessing the ability to modulate the circadian clock. This CLOCK-like protein, PASD1, is not expressed in healthy somatic tissues, but is instead limited to gametogenic tissues where there are no functional clocks. Using transcriptional reporter assays I confirmed initial results that PASD1 inhibits transactivation of genes by the core circadian transcription factor, CLOCK:BMAL1. Through series of truncation experiments I found that the C-terminus of PASD1 is sufficient to repress CLOCK:BMAL1 in the nucleus. Furthermore, deletion of one region that is highly conserved with CLOCK alleviates repression by PASD1 to suggest it utilizes molecular mimicry to interfere with CLOCK:BMAL1 function. Furthermore, knockdown of PASD1 in both colon and lung cancer cell lines improves the amplitude of cycling, indicative of a more robust oscillator. These data together provide a tool to rescue the clock in PASD1+ tumors. In summary, I have used biochemistry and biophysics to describe how the essential circadian protein, CRY1, serves as a potent repressor of CLOCK:BMAL1 activity to establish circadian rhythms. This work provides molecular details of the CRY1-CLOCK:BMAL1 interaction that are being actively used to create circadian-based therapeutics and study the structure of circadian repressive complexes. I have also used cell biology to identify a novel repressor of CLOCK:BMAL1 that is highly up-regulated in many forms of cancer and suppresses circadian cycling
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The Cellular Consequences of PASD1 Expression in Human Cancer
Full text linkABSTRACTThe Cellular Consequences of PASD1 Expression in Human CancerAshley M. KernThere are nearly 1,000 human proteins that are expressed only in the germline. Cancer /Testis antigens (CT antigens) represent a subset of germline-specific proteins that become reactivated in somatic cells that have undergone oncogenic transformation. Human PAS Domain containing protein 1 (PASD1) is an X-linked CT Antigen. In 2015, the Partch Laboratory established that PASD1 prevents the core circadian transcription factor, CLOCK:BMAL1, from rhythmically transcribing its target genes to suppress circadian rhythms after it becomes upregulated in cancer cells. In addition to this striking phenotype, PASD1 also promotes mitotic defects that could favor tumor progression. These phenotypes may go hand in hand; over 43% of the mammalian genome, including many cell cycle genes, is under circadian control. In this study, we found that expression of PASD1, promotes mitotic arrest, slows DNA replication, and diminishes the effects of mitotic spindle poisoning. Interestingly, preliminary data demonstrate that PASD1 is natively expressed in spermatogonia, which are testicular stem cells that undergo repeated asymmetric mitotic divisions to give rise to progenitor cells. These data support the hypothesis that expression of PASD1 in somatic cancer cells promotes premature mitotic entry, which could promote oncogenic transformation in somatic cells. The cell cycle phenotypes we observe in our study could be a consequence of reactivating the native functions of PASD1. Further studies will be required to determine if these phenotypes are due to PASD1-mediated clock repression, or whether they arise from distinct cellular pathways regulated by PASD1 in somatic cancer cells
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Mechanism and quantitation of cooperative interactions in the cyanobacterial circadian oscillator
Full text linkCyanobacteria are photosynthetic microbes that have shaped the very environment of the world we live in over several billion of years. This is, in large part, because of their ability to perform the chemically exclusive processes of photosynthesis and nitrogen fixation. To segregate these processes temporally, they have evolved an elegant and complex circadian clock that aligns their physiology with the solar day to maximize biological fitness. This clock keeps time through the action of a biochemical oscillator comprising just three proteins: KaiA, KaiB and KaiC, that, along with ATP, can recapitulate a 24-h pacemaking activity in vitro. This process is achieved by a negative feedback loop akin to a biochemical game of Rock, Paper, Scissors, whereby the phosphorylation state of the hexameric ATPase KaiC is controlled by the nucleotide exchange factor KaiA, the ability of KaiA to stimulate KaiC repression is controlled by the metamorphic protein KaiB, and the ability of KaiB to inactivate KaiA is controlled by KaiC phosphorylation state. This creates a repetitive biochemical cycle that takes just bout 24 hours to complete. Additionally, the output proteins SasA and CikA interact with the clock in various phases of the biochemical oscillation that influence their ability to activate the master circadian transcription factor RpaA and orchestrate circdain gene expression throughout the cyanobacterial cell.This thesis focuses on the relationship between KaiC and KaiB. In particular, on positive cooperativity that increases KaiB’s affinity for KaiC, or in other words, the process by which initial binding of KaiB to the KaiC hexamer enables more efficient recruitment of KaiB to the remaining 5 binding sites. While this effect is well documented when considering KaiB and KaiC on their own, in Chapter 2 we expand this concept in the in the context of the entire reconstituted clock system including output pathways that link biochemical oscillation to DNA binding by the transcription factor RpaA. In doing so, we uncovered the unexpected result that SasA expands the range of permissive KaiB concentrations for biochemical oscillation to occur. I showed that SasA does this by binding to KaiC analogously to how KaiB does, and then recruiting additional KaiB molecules through positive heterotropic cooperativity. Integrating a novel crystal structure of the interacting domains of KaiC and SasA with existing crystal structures of the KaiC hexamer, I identified SasA mutations that abrogate heterotropic cooperativity but have only minor effects on the output signaling function of SasA. Remarkably, cyanobacteria bearing these mutations have defective circadian rhythms, demonstrating that SasA’s ability to bolster KaiB recruitment is an evolved aspect of biochemical oscillation.Chapter 3 describes our structural analysis of phosphomimetic variants of KaiC that differ in their ability to bind KaiB. Because crystallography has failed to identify the structural basis of this discrimination previously, we employed cryo-electron microscopy to analyze KaiC particles as a structural ensemble frozen in ice. Each KaiC protomer is composed of two ATPase domains, termed CI and CII. The phase-determining phosphosites are located on CII, and KaiB binds to KaiC over 70 Å away to the ADP-bound form of CI. Our data corroborated previous studies that daytime KaiC, which does not bind KaiB, loses interactions amongst the CII domain protomers within the hexamer, while maintaining a hexameric CI domain. Additionally, we obtained a relatively high resolution (3.2 Å) structure of a compressed form of KaiC where the CII domain breaks into a split washer with 2-fold symmetry, causing two of the CII domain subunits to interact more tightly with their respective CI domains. Using mutagenesis and various functional assays, I identified an allosteric conduit that connects the ATPase domains of the CI and CII domains of KaiC in both intra-protomer and inter-protomer contexts. Importantly, I trace these interactions back to cooperativity in KaiB association, with mutants along the pathway disrupting both KaiB affinity and cooperativity. Furthermore, I link KaiB cooperativity to ATP hydrolysis in the CI domain by identifying a key residue that senses CI nucleotide state. This residue is dispensable for both ATP hydrolysis as well as KaiB association, but is critical for both KaiB cooperativity and in vivo circadian rhythms. This, along with additional nucleotide dependence studies reported in Chapter 4, suggests that allosteric control of cooperativity through the CI active site is the structural basis for restriction of KaiB association to the nighttime KaiC phosphostates.Finally, in the latter half of Chapter 4 I summarize experiments that used the solvatocromatic dye Sypro Orange to detect changes in KaiC structure as a function of temperature and mutagenesis. I describe our preliminary efforts to build on these results by identifying additional solvatochromatic dyes that can leverage this effect to report continuously on the phase of biochemical oscillation by discriminate binding to different KaiC phosphostates at constant temperature
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Modulation of circadian cycling by the C-terminal transactivation domain of BMAL1
Full text linkNearly all terrestrial organisms possess an intrinsic biological timekeeping system that functions to align key physiological processes with the solar day. In mammals, this circadian cycling is driven by temporally specific interactions between the heterodimeric transcription factor, CLOCK:BMAL1 and its cognate transcriptional regulators. The biochemical processes that drive the handoff between active and repressive complexes tunes the phase, period and robustness of circadian cycling. In this study, we show that the unstructured C-terminal transactivation domain (TAD) of BMAL1 is a regulatory hub where transcriptional activators and repressors compete for binding. Using NMR spectroscopy and real time monitoring of circadian cycling in mammalian cells we studied how interactions of the TAD with transcriptional regulators control the generation of circadian rhythms. We show that the KIX and TAZ1 domains of CBP and the CC helix of CRY1 interact with the BMAL1 TAD at overlapping sites. Perturbations of these regions of BMAL1 by site-directed mutagenesis have direct consequences for the balance of transcriptional activation and repression and elicit large changes the period of circadian oscillations. Using paramagnetic resonance spectroscopy we show that the BMAL1 TAD undergoes significant and differing conformational rearrangements upon binding CBP KIX and CRY CC. Furthermore, we identified a slow conformational change in the extreme C-terminus of BMAL1, a region of the TAD that is essential for normal circadian timekeeping. Using NMR spectroscopy, we performed structural and kinetic analysis of the conformational switch to identify a cis/trans isomerization about a Trp-Pro imide bond. Using NMR, fluorescence polarization and cell-based studies, we further characterized the switch to assess its role in circadian cycling and identified cyclophilins capable of regulating the rate of switch interconversion in vitro. Together, these data highlight the importance of structural dynamics of the BMAL1 TAD in tuning the molecular circadian clock
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Casein Kinase 1δ Splice Isoforms Differentially Regulate Kinase Activity Through Their Intrinsically Disordered C-terminus
Full text linkCasein Kinase 1δ influences the timing of the mammalian clock by regulating PER2 stability through post-translational modifications. CK1δ regulates PER2 through a phosphoswitch mechanism, in which phosphorylation of the FASP (Familial Advanced Sleep Phase) region of PER2 stabilizes the protein to lengthen clock period, while phosphorylation of the degron promotes its degradation to shorten clock period. This phosphoswitch is held in delicate balance to control proper timing of the clock; mutations in CK1δ and PER2 influence the selectivity of the kinase on this switch and result in sleep phase disorders. Two CK1δ splice variants, δ1 and δ2, that differ only in the last 15 residues, show different kinase activity towards the FASP region, which suggests that the extreme C-terminus of CK1δ is a regulator of this switch. Although autophosphorylation of the disordered C-terminus of CK1δ is known to inhibit its kinase activity, it is not clear how this might differ between the CK1δ splice variants. Using Hydrogen/Deuterium Exchange-Mass Spectrometry (HDX-MS), we have begun to map the differences in tail interaction with the catalytic kinase domain of CK1δ. Nuclear Magnetic Resonance (NMR) studies have also been used to measure autophosphorylation rates of the δ1 tail in trans and in cis, and to shed light on potential differences in intramolecular interactions of the tail isoforms. Coupled with biochemical assays using full length kinase mutants, we demonstrate that anion binding sites on the kinase domain may influence the difference in activity between the splice isoforms. Inhibition assays with tail phosphopeptides and full-length tail mutants have also shown a difference in phosphorylation dependence between the splice variants. A deeper comparison of intermolecular interactions in CK1δ and the effects of phosphorylation in splice variants will shed light on how they exhibit differential activity on the PER2 phosphoswitch to control circadian timing
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Interactions with and within mammalian cryptochromes regulate circadian rhythms
Full text linkOrganisms across all kingdoms of life have an internal 24-hour timekeeping mechanism known as circadian rhythms. In mammals, circadian rhythms arise from interlocking transcription-translation feedback loops which include the transcription factor CLOCK:BMAL1 driving the transcription of key repressors such as cryptochrome (CRY1 and CRY2) and period (PER1 and PER2). CRY1 and CRY2 are both similarly composed of a conserved structured domain known as the photolyase homology region (PHR) that is tethered to an intrinsically disordered C-terminal tail. While the PHR is necessary and sufficient to directly interact with CLOCK:BMAL1 to induce repression, the C-terminal tails also play a role in regulating circadian timing. In other CRY homologs such as Drosophila CRY, the PHR and C-terminal tail reversibly bind each other to create an autoinhibited conformation. In this study, we demonstrate how interactions with CRY molecules (e.g. CLOCK interacting with CRY1/2) and interactions within a CRY protein molecule (e.g. the PHR-tail interaction) regulate circadian rhythms interactions. In Chapter 2, we describe how CRY1 and CRY2 play divergent roles in circadian timing due to structural differences in the secondary pockets of CRY1 and CRY2 that lead to differences in how strongly CRY1 or CRY2 interact with CLOCK. In Chapter 3, we determined that the CRY1 C-terminal tail makes an autoinhibitory interaction with the CRY1 PHR and inhibits the CRY1–CLOCK interaction at the secondary pocket. We also found that CRY1Δ11 (a prevalent mutation that extends circadian period and causes delayed sleep phase disorder) enhances the interaction between CRY1 and CLOCK by removing an autoinhibitory region on the CRY1 tail. In Chapter 4, we describe work with collaborators and identify how a cancer-related CRY2 mutation alters circadian timing by weakening the interaction between CRY2 and CLOCK at the secondary pocket. In the final chapters of this dissertation, we describe how small molecules that target interactions “with and within” CRYs can modulate circadian rhythms
Casein Kinase 1δ and PERIOD2 regulate circadian rhythms through a combination of substrate selectivity and feedback inhibition
Full text linkBiological clocks such as circadian rhythms are perhaps among the most fundamentally conserved adaptations of organisms that have evolved under the light/dark cycle of earth. These rhythms confer an advantage to organisms by allowing them to anticipate daily environmental changes. While the genetic networks that govern circadian rhythms in mammals are now fairly well-established, a picture of the molecular mechanisms that regulate the intrinsic timing of circadian rhythms is only recently beginning to emerge. In this dissertation, I discuss the molecular level details of the mammalian clock and provide new insights that shed light on the biochemical mechanisms of period control within.Chapter 2 describes how Casein Kinase 1δ and ε (CK1) post- translationally control PERIOD (PER) stability. CK1 is a deeply conserved circadian protein, yet little is known about its regulation of functionally antagonistic sites in PER that control circadian timing. The balance of CK1 activity within these two regions defines a model of PER2 stability known as the phosphoswitch. In this chapter, we discover an allosteric conformational
switch in the CK1 activation loop segment that influences substrate specificity on PER2 to directly regulate its stability. We further show that period-altering mutations of the kinase across organisms differentially modulate the activation loop switch and provide a framework to understand and manipulate CK1 regulation of circadian period.
PER proteins are fundamental in defining the phase and timing of circadian rhythms, likely due to their role as stoichiometrically limiting factors in the assembly of repressive complexes that provide feedback inhibition of transcription within the clock. CK1-dependent changes in PER abundance are therefore central to circadian timing. CK1 phosphorylation of PER2 is mediated by the stable anchoring of CK1 to PER2 via the Casein Kinase 1 Binding Domain (CK1BD). This stable interaction is also required for CK1-mediated displacement of CLOCK from DNA. Chapter 3 describes the role of CK1 phosphorylation of the PER2 FASP region in the regulation of PER2 stability and repressive activity. We show that the phosphorylated FASP region (pFASP) directly interacts with and inhibits CK1δ, and that stable anchoring to the CK1BD increases the kinetics of FASP phosphorylation and product inhibition. We solve multiple crystal structures of CK1δ bound to pFASP and conduct accelerated molecular dynamics simulations to reveal a mechanism of inhibition where phosphoserines in pFASP anchor into conserved anion binding sites along the substrate binding cleft and active site of the kinase. We further show how limiting phosphorylation within the FASP region reduces product inhibition and find that feedback inhibition is a conserved mechanism within Drosophila PER.
Much of the work in this dissertation focuses on the molecular determinants that regulate the stabilizing arm of the PER2 phosphoswitch. Chapter 4 discusses the molecular features of PER2 degradation and provides a survey of the current state of my contributions to this area. I provide a framework for extending previous studies using reagents from mPER2 into hPER2, and further discuss future directions to shed light on mechanisms of regulation for CK1 activity within the PER2 Degron and the recruitment of b- TrCP.
In summary, throughout this dissertation I have used an integrative approach of utilizing biochemistry, biophysics, molecular dynamics, and tissue culture to describe how CK1 and PER2 form a critical regulatory nexus within the mammalian circadian clock. In addition to the findings discussed herein, this work has provided a framework for targeted mutations to further develop a molecular level understanding of circadian timekeeping, as well as an avenue to develop novel therapeutics to target the clock and modulate circadian period
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Temporal Regulation of Nematode Development from a Biochemical, Circadian Perspective
No full textTiming mechanisms are utilized by organisms in a variety of biological functions. From circadian rhythms to nematode development, the genetic networks that underly the keeping of time are complex and thoroughly regulated. Circadian rhythms allow organisms to anticipate daily environmental changes and thus confer an adaptive advantage and the genetic network that governs them is well-established. While much is still to be gleaned about the molecular basis of circadian timekeeping, a model of a rewired developmental timer based on conserved circadian clock orthologs C. elegans, is beginning to emerge. In this dissertation, I discuss the conservation of specific factors and provide new insights that highlight the biochemical mechanisms that regulate C. elegans development. C. elegans are a widely studied model organism, yet little is known about the molecular basis for its temporal control of development. Two intricately linked timers, the molting cycle and heterochronic pathway, coordinate cuticle regeneration and growth with stage-specific cellular events. Several circadian orthologs have established roles in regulating these processes. Chapter 2 describes the homology between nuclear hormone receptors (NHRs), retinoic acid-related nuclear receptor (RORα/β/γ) and NHR-23, transcription factors that activate the expression of a core clock component and drives the transcriptional network that governs nematode molting, respectively. We lay the groundwork for a conserved mode of ligand-binding, as well as identify a separate class of small molecules that bind to NHR-23.
The interaction between PERIOD (PER) proteins and its cognate kinase, Casein Kinase 1 and ε (CK1), is integral to determining the phase and timing of circadian rhythms. PER is a stoichiometrically limiting factor in the repressive complex that provides the inhibition of circadian transcription. Stable anchoring of CK1 to PER2 mediates phosphorylation of PER that regulates its stability and abundance in the cell. This interaction is also required for CK1-dependent displacement of the core clock transcription factor from DNA. Chapter 3 demonstrates the C. elegans homologs to PER and CK1, LIN-42 and KIN-20, respectively, interact in a similar mode to regulate C. elegans development. We show that two kinase-binding motifs within the CK1-binding domain (CK1BD; CK1BD-A and CK1BD-B) are conserved enough in LIN-42 to mediate binding to CK1 in vitro. We determine that the expression of LIN-42 and KIN-20 temporally and spatially overlaps and that the CK1BD as well as KIN-20 kinase activity are required from proper molting timing. We further show that phosphorylation of LIN-42 by CK1 leads to kinase inhibition, suggesting a conserved mode of product inhibition whereby phosphoserine(s) anchor into conserved anion binding sites along the kinase active site.
In chapter 4, we discuss our recent work to identify a novel regulator of the C. elegans molt cycle. Through in vivo techniques, we show that KIN-20 and a previously uncharacterized ankyrin repeat domain-containing protein (ANKRD49), are similarly expressed temporally and spatially, and interact. We show that C. elegans ANKRD49 binds to human CK1 with nanomolar affinity in vitro, and that this interaction influences kinase activity on LIN-42. An AlphaFold binding model of the complex predicts that stable binding is mediated through the ANKRD49 structured C-terminus and a flexible CK1 helix near the active site that is important for substrate recognition and processing. This model also predicts that the interaction is enhanced via binding of the ANKRD49 unstructured N-terminus to the CK1 substrate binding cleft. We show that deletion of the ANKRD49 unstructured N-terminus as well as mutations near and on the flexible CK1 helix that alter circadian period in mammals, reduce the C. elegans ANKRD49/human CK1 affinity >10-fold. Depletion of ANKRD49 in vivo leads to asynchronous and delayed molting similar to kin-20(null) phenotypes. Given the high conservation of CK1 across organisms as well as several proteome-scale studies that also identify a human ANKRD49/CK1 interaction, this work potentially has broader implications for understanding circadian rhythms and temporal regulation in diverse organisms.
In summary, throughout this dissertation I have used an interdisciplinary approach of utilizing biochemistry and in vivo C. elegans genetics, to describe the molecular basis for circadian homolog function in C. elegans development. In addition to the findings discussed herein, this work provides a framework elucidating the molecular underpinnings of nematode timing mechanisms as well as an additional insight into evolutionary conservation of timekeeping mechanisms
Signal transduction mechanisms of cryptochrome
Full text linkPhotolyase and cryptochrome flavoproteins help living organisms manage the deleterious and beneficial effects of sunlight. Photolyase maintains genome integrity by reversing UV-induced DNA damage with near-UV/blue-light, and cryptochromes act as bluelight photosensory receptors to regulate growth in plants and entrainment of circadian rhythms in both plants and animals. Although photolyase and cryptochrome are highly structurally homologous and the photocycle of photolyase is known in great detail, we do not currently understand how cryptochromes signal in response to light. It is hypothesized that cryptochrome, like photolyase, employs light-driven electron transfer to initiate signaling, although the photocycle and other downstream signaling events remain to be described in detail. The studies described here were designed to take advantage of differences and similarities in the known functions of photolyases and cryptochromes in order to characterize the signaling mechanisms of cryptochromes. An examination of the structural and biochemical properties of plant and animal cryptochromes demonstrates that although they evolved independently from functionally distinct photolyase progenitors, they possess several unexpected similarities, demonstrating convergence in the evolution of cryptochromes. The implications of these results for the cryptochrome photocycle are discussed. Metazoan cryptochromes additionally have a critical, light-independent function in the molecular clock that engenders circadian rhythms. Other studies have shown that iv cryptochromes act as transcriptional repressors in the major transcription/translation feedback loop of the clock. I studied the interaction of mammalian cryptochromes with protein phosphatase 5 (PP5) and show that inhibition of PP5 by cryptochrome modulates the activity of the major clock kinase, casein kinase I epsilon. PP5 is required for proper cycling of the clock; therefore, these data provide the first demonstration of the role of a phosphatase in the mammalian circadian clock. Furthermore, they suggest that cryptochromes regulate the molecular clock by both transcriptional and posttranslational mechanisms
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