In mammals, a master circadian "clock" resides in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN clock is composed of multiple, single-cell circadian oscillators, which, when synchronized, generate coordinated circadian outputs that regulate overt rhythms. Eight clock genes have been cloned that are involved in interacting transcriptional-/translational-feedback loops that compose the molecular clockwork. The daily light-dark cycle ultimately impinges on the control of two clock genes that reset the core clock mechanism in the SCN. Clock-controlled genes are also generated by the central clock mechanism, but their protein products transduce downstream effects. Peripheral oscillators are controlled by the SCN and provide local control of overt rhythm expression. Greater understanding of the cellular and molecular mechanisms of the SCN clockwork provides opportunities for pharmacological manipulation of circadian timing.

Molecular Analysis of Mammalian Circadian Rhythms

Familial advanced sleep phase syndrome (FASPS) is an autosomal dominant circadian rhythm variant; affected individuals are "morning larks" with a 4-hour advance of the sleep, temperature, and melatonin rhythms. Here we report localization of the FASPS gene near the telomere of chromosome 2q. A strong candidate gene (hPer2), a human homolog of the period gene in Drosophila, maps to the same locus. Affected individuals have a serine to glycine mutation within the casein kinase Iepsilon (CKIepsilon) binding region of hPER2, which causes hypophosphorylation by CKIepsilon in vitro. Thus, a variant in human sleep behavior can be attributed to a missense mutation in a clock component, hPER2, which alters the circadian period.

https://science.sciencemag.org/content/sci/291/5506/1040.full.pdf

An hPer2 Phosphorylation Site Mutation in Familial Advanced Sleep Phase Syndrome

https://www.cell.com/cell/pdf/S0092-8674(01)00610-9.pdf

Posttranslational mechanisms regulate the mammalian circadian clock.

The circadian clock is a widespread cellular mechanism that underlies diverse rhythmic functions in organisms from bacteria and fungi, to plants and animals. Intense genetic analysis during recent years has uncovered many of the components and molecular mechanisms comprising these clocks. Although autoregulatory genetic networks are a consistent feature in the design of all clocks, the weight of evidence favours their independent evolutionary origins in different kingdoms.

Time zones: a comparative genetics of circadian clocks.

During the past decade, the molecular mechanisms underlying the mammalian circadian clock have been defined. A core set of circadian clock genes common to most cells throughout the body code for proteins that feed back to regulate not only their own expression, but also that of clock output genes and pathways throughout the genome. The circadian system represents a complex multioscillatory temporal network in which an ensemble of coupled neurons comprising the principal circadian pacemaker in the suprachiasmatic nucleus of the hypothalamus is entrained to the daily light/dark cycle and subsequently transmits synchronizing signals to local circadian oscillators in peripheral tissues. Only recently has the importance of this system to the regulation of such fundamental biological processes as the cell cycle and metabolism become apparent. A convergence of data from microarray studies, quantitative trait locus analysis, and mutagenesis screens demonstrates the pervasiveness of circadian regulation in biological systems. The importance of maintaining the internal temporal homeostasis conferred by the circadian system is revealed by animal models in which mutations in genes coding for core components of the clock result in disease, including cancer and disturbances to the sleep/wake cycle.

/pdf/mammalian-circadian-biology-elucidating-genome-wide-levels-5fs9zyaexz.pdf

Mammalian circadian biology: elucidating genome-wide levels of temporal organization.

The B56 Family of Protein Phosphatase 2A (PP2A) Regulatory Subunits Encodes Differentiation-induced Phosphoproteins That Target PP2A to Both Nucleus and Cytoplasm

The circadian rhythm is an intrinsic 24-h cycle that, in species from Neurospora to humans, is generated by an intracellular oscillating negative feedback loop that controls the periodic transcription of both regulatory and output genes. The molecular mechanism generating the circadian rhythm has been the object of intense study (reviewed in references 12, 42, and 57). Genetic investigations in the fruit fly Drosophila melanogaster, augmented by studies of circadian rhythm mutants in mammals, have led to a rapidly evolving understanding of the workings of the metazoan central clock. In Drosophila, a heterodimeric transcription factor composed of CLOCK and CYCLE binds to E-box-containing promoters and drives expression of the negative regulators PERIOD (dPER) and TIMELESS (dTIM). dPER and dTIM accumulate in the cytoplasm until they heterodimerize. Heterodimerization serves to mask their cytoplasmic localization domains, allowing the complex to enter the nucleus (46). Nuclear dPER-dTIM heterodimers repress the activity of the CLOCK/CYCLE transcription factor, thus causing a decrease in dPER and dTIM expression (10).

Although the mammalian circadian clock is composed of proteins homologous to those found in Drosophila, the mechanisms for regulating circadian rhythm in mammals appear to be more complex and in many aspects quite different from those in Drosophila. The increased complexity in the mammalian system is due in part to the expansion of the per gene family. Three mammalian period genes have been cloned; all are rhythmically expressed in the anatomic location of the central clock, the suprachiasmatic nucleus (SCN), as well as in diverse peripheral tissues (including heart, liver, and muscle) and cultured fibroblasts, with peak levels of transcripts detected during the circadian day in the mouse (1, 2, 47, 55, 62). The three mper genes differ in their transcriptional regulation. Several reports suggest that mper1 is expressed 4 to 8 h before mper2 and mper3 (1, 25, 53).

Regulated nuclear entry of the PER proteins is a common element in many but not all (50) of the observed circadian regulators (12, 42, 57). In the mouse, periodic nuclear accumulation of mPER1 protein has been demonstrated in the mouse SCN, peaking 4 to 6 h after mper1 mRNA expression (19). How and if mPER nuclear entry is regulated is less clear. In the murine system, heterodimerization of mPER proteins with mTIM has been controversial, being found by some but not all observers (48, 54, 61). However, each of the mPER proteins can homodimerize with itself and heterodimerize with other mPER proteins. Forced expression of mPER proteins alone can partially repress CLOCK/BMAL1-activated transcription (BMAL1 is the mammalian homolog of CYCLE) in the absence of coexpressed mTIM (25, 48). Recently, Kume et al. (31) reported that coexpression of cryptochrome proteins mCRY1 and mCRY2 facilitated the nuclear entry of mPER proteins and fully repressed transcription from CLOCK/BMAL1-driven promoters. Thus, PER nuclear entry seems to be periodic and regulated in mammals as well as in Drosophila.

Phosphorylation of the components of the circadian clock has been postulated to regulate the duration of the cycle. Treatment of the dinoflagellate Gonyaulax polyedra with either serine/threonine phosphatase or kinase inhibitors alters its circadian rhythm (6, 7). The frequency gene product, a negative regulator of the Neurospora circadian clock, is rhythmically phosphorylated (12), and its phosphorylation regulates both its stability and period duration (34). dCLOCK, dPER, and dTIM are phosphoproteins, and the level of dPER and dTIM phosphorylation increases steadily from the time of their synthesis until their degradation at dawn (13, 32). The first genetic evidence that a specific protein kinase regulates the Drosophila circadian rhythm was the discovery of the novel gene double-time (dbt), encoding a protein serine/threonine kinase (27, 41). dbt is coexpressed with per and tim in the fly brain lateral neurons that regulate circadian rhythm. Different missense alleles of dbt cause marked lengthening or shortening of the circadian period, while homozygosity for the null allele causes pupal lethality (27).

Examination of flies with mutations in the dbt gene led Kloss and coworkers (27) to conclude that the DBT kinase phosphorylated and regulated dPER. Drosophila larvae homozygous for the dbt-null allele manifest several distinct phenotypes. First, they accumulate high levels of dPER but not dTIM, suggesting a role for phosphorylation in the degradation of dPER. Second, the dPER that accumulates is hypophosphorylated, indicating a major role for DBT in the phosphorylation of dPER. In a final indication that DBT directly regulates dPER, DBT binds to an amino-terminal fragment of dPER (27).

Drosophila DBT is most similar in sequence (86% identical) in its kinase domain to the kinase domains of mammalian casein kinase I ɛ and δ (CKIɛ and CKIδ). The CKI gene family encodes a number of widely expressed kinases that localize to membranes, cytoplasm, and nucleus; and various members of the CKI family have been identified in plants, fungi, and mammals (14, 18, 44, 45). CKIɛ and CKIδ belong to a branch of the family that includes the yeast kinases HRR25 and Hhp1 and Hhp2, implicated in the response to DNA damage (11, 14, 21). Mammalian CKIδ and CKIɛ have closely related 123-amino-acid carboxy-terminal domains that can autoregulate kinase activity in a phosphorylation-dependent manner (5, 16, 17, 44). However, the carboxy-terminal domains of DBT and CKIɛ are unrelated.

Accumulating evidence suggests CKI family members can regulate the intracellular localization of specific substrates. For example, mammalian CKIα (71% identical to DBT over the kinase domain) binds to, phosphorylates, and inhibits the nuclear import of the transcription factor NF-AT4 (60). In Drosophila, a CKIα homolog shuttles from the cytoplasm into the larval nuclei in response to gamma irradiation (49). Finally, one of the few identified substrates of HRR25 is the yeast transcriptional regulator SWI6, a protein whose cytoplasmic retention is dependent on phosphorylation (20, 52).

Given the differences between the Drosophila and mammalian PER and TIM proteins and the higher level of complexity in the regulation and interactions of the mPER proteins, the interaction between CKI and the mammalian mPER1 protein was investigated. We find that specific and closely related isoforms of CKI bind to and phosphorylate mPER1 both in vitro and in vivo. Unexpectedly, overexpressed mPER1 was found to accumulate in the nuclei of transfected HEK 293 cells. Two distinct mechanisms appear to be capable of regulating mPER1 nuclear entry. First, coexpressed mPER2 prevents mPER1 nuclear accumulation. Second, CKIɛ or CKI∂ coexpression blocks mPER1 nuclear accumulation in a kinase-dependent manner, by masking its nuclear localization signal (NLS). These results suggest that a critical function of both mPER2 and CKI in circadian rhythm is to control the nuclear entry of mPER1.

Ann Rivers

Papers

The B56 Family of Protein Phosphatase 2A (PP2A) Regulatory Subunits Encodes Differentiation-induced Phosphoproteins That Target PP2A to Both Nucleus and Cytoplasm

Nuclear Entry of the Circadian Regulator mPER1 Is Controlled by Mammalian Casein Kinase I ɛ

Autoinhibition of Casein Kinase I e (CKIe) Is Relieved by Protein Phosphatases and Limited Proteolysis

Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle.