Both casein kinase 1 delta (CK1␦) and epsilon (CK1) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1␦ and CK1 in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1␦ ⌬2 ). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1␦. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by ϳ20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1␦-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1 did not alter these circadian endpoints. These results reveal important functional differences between CK1␦ and CK1: CK1␦ plays an unexpectedly important role in maintaining the 24-h circadian cycle length.Circadian rhythms are rhythms in gene expression, metabolism, physiology, and behavior that persist in constant environmental conditions with a cycle length near 24 h. In mammals, the circadian timing system is hierarchical. The primary pacemaker regulating circadian behavioral rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Most cell types express circadian clock genes and will express rhythmicity in vitro. In vivo, the SCN entrains peripheral oscillators through a complex set of physiological and hormonal rhythms (31,32,36).At the molecular level, circadian oscillations are governed by a cell-autonomous negative-feedback loop in which transcription factors drive the expression of their own negative regulators, leading to oscillation between periods of transcriptional activation and repression (reviewed in references 32 and 36). The bHLH-PAS containing transcription factors CLOCK or NPAS2 form heterodimers with BMAL1. These heterodimers binds to E-box elements within regulatory regions of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes to stimulate their transcription. Approximately 12 h after transcriptional activation, PER and CRY proteins reach concentrations sufficient to form repressor complexes that inhibit the activity of the CLOCK/NPAS2:BMAL1 heterodimer, reducing the transcription of Per and Cry genes and subsequently relieving PER/CRY-mediated negative feedback. E-box-mediated expression of other transcription factors, including members of the DBP/HLF/TEF and nuclear orphan receptor families (e.g., Rev-Erb␣ and ROR-A), provides a mechanism for clock control of genes with diverse promoters and with gene expression peaks occurring at a variety of phases.Posttranslational modifications of circadian clock proteins play a well-established role in the re...

The ability to generate intrinsic circadian rhythms in electrical activity appears to be a key property of central pacemaker neurons and one essential to the function of the circadian timing system. Previous work has demonstrated that suprachiasmatic nucleus (SCN) neurons express the fast delayed rectifier (FDR) potassium current and raise questions about the function of this current. Here, we report that mice lacking both Kcnc1 and Kcnc2 genes (double knockout, dKO) fail to express the Kv3.1 and 3.2 channels in the SCN as well as exhibit a greatly reduced FDR current. SCN neurons from these dKO mice exhibit reduced spontaneous activity during the day as well as reduced NMDA-evoked excitatory responses during the night. Interestingly, the daily rhythm in PER2 expression in the SCN was not altered in the dKO mice though the photic induction of c-FOS was attenuated. Behaviorally, the dKO mice exhibited extremely disrupted daily rhythms in wheel running behavior. In a light/dark cycle, some of the dKO mice were arrhythmic while others expressed a diurnal rhythm with low amplitude and significant activity during the day. When placed in constant darkness, the dKO mice exhibited low amplitude, fragmented rhythms and attenuated light-responses. Together, this data is consistent with the hypothesis that the FDR current is critical for the generation of robust circadian rhythms in behavior as well as the synchronization of the circadian system to the photic environment.

CCAAT/enhancer-binding protein alpha (C/EBP␣) is expressed in postmitotic, differentiated adipocytes and is required for adipose conversion of 3T3-L1 cells in culture. Temporal misexpression of C/EBP␣ in undifferentiated adipoblasts leads to mitotic growth arrest. We report here that growth arrest-and DNA damageinducible gene 45 (gadd45) is preferentially expressed in differentiated 3T3-L1 adipocytes similar to phenotypeassociated genes. Furthermore, C/EBP␣ transactivates a reporter plasmid containing 1.5 kb of the gadd45 promoter region. The proto-oncogene myc, which inhibits adipocyte differentiation, abrogates C/EBP␣ activation of gadd45. gadd45 is known to be a target of the tumor suppressor p53 in a G 1 checkpoint activated by DNA damage. Immunoprecipitation of radiolabeled proteins with conformation-specific antibodies revealed that wild-type p53 is expressed throughout 3T3-L1 adipocyte development, including the postmitotic period characterized by the accumulation of gadd45 and C/EBP␣. A stable 3T3-L1 subline was engineered to express a dominant negative p53, human p53 143ala . The p53 143ala subline differentiated to adipocytes and showed appropriate developmental expression of gadd45. These findings suggest that postmitotic growth arrest is coupled to adipocyte differentiation via C/EBP␣ stimulation of growth arrest-associated and phenotype-associated genes.

Targeted deletion of IA-2 and IA-2beta, major autoantigens in type 1 diabetes and transmembrane secretory vesicle proteins, results in impaired secretion of hormones and neurotransmitters. In the present study, we evaluated the effect of these deletions on daily rhythms in blood pressure, heart rate, core body temperature, and spontaneous physical and neuronal activity. We found that deletion of both IA-2 and IA-2beta profoundly disrupts the usual diurnal variation of each of these parameters, whereas the deletion of either IA-2 or IA-2beta alone did not produce a major change. In situ hybridization revealed that IA-2 and IA-2beta transcripts are highly but nonrhythmically expressed in the suprachiasmatic nuclei, the site of the brain's master circadian oscillator. Electrophysiological studies on tissue slices from the suprachiasmatic nuclei showed that disruption of both IA-2 and IA-2beta results in significant alterations in neuronal firing. From these studies, we concluded that deletion of IA-2 and IA-2beta, structural proteins of secretory vesicles and modulators of neuroendocrine secretion, has a profound effect on the circadian system.