Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms1. While attention has been focused on transcriptional and posttranslational mechanisms1–3, other posttranscriptional modes have been less clearly delineated. Here we report mutants of a novel Drosophila gene twenty-four (tyf) that display weak behavioural rhythms. Weak rhythms are accompanied by dramatic reductions in the levels of the clock protein PERIOD (PER) as well as more modest effects on TIMELESS (TIM). Nonetheless, PER induction in pacemaker neurons can rescue tyf mutant rhythms. TYF associates with a 5′-cap binding complex, poly(A)-binding protein (PABP) as well as per and tim transcripts. Furthermore, TYF activates reporter expression when tethered to reporter mRNA even in vitro. Taken together, these data suggest that TYF potently activates PER translation in pacemaker neurons to sustain robust rhythms, revealing a novel and important role for translational control in the Drosophila circadian clock.

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Rhythmic histone acetylation underlies the oscillating expression of clock genes in the mammalian circadian clock system. Cellular factors that contain histone acetyltransferase and histone deacetylase activity have been implicated in these processes by direct interactions with clock genes, but their functional relevance remains to be assessed by use of appropriate animal models. Here, using transgenic fly models, we show that CREBbinding protein (CBP) participates in the transcriptional regulation of the Drosophila CLOCK/CYCLE (dCLK/ CYC) heterodimer. CBP knockdown in pigment dispersing factor-expressing cells lengthens the period of adult locomotor rhythm with the prolonged expression of period and timeless genes, while CBP overexpression in timeless-expressing cells causes arrhythmic circadian behaviors with the impaired expression of these dCLK/ CYC-induced clock genes. In contrast to the mammalian circadian clock system, CBP overexpression attenuates the transcriptional activity of the dCLK/CYC heterodimer in cultured cells, possibly by targeting the PER-ARNT-SIM domain of dCLK. Our data suggest that the Drosophila circadian clock system has evolved a distinct mechanism to tightly regulate the robust transcriptional potency of the dCLK/CYC heterodimer.The circadian clock is an evolutionarily conserved system that perceives environmental time cues, synchronizes the organism's inherent clock with external time, and exhibits the circadian physiology of organisms (e.g., diurnal or nocturnal locomotor activities) (14,40,52,59). At the cellular level of circadian clock systems in animals, pacemaker cells in a small subset of brain neurons display a robust oscillation of clock gene products and dominate the circadian behaviors of the organism by governing the peripheral clock systems. These core clock cells correspond to ventral lateral neurons (LN v s) and the suprachiasmatic nucleus in Drosophila and mouse circadian clock systems, respectively (21,23,58). Drosophila LN v s express the neuropeptide pigment dispersing factor (pdf) gene, which is implicated in the synchronization of clock cells (33,41,42,44). At the molecular level, some core clock genes are periodically expressed, and their rhythmic expression is maintained under free-running conditions (i.e., exclusion of external time cues). This molecular clock system involves several transcription factors, protein kinases, phosphatases, and proteosomal pathway components, which together mediate the transcriptional regulation of clock transcripts and control the posttranslational localization, quantity, and quality of clock proteins (19,45).Published data suggest that two interlocking feedback loops maintain the oscillating expression of core clock genes in Drosophila melanogaster and mouse. In Drosophila, a heterodimer of the dClock (dClk) and cycle (cyc) gene products activates the transcription of the period (per), timeless (tim), vrille (vri), and Par domain protein 1ε (Pdp1ε) genes during subjective night by binding to E-box sequences within their promoter...

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the persistence of the viral genome during latent infection. It has been suggested that LANA1 tethers the viral genome to the host chromosome and also participates actively in DNA replication from the terminal repeat of KSHV. Here we show by mutational analysis that the mitotic chromosome-binding activity of LANA1 is tightly coupled to its replication activity. Thus, KSHV appears to have evolved a unique tactic for its stable maintenance.Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma and some lymphoproliferative diseases (9,11,47); it is closely related to Epstein-Barr virus (EBV) and herpesvirus saimiri. The circularized viral genome is maintained as an extrachromosomal element in latently infected cells, whereas the linearized form of the viral genome is packaged into infectious virus particles during lytic infection (10, 16). Latency-associated nuclear antigen 1 (LANA1) is a viral protein expressed during latent infection (22,33,51,55). As a transcriptional modulator, LANA1 interacts with several cellular transcription factors and influences the activity of viral and cellular promoters (3,20,23,25,30,35,36,40,41,50,54). When tethered to promoters via a heterologous DNA-binding domain, LANA1 acts as a transcriptional repressor, possibly by interacting with the mSin3 complex and heterochromatin protein 1 (36, 43, 57). As a replication factor, LANA1 colocalizes with the viral genome on the host chromosome and has been shown to be responsible for the stable maintenance of plasmids containing the KSHV terminal repeat (TR); it may therefore link the viral genome to a host chromosome, retaining the viral genome in the nucleus during mitosis and permitting equipartition to the progeny (5, 14). The chromosome-binding activity of LANA1 has been mapped to its N-terminal 22 amino acids and has been designated the chromosome-binding sequence (CBS) (49). Its C terminus binds to sequences within the TRs located at both ends of the KSHV genome and represses TR-dependent transcription (6,15,24,42). As expected from the chromosome-tethering model, the LANA1 CBS is necessary for long-term replication of a KSHV TR-containing plasmid (59). In addition, we and others have shown that LANA1 is required for the transient replication of KSHV TR-containing plasmids, indicating that it may play an essential role not only in plasmid maintenance but also in DNA replication from the KSHV TR (26,28,42). Using a panel of LANA1 deletion mutants, we found that the CBS is necessary and sufficient for the C-terminal DNA-binding domain to support the replication of a KSHV TR-containing plasmid, suggesting that LANA1 must bind to the chromosome to fulfill its replication function (C. Lim, T. Seo, J. Jung, and J. Choe, submitted for publication). To examine this possibility further, we have generated several LANA1 derivatives with point mutations in their CBS and characterized their activities....

Reversible phosphorylation of clock proteins plays an important role in circadian timekeeping as it is a key post‐translational mechanism that regulates the activity, stability and subcellular localization of core clock proteins. The kinase DOUBLETIME (DBT), a Drosophila ortholog of mammalian casein kinase Iε, regulates circadian phosphorylation of two essential clock proteins, PERIOD and dCLOCK. We present evidence that Par Domain Protein 1ε (PDP1ε), a transcription factor and mediator of clock output in Drosophila, is phosphorylated in vivo and in cultured cells by DBT activity. We also demonstrate that DBT interacts with PDP1ε and promotes its degradation by the ubiquitin‐proteasome pathway in cultured cells. In addition, PDP1ε nuclear localization is decreased by dbt RNA interference in S2 cell system. These results suggest that DBT regulates phosphorylation, stability and localization of PDP1ε, and that it has multiple targets in the Drosophila circadian system.