The novel gene twenty-four defines a critical translational step in the Drosophila clock.

Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms. While attention has been focused on transcriptional and post-translational mechanisms, other post-transcriptional modes have been less clearly delineated. Here we report mutants of a novel Drosophila gene twenty-four (tyf) that show weak behavioural rhythms. Weak rhythms are accompanied by marked 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 messenger RNA even in vitro. Taken together, these data indicate that TYF potently activates PER translation in pacemaker neurons to sustain robust rhythms, revealing a new and important role for translational control in the Drosophila circadian clock.

The DOUBLETIME protein kinase regulates phosphorylation of the Drosophila PDP1epsilon.

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 Iepsilon, regulates circadian phosphorylation of two essential clock proteins, PERIOD and dCLOCK. We present evidence that Par Domain Protein 1epsilon (PDP1epsilon), 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 PDP1epsilon and promotes its degradation by the ubiquitin-proteasome pathway in cultured cells. In addition, PDP1epsilon nuclear localization is decreased by dbt RNA interference in S2 cell system. These results suggest that DBT regulates phosphorylation, stability and localization of PDP1epsilon, and that it has multiple targets in the Drosophila circadian system.

Functional role of CREB-binding protein in the circadian clock system of Drosophila melanogaster.

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 CREB-binding 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.

Mitotic chromosome-binding activity of latency-associated nuclear antigen 1 is required for DNA replication from terminal repeat sequence of Kaposi's sarcoma-associated herpesvirus.

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.

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus functionally interacts with heterochromatin protein 1.

Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus plays an important role in maintenance of the viral genome during latent infection. LANA additionally participates in the transcriptional regulation of several viral and cellular promoters. When tethered to constitutively active promoters, the protein exhibits transcriptional repressor activity. In this report, we further characterized cell type-, promoter-, and domain-specific transcriptional repression by LANA. We additionally speculated on the mechanism underlying transcriptional repression by the C terminus of the protein. Subnuclear localization patterns and association with heterochromatin suggested a possible link between LANA and heterochromatin protein 1, a representative heterochromatin-associated protein. In vivo and in vitro binding and immunofluorescence assays revealed that LANA associates with heterochromatin protein 1 in an isotype-specific manner. Furthermore, biochemical fractionation and transient replication assays supported the possibility that this interaction contributes to transcriptional repression, targeting to subnuclear structures, and latent DNA replication activity of LANA.