Transcriptional activation by CLOCK-CYCLE (CLK-CYC) heterodimers and repression by PERIOD-TIMELESS(PER-TIM) heterodimers are essential for circadian oscillator function in Drosophila. PER-TIM was previously found to interact with CLK-CYC to repress transcription, and here we show that this interaction inhibits binding of CLK-CYC to E-box regulatory elements in vivo. Coincident with the interaction between PER-TIM and CLK-CYC is the hyperphosphorylation of CLK. This hyperphosphorylation occurs in parallel with the PER-dependent entry of DOUBLE-TIME (DBT) kinase into a complex with CLK-CYC, where DBT destabilizes both CLK and PER. Once PER and CLK are degraded, a novel hypophosphorylated form of CLK accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that PER-dependent rhythms in CLK phosphorylation control rhythms in E-box-dependent transcription and CLK stability, thus linking PER and CLK function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals.

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The Drosophila circadian oscillator consists of interlocked period (per)/timeless (tim) and Clock (Clk) transcriptional/translational feedback loops. Within these feedback loops, CLK and CYCLE (CYC) activate per and tim transcription at the same time as they repress Clk transcription, thus controlling the opposite cycling phases of these transcripts. CLK-CYC directly bind E box elements to activate transcription, but the mechanism of CLK-CYC-dependent repression is not known. Here we show that a CLK-CYC-activated gene, vrille (vri), encodes a repressor of Clk transcription, thereby identifying vri as a key negative component of the Clk feedback loop in Drosophila's circadian oscillator. The blue light photoreceptor encoding cryptochrome (cry) gene is also a target for VRI repression, suggesting a broader role for VRI in the rhythmic repression of output genes that cycle in phase with Clk.

Ubiquitination and deubiquitination have emerged as critical post-translational regulatory mechanisms for activation or attenuation of the virus-triggered type I interferon (IFN) 2 induction pathways. In this study, we identified two deubiquitinating enzymes, OTUB1 and OTUB2, as negative regulators of virus-triggered type I IFN induction. Overexpression of OTUB1 and OTUB2 inhibited virus-induced activation of IRF3 and NF-B, transcription of the IFNB1 gene as well as cellular antiviral response, whereas knockdown of OTUB1 and OTUB2 had opposite effects. Coimmunoprecipitations indicated OTUB1 and -2 interacted with TRAF3 and TRAF6, two E3 ubiquitin ligases required for virus-triggered IRF3 and NF-B activation, respectively. Furthermore, we found that OTUB1 and OTUB2 mediated virus-triggered deubiquitination of TRAF3 and -6. These findings suggest that OTUB1 and OTUB2 negatively regulate virus-triggered type I IFN induction and cellular antiviral response by deubiquitinating TRAF3 and -6. Viral infections triggered a series of signaling events that lead to induction of type I interferons (IFNs). Type I IFNs then activate the JAK-STAT signal transduction pathways, leading to transcriptional induction of a wide range of downstream antiviral genes and subsequent innate antiviral response (1-4). Transcriptional induction of type I IFN genes requires the coordinate activation of multiple transcription factors and their cooperative assembly into transcriptional enhancer complexes in vivo. For example, the IFNB1 gene promoter contains conserved enhancer elements recognized by NF-B (B site) and phosphorylated IRF3 (ISRE site, also known as PRDIII or IRF-E). It has been shown that transcriptional activation of the IFNB1 gene requires coordinate and cooperative assembly of an enhanceosome that contains all of these transcription factors (2, 5, 6).The innate immune system has developed at least two types of pathogen recognition receptors for the recognition of viral RNAs (7).One is mediated by membrane-bound Toll-like receptors (TLRs) such as TLR3. Engagement of TLR3 by double-stranded RNA triggers TRIF-mediated signaling pathways, leading to IRF3 and NF-B activation (8). The second one involves the cytosolic RIG-I-like receptor family members RIG-I, MDA5, and Lgp2. Both RIG-I and MDA5 contain two CARD modules at their N terminus and a DexD/H-box RNA helicase domain at their C terminus (9, 10). Upon viral infection, the RNA helicase domains of RIG-I and MDA5 serve as intracellular viral RNA receptors, whereas their CARD modules are associated with the downstream CARD-containing adapter protein VISA (also known as MAVS, IPS-1, and Cardif) (11-15). The essential roles of VISA in antiviral innate immune response were demonstrated by the observations that VISAdeficient mice failed to mount a proper IFN response to viral infections (14,16). Various studies have demonstrated that VISA plays a central role in assembling a complex that activates distinct signaling pathways leading to NF-B and IRF3 activation, respectively. VISA is as...