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...

Highlight AtMYB74, a R2R3-MYB gene, is transcriptionally regulated through RdDM for response to salt stress. The accumulation of siRNA targeting to the AtMYB74 promoter region is essential for maintaining AtMYB74 expression.

The Drosophila circadian oscillator is composed of autoregulatory period/timeless (per/tim) and Clock (Clk) feedback loops that control rhythmic transcription. In the Clk loop, CLOCK-CYCLE heterodimers activate vrille (vri) and PAR domain protein 1 (Pdp1) transcription, then sequential repression by VRI and activation by PDP1 mediate rhythms in Clk transcription. Because VRI and PDP1 bind the same regulatory element, the VRI/PDP1 ratio is thought to control the level of Clk transcription. Thus, constant high or low PDP1 levels in clock cells should eliminate Clk mRNA cycling and disrupt circadian oscillator function. Here we show that reducing PDP1 levels in clock cells by ϳ70% via RNA interference or increasing PDP1 levels by ϳ10-fold in clock cells does not alter Clk mRNA cycling or circadian oscillator function. However, constant low or high PDP1 levels in clock cells disrupt locomotor activity rhythms despite persistent circadian oscillator function in brain pacemaker neurons that extend morphologically normal projections into the dorsal brain. These results demonstrate that the VRI/PDP1 ratio neither controls Clk mRNA cycling nor circadian oscillator function and argue that PDP1 is not essential for Clk activation. PDP1 is nevertheless required for behavioral rhythmicity, which suggests that it functions to regulate oscillator output.

The synergism between cardiomyogenesis and angiogenesis is essential for cardiac regeneration. Circular RNAs (circRNAs) play pivotal roles in cell growth and angiogenesis, but their functions in cardiac regeneration are not yet known. In this study, we investigated the role and underlying mechanisms of circRNA Hipk3 (circHipk3) in both cardiomyogenesis and angiogenesis during cardiac regeneration. We found that circHipk3 was overexpressed in the fetal or neonatal heart of mice. The transcription factor Gata4 bound to the circHipk3 promoter and increased circHipk3 expression. Cardiomyocyte (CM) proliferation in vitro and in vivo was inhibited by circHipk3 knockdown and increased by circHipk3 overexpression. Moreover, circHipk3 overexpression promoted coronary vessel endothelial cell proliferation, migration, and tube-forming capacity and subsequent angiogenesis. More importantly, circHipk3 overexpression attenuated cardiac dysfunction and decreased fibrotic area after myocardial infarction (MI). Mechanistically, circHipk3 promoted CM proliferation by increasing Notch1 intracellular domain (N1ICD) acetylation, thereby increasing N1ICD stability and preventing its degradation. In addition, circHipk3 acted as a sponge for microRNA (miR)-133a to promote connective tissue growth factor (CTGF) expression, which activated endothelial cells. Our findings suggested that circHipk3 might be a novel therapeutic target for preventing heart failure post-MI.

Recently pseudorabies outbreaks have occurred in many vaccinated farms in China. To identify genetic characteristics of pseudorabies virus (PRV) strains, we obtained the genomic sequences of PRV strains HeN1 and JS, which were compared to 4 PRV genomes and 729 partial gene sequences. PRV strains isolated in China showed marked sequence divergence compared to European and American strains. Phylogenetic analysis revealed that for the first time PRV can be divided into 2 distinct clusters, with Chinese strains being genotype II and PRVs isolated from other countries being genotype I. Restriction fragment length polymorphism analysis confirmed differences between HeN1 and Bartha strains, as did the presence of unique insertion/deletion polymorphisms and microsatellites. This divergence between the two genotypes may have been generated from long-term, independent evolution, which could also explain the low efficacy of the Bartha vaccine in protecting pigs infected with genotype II PRV.