Background: Gene set analysis (GSA) is a widely used strategy for gene expression data analysis based on pathway knowledge. GSA focuses on sets of related genes and has established major advantages over individual gene analyses, including greater robustness, sensitivity and biological relevance. However, previous GSA methods have limited usage as they cannot handle datasets of different sample sizes or experimental designs.

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKI (casein kinase I) has been postulated to prime PER2 for degradation. Supporting this idea, CKI inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKI inhibition also slows the degradation of PER2 in cells. CKI-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein ␤-TrCP to a specific site, and dominant negative ␤-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.Diverse organisms from prokaryotes to mammals coordinate behavioral and physiological rhythms with the daily dark-light cycle by means of a circadian clock. In mammals, the master circadian clock is located in the suprachiasmatic nucleus of the brain, and it entrains peripheral cell-autonomous clocks throughout the body. In mice, a positively acting heterodimeric transcription factor composed of the PAS-bHLH proteins CLOCK (CLK) and BMAL1 drives transcription of tissuespecific circadian output genes, as well as its own negative regulators, the Period (denoted mPer1, mPer2, and mPer3), and Cryptochrome (mCry1 and mCry2) genes. The mammalian PER and CRY proteins form multimeric complexes that enter the nucleus and repress the transcriptional activity of CLK/ BMAL1, modulating circadian output (reviewed in references 29 and 41). Additional stabilizing feedback loops, including inhibition of Bmal1 transcription by REV-ERB␣ (37), further contribute to the timing and robustness of the cycle. The daily rhythmic degradation of PERIOD proteins leading to derepression of CLK/BMAL1 is postulated to be critical to the proper functioning of the clock. Therefore, the mechanism and control of this process are of great interest.Genetic studies have identified CKIε (casein kinase Iε) as a key regulator of metazoan circadian rhythm and both genetic and biochemical studies suggest that the PER proteins are important substrates (reviewed in reference 10). CKIε was first implicated as a circadian regulator in Drosop...

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network.

Exposure to influenza viruses is necessary, but not sufficient, for healthy human hosts to develop symptomatic illness. The host response is an important determinant of disease progression. In order to delineate host molecular responses that differentiate symptomatic and asymptomatic Influenza A infection, we inoculated 17 healthy adults with live influenza (H3N2/Wisconsin) and examined changes in host peripheral blood gene expression at 16 timepoints over 132 hours. Here we present distinct transcriptional dynamics of host responses unique to asymptomatic and symptomatic infections. We show that symptomatic hosts invoke, simultaneously, multiple pattern recognition receptors-mediated antiviral and inflammatory responses that may relate to virus-induced oxidative stress. In contrast, asymptomatic subjects tightly regulate these responses and exhibit elevated expression of genes that function in antioxidant responses and cell-mediated responses. We reveal an ab initio molecular signature that strongly correlates to symptomatic clinical disease and biomarkers whose expression patterns best discriminate early from late phases of infection. Our results establish a temporal pattern of host molecular responses that differentiates symptomatic from asymptomatic infections and reveals an asymptomatic host-unique non-passive response signature, suggesting novel putative molecular targets for both prognostic assessment and ameliorative therapeutic intervention in seasonal and pandemic influenza.