Glucocorticoids can inhibit inflammation by abrogating the activity of NF-?B, a family of transcription factors that regulates the production of proinflammatory cytokines. To understand the molecular mechanism of repression of NF-?B activity by glucocorticoids, we performed a high-throughput siRNA oligo screen to identify novel genes involved in this process. Here, we report that loss of p53, a tumor suppressor protein, impaired repression of NF-?B target gene transcription by glucocorticoids. Additionally, loss of p53 also impaired transcription of glucocorticoid receptor (GR) target genes, whereas upstream NF-?B and glucocorticoid receptor signaling cascades remained intact. We further demonstrate that p53 loss severely impaired glucocorticoid rescue of death in a mouse model of LPS shock. Our findings unveil a new role for p53 in the repression of NF-?B by glucocorticoids and suggest important implications for treatment of the proinflammatory microenvironments found in tumors with aberrant p53 activity.
One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened approximately 10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.
Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.
Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-?B signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.
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