Regulation of endosome fusion

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53(+/+) cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53.

Regulation of TLR2, TLR4 and CD14 mRNA in gingival fibroblasts.

Gingival fibroblasts constitutively express pattern recognition molecules including the Toll-like receptors (TLRs) and produce various cytokines following interaction with bacterial ligands including LPS. Hence gingival fibroblasts are thought to play an important role in the pathogenesis of chronic inflammatory periodontal disease.
Objectives: The aim of this study was to investigate the regulation of expression of TLRs and CD-14 mRNA by gingival fibroblasts, and subsequently the responsiveness of these cells to bacterial stimulation Methods: Gingival fibroblasts were stimulated with IL-1ß (10ng/ml), IFN-g (1000IU/ml), P. gingivalis LPS (1µg/ml), E. coli LPS (1µg/ml) or P. gingivalis sonicate (10µg/ml) for 6 and 24 hr. TLR2, TLR4 and CD14 mRNA expression was subsequently determined by Q-PCR utilising Taqman chemistry. The effects of each factor on mRNA expression was analysed by ANOVA. Cells were pre-incubated with IFN-g (1000IU/ml) for 48hr followed by stimulation with E. coli LPS over the concentration range 0 - 10.0 µg/ml for a further 48 hr. IL-8 production by fibroblasts was subsequently determined by ELISA. Results: After 24 hr IFN-g induced a statistically significant increase in TLR2, TLR4 and CD14 mRNA expression. In contrast, IL-1ß, P. gingivalis LPS, E. coli LPS and P. gingivalis sonicate had no significant effect on mRNA expression at either timepoint. Following pre-stimulation with IFN-g, E. coli LPS increased IL-8 production by gingival fibroblasts in a concentration-dependent manner. Conclusion: IFN-g stimulates mRNA expression levels of TLR2, TLR4 and CD14 in gingival fibroblasts, which may subsequently lead to an increased responsiveness of fibroblasts to bacterial stimulation.

Quantitative regulation of FLC via coordinated transcriptional initiation and elongation

The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼ 25-fold with elongation rate varying ∼ 8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context.

DED or alive: assembly and regulation of the death effector domain complexes.

Death effector domains (DEDs) are protein-protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.