Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired‐end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories’. Some factories further specialize in transcribing responsive genes encoding micro‐RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.
The tumor suppressor p53 regulates cell cycle progression and apoptosis in response to various types of stress, whereas excess p53 activity creates unwanted effects. Tight regulation of p53 is essential for maintaining normal cell growth. p53-associated cellular protein-testes derived (PACT, also known as P2P-R, RBBP6) is a 250-kDa Ring finger-containing protein that can directly bind to p53. PACT is highly up-regulated in esophageal cancer and may be a promising target for immunotherapy. However, the physiological role of the PACT-p53 interaction remains largely unclear. Here, we demonstrate that the disruption of PACT in mice leads to early embryonic lethality before embryonic day 7.5 (E7.5), accompanied by an accumulation of p53 and widespread apoptosis. p53-null mutation partially rescues the lethality phenotype and prolonged survival to E11.5. Endogenous PACT can interact with Hdm2 and enhance Hdm2-mediated ubiquitination and degradation of p53 as a result of the increase of the p53-Hdm2 affinity. Consequently, PACT represses p53-dependent gene transcription. Knockdown of PACT significantly attenuates the p53-Hdm2 interaction, reduces p53 polyubiquitination, and enhances p53 accumulation, leading to both apoptosis and cell growth retardation. Taken together, our data demonstrate that the PACT-p53 interaction plays a critical role in embryonic development and tumorigenesis and identify PACT as a member of negative regulators of p53.apoptosis ͉ embryonic lethality ͉ ubiquitination
Human nuclei contain three RNA polymerases (I, II, and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here, we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory; for example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors, but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo.
The Ras-MAPK and PI3K-AKT pathways are conserved in metazoan organisms, which involve a series of signaling cascades and form the basis for numerous physiological and pathological processes. Here we report on yeast two hybrid screening results of a protein interaction network around the known components of human Ras-MAPK/PI3K pathways. A total of 42 independent cDNA library screenings resulted in 200 protein-protein interaction (PPI) pairs among 180 molecules. Most of the proteins formed a large cluster that contains 193 PPIs between 169 proteins. Seventy-four interactions indicate high-confidence according to bioinformatics analysis. The prey list contains high enrichment genes with specific Gene Ontology (GO) terms such as response to stress and response to external stimulus. Most interactions link the Ras signaling pathway with various cellular processes. Five interactions were validated by coimmunoprecipitation and colocalization assays in mammalian cells to confirm their in vivo interactions. This protein interaction network provides further insights into the molecular mechanism of Ras-MAPK/PI3K signaling pathways.
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