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TNFalpha signaling primes chromatin for NF-kappaB binding and induces rapid and widespread nucleosome repositioning

ABSTRACT: Background The rearrangement of nucleosomes along the DNA fiber profoundly affects gene expression, but little is known about how signaling reshapes the chromatin landscape, in 3D space and over time, to allow the establishment of new transcriptional programs. Results Using micrococcal nuclease treatment and high-throughput sequencing, we map genome-wide changes in nucleosome positioning in primary human endothelial cells stimulated with tumour necrosis factor alpha (TNFalpha) - a proinflammatory cytokine that signals through nuclear factor kappaB (NF-kappaB). Within 10 minutes, nucleosomes reposition at regions both proximal and distal to NF-kappaB binding sites, before the transcription factor quantitatively binds thereon. Similarly, in long TNFalpha-responsive genes, repositioning precedes transcription by pioneering elongating polymerases and appears to nucleate from intragenic enhancer clusters resembling super-enhancers. By 30 minutes, widespread repositioning occurs throughout Mbp-long chromosomal segments, with consequential effects on 3D structure, detected using chromosome conformation capture. Conclusions Whilst nucleosome repositioning is viewed as a local phenomenon, our results point to effects occurring over multiple scales. Here, we present data in support of a TNFalpha-induced priming mechanism, mostly independent of NF-kappaB binding and/or elongating RNA polymerases, leading to a plastic network of interactions that affects DNA accessibility over large domains.

ORGANISM(S): Homo sapiens

PROVIDER: GSE53343 | GEO | 2014/11/05

SECONDARY ACCESSION(S): PRJNA231765

REPOSITORIES: GEO

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Project description:Prostaglandin E2 (PGE2) is involved in several inflammatory conditions including periodontitis. The aim of this study was to investigate the global gene expression profile of tumor necrosis factor alpha (TNFalpha) stimulated human gingival fibroblasts, focusing on signal pathways related to PGE2 production and the new PGE2-synthesizing enzymes, prostaglandin E synthases (PGES). The expression of microsomal prostaglandin E synthase-1 (mPGES-1) as well as the upstream cyclooxygenase-2 (COX-2) was up-regulated by TNFalpha, accompanied by increased PGE2 production. In contrast, the expression of microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNFalpha. Using microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, we identified differentially expressed genes in response to TNFalpha treatment. Enrichment analysis of microarray data identified two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-kappaB (NF-kappaB). We used specific inhibitors and phosphorylation analysis to confirm their role in PGE2 regulation. Both JNK and NF-kappaB inhibitors reduced the TNFalpha-stimulated up-regulation of mPGES-1 and COX-2 as well as subsequent PGE2 production. The novel finding that TNFalpha-stimulated mPGES-1 is regulated by JNK suggests this kinase as a potential future target for treatment strategies in inflammatory disorders, including periodontitis. Keywords: Time course, gene expression, factorial design. Three human gingival fibroblast cell lines were established from gingival biopsies obtained from 3 healthy patients, 3 to 12 years of age, with no clinical signs of periodontal disease. Cells were incubated with or without TNF-alpha (20 ng/ml) for 1, 3 or 6h. After incubation for 1, 3 or 6 h in the two conditions, the cells were immediately frozen in liquid nitrogen and then stored at -70°C for subsequent isolation of total RNA. The experimental design of the microarray study was set up as a time-course factorial design, to best observe the TNF-alpha induced gene expression changes over time. C++ program (G.F. Glonek, P.J. Solomon, Factorial and time course designs for cDNA microarray experiments, Biostatistics 5 (2004) 89-111) was used to determine the exact layout of the design in order to estimate the interaction effect between treatment and time, i.e. genes that are differentially expressed over time, with optimal statistical efficiency. Cyanine 5 and cyanine 3 were used for labeling. For each cell line 12 hybridizations were performed in a time-course factorial design. In total, 36 hybridizations were performed.

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TNFalpha signaling primes chromatin for NF-kappaB binding and induces rapid and widespread nucleosome repositioning

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