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

Project description: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. MNase-Seq of HUVEC cells after stimulation with TNFÎ± for 0, 10 or 30 min. Biological replicates for 0 and 30 min.

2014-11-05 | E-GEOD-53343 | biostudies-arrayexpress

Homo sapiens

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

| PRJNA231765 | ENA

S.cerevisiae INO80 is required for promoter proximal nucleosome remodeling.

Project description:During transcription, nucleosomes are evicted from regulatory and coding regions yet chromatin structure is stable. Restoration of chromatin structure involves concerted action of chromatin modifying activities. Our analysis demonstrates a genome wide function of the INO80 remodeling complex for stable repositioning of the nucleosome immediately proximal to the transcription initiation site. INO80 dependent remodeling of the promoter proximal nucleosomes has a global repressive role. Recruitment of INO80 to proximal nucleosomes overlaps with the elongating Polymerase II complex assembly. The amount of associated Polymerase II at start sites correlates with INO80 recruitment for inducible and constantly transcribed genes. Furthermore, at highly inducible promoters INO80 is required for repression of bidirectional transcription. Therefore, we suggest a function for INO80 after transcription initiation to achieve Polymerase II dependent reassembly of promoter proximal nucleosomes.

2016-12-24 | E-MTAB-1810 | biostudies-arrayexpress

Nucleosome Repositioning: Nicotine- and Cocaine-induced Changes

Project description:SH-SY5Y, neuroblastoma cells, were exposed to nicotine for 10, 60, and 90 minutes or cocaine for 5, 20, 40, and 60 minutes. Drugs of abuse modify behavior by altering gene expression in the brain. Gene expression can be regulated by changes in DNA methylation as well as by histone modifications, which alter chromatin structure, DNA compaction and DNA accessibility. In order to better understand the molecular mechanisms directing drug-induced changes in chromatin structure, we examined DNA-nucleosome interactions within promoter regions of 858 genes in human neuroblastoma cells (SH-SY5Y) exposed to nicotine or cocaine. Widespread, drug- and time-resolved repositioning of nucleosomes was identified at the transcription start site and promoter region of multiple genes. Nicotine and cocaine produced unique and shared changes in terms of the numbers and types of genes affected, as well as repositioning of nucleosomes at sites which could increase or decrease the probability of gene expression based on DNA accessibility. Half of the drug-induced nucleosome positions approximated a theoretical model of nucleosome occupancy based on physical and chemical characteristics of the DNA sequence, whereas the basal or drug-naive positions were generally DNA sequence independent. Thus we suggest that nucleosome repositioning represents an initial dynamic genome-wide alteration of the transcriptional landscape preceding more selective downstream transcriptional reprogramming, which ultimately characterizes the cell- and tissue-specific responses to drugs of abuse. SH-SY5Y, neuroblastoma cells, were exposed to nicotine for 10, 60, and 90 minutes or cocaine for 5, 20, 40, and 60 minutes. Two independently grown biological replicates for each time point and condition, and two controls were processed.

2015-08-07 | E-GEOD-71795 | biostudies-arrayexpress

Nucleosome Repositioning: Nicotine- and Cocaine-induced Changes

2015-08-07 | GSE71795 | GEO

Microarray analysis of the regulation of TNFalpha-stimulated PGE2 production in gingival fibroblasts

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.

2008-12-11 | GSE13903 | GEO

Transcription profiling by array of mouse C2C12 muscle cells overexpressing PGC-1alpha, PGC-1beta or GFP to investigate differential expression of TNFalpha-induced genes

Project description:Differentiated C2C12 cells adenovirally overexpressing either PGC-1alpha or PGC-1beta or GFP as control were treated with TNFalpha for 2h or left untreated. NF-kappaB- and stress-depedent gene expression was determined by a customized array.

2012-12-14 | E-MEXP-3676 | biostudies-arrayexpress

p65-HaloTag copurifying proteins in human 293T cells

Project description:In effort to develop methodology for targeted top down mass spectrometry of NF kappa B p65 from human cells, we evaluated the utility of HaloTag for purification and analysis of recombinant protein. During our study, two datasets of bottom up LC-MS/MS were generated: one from in-gel digestion of the predominant band following p65-HaloTag purification, another from in-solution digestion of all the proteins present in a p65-HaloTag purification. p65-HaloTag copurifying proteins identified in our datasets include the known interactors c-Rel, NF-kappaB p105, NF-kappaB p100, and NF-kappaB inhibitor beta. Over 100 proteins were identified by at least two peptides using a Mascot ion cut-off score of 30.

2018-10-19 | PXD001932 | Pride

Genome-wide maps of NME2 in A549 lung adenocarcinoma cells.

Project description:It is widely believed that reorganization of nucleosomes result in availability of transcription factor (TF) binding sites for eukaryotic gene regulation. Recent findings also show TFs induced during physiological perturbations can alter nucleosome occupancy to facilitate DNA binding. Although, these suggest a close relationship between TF binding and nucleosomes, the nature of this interaction, or to what extent it influences transcription is not clear. Moreover, since physiological perturbations induced multiple TFs, relatively direct effect of any TF on nucleosome occupancy remains poorly addressed. With these in mind, we used a single TF to induce physiological changes and following characterization of the two states (before and after induction of the TF) we determined: (a) genome wide binding sites of the TF, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. We find only ~20% of TF binding results from nucleosome repositioning - interestingly, almost all corresponding genes were transcriptionally altered. Whereas, when TF-occupancy was independent of nucleosome repositioning only a small fraction of corresponding genes were expressed/repressed. These observations suggest a model where TF occupancy leads to transcriptional change only when coupled with nucleosome repositioning in close proximity. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-sequencing) typically overlaps with only a small fraction of genes that change expression. The nature of interaction between TF binding and nucleosomes and what extent it influences transcription

2014-08-04 | E-GEOD-40300 | biostudies-arrayexpress

Expanding the substantial interactome of NEMO using protein microarrays

Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.

2010-01-20 | GSE19841 | GEO

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