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Fold change of nuclear NF-?B determines TNF-induced transcription in single cells.

ABSTRACT: In response to tumor necrosis factor (TNF), NF-?B enters the nucleus and promotes inflammatory and stress-responsive gene transcription. Because NF-?B deregulation is associated with disease, one might expect strict control of NF-?B localization. However, nuclear NF-?B levels exhibit considerable cell-to-cell variability, even in unstimulated cells. To resolve this paradox and determine how transcription-inducing signals are encoded, we quantified single-cell NF-?B translocation dynamics and transcription in the same cells. We show that TNF-induced transcription correlates best with fold change in nuclear NF-?B, not absolute nuclear NF-?B abundance. Using computational modeling, we find that an incoherent feedforward loop, from competition for binding to ?B motifs, could provide memory of the preligand state necessary for fold-change detection. Experimentally, we observed three gene-specific transcriptional patterns that our model recapitulates by modulating competition strength alone. Fold-change detection buffers against stochastic variation in signaling molecules and explains how cells tolerate variability in NF-?B abundance and localization.

SUBMITTER: Lee RE

PROVIDER: S-EPMC3977799 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Fold change of nuclear NF-κB determines TNF-induced transcription in single cells.

Lee Robin E C RE Walker Sarah R SR Savery Kate K Frank David A DA Gaudet Suzanne S

Molecular cell 20140213 6

In response to tumor necrosis factor (TNF), NF-κB enters the nucleus and promotes inflammatory and stress-responsive gene transcription. Because NF-κB deregulation is associated with disease, one might expect strict control of NF-κB localization. However, nuclear NF-κB levels exhibit considerable cell-to-cell variability, even in unstimulated cells. To resolve this paradox and determine how transcription-inducing signals are encoded, we quantified single-cell NF-κB translocation dynamics and tra ...[more]

PMID: 24530305

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Project description:BackgroundChronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N-glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate-ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression.MethodsR-based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real-time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation-specific PCR. Phospho-p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN-induced and DEN only-induced transgenic murine models of HCC.ResultsWe identified PSD4 as a hypermethylated, suppressed gene in alcohol-related HCC tumors; however, PSD4 was not dysregulated in all-cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF-α-induced phospho-p65(S276)'s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro-EMT CDC42 activity, resulting in downregulation of E-cadherin and upregulation of N-cadherin and vimentin. Hepatocyte-specific PSD4 overexpression reduced ethanol/DEN-induced HCC tumor progression and EMT marker expression in vivo.ConclusionsPSD4 is a hypermethylated, suppressed gene in alcohol-related HCC tumors that negatively modulated pro-EMT CDC42 activity. Furthermore, we present a novel phospho-NF-κB p65(S276)/DNMT1-mediated promoter methylation mechanism by which TNF-α/NF-κB signaling represses PSD4 transcription in HCC cells.

Dataset Information

Fold change of nuclear NF-?B determines TNF-induced transcription in single cells.

Publications

Fold change of nuclear NF-κB determines TNF-induced transcription in single cells.

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