Identification of direct genomic targets downstream of the nuclear factor-κB transcription factor mediating tumor necrosis factor signaling

Bing Tian, David E. Nowak, Mohammad Jamaluddin, Shaofei Wang, Allan R. Brasier

Research output: Contribution to journalArticle

173 Citations (Scopus)

Abstract

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-κB (NF-κB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-κB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-κB inhibitor to systematically identify NF-κB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-κB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F) <0.001) and demonstrated a change in signal intensity of ± 3-fold relative to control. Of these, 28 were NF-κB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time FCR assays of eight characterized NF-κB-dependent genes and five genes not previously known to be NF-κB-dependent (Gro-β and-γ, IκBε, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-κB regulated. Expression of constitutively active enhanced green fluorescent-NF-κB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-κB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IκBα/ε, Gro-β/γ and Naf-1 promoters directly bound NF-κ/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-κB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

Original languageEnglish (US)
Pages (from-to)17435-17448
Number of pages14
JournalJournal of Biological Chemistry
Volume280
Issue number17
DOIs
StatePublished - Apr 29 2005

Fingerprint

Transcription Factors
Tumor Necrosis Factor-alpha
Genes
Assays
Interleukins
Biological Phenomena
Electrophoretic mobility
Proteins
3'-(1-butylphosphoryl)adenosine
Gene Regulatory Networks
Chromatin Immunoprecipitation
Electrophoretic Mobility Shift Assay
Microarrays
Tetracycline
Chromatin
Interleukin-6
Fusion reactions
Binding Sites
Cells
Cytokines

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of direct genomic targets downstream of the nuclear factor-κB transcription factor mediating tumor necrosis factor signaling. / Tian, Bing; Nowak, David E.; Jamaluddin, Mohammad; Wang, Shaofei; Brasier, Allan R.

In: Journal of Biological Chemistry, Vol. 280, No. 17, 29.04.2005, p. 17435-17448.

Research output: Contribution to journalArticle

Tian, Bing ; Nowak, David E. ; Jamaluddin, Mohammad ; Wang, Shaofei ; Brasier, Allan R. / Identification of direct genomic targets downstream of the nuclear factor-κB transcription factor mediating tumor necrosis factor signaling. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 17. pp. 17435-17448.
@article{e5001a56e228405fbd6e802b32c15d1e,
title = "Identification of direct genomic targets downstream of the nuclear factor-κB transcription factor mediating tumor necrosis factor signaling",
abstract = "Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-κB (NF-κB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-κB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-κB inhibitor to systematically identify NF-κB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-κB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F) <0.001) and demonstrated a change in signal intensity of ± 3-fold relative to control. Of these, 28 were NF-κB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time FCR assays of eight characterized NF-κB-dependent genes and five genes not previously known to be NF-κB-dependent (Gro-β and-γ, IκBε, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-κB regulated. Expression of constitutively active enhanced green fluorescent-NF-κB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-κB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IκBα/ε, Gro-β/γ and Naf-1 promoters directly bound NF-κ/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-κB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.",
author = "Bing Tian and Nowak, {David E.} and Mohammad Jamaluddin and Shaofei Wang and Brasier, {Allan R.}",
year = "2005",
month = "4",
day = "29",
doi = "10.1074/jbc.M500437200",
language = "English (US)",
volume = "280",
pages = "17435--17448",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "17",

}

TY - JOUR

T1 - Identification of direct genomic targets downstream of the nuclear factor-κB transcription factor mediating tumor necrosis factor signaling

AU - Tian, Bing

AU - Nowak, David E.

AU - Jamaluddin, Mohammad

AU - Wang, Shaofei

AU - Brasier, Allan R.

PY - 2005/4/29

Y1 - 2005/4/29

N2 - Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-κB (NF-κB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-κB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-κB inhibitor to systematically identify NF-κB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-κB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F) <0.001) and demonstrated a change in signal intensity of ± 3-fold relative to control. Of these, 28 were NF-κB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time FCR assays of eight characterized NF-κB-dependent genes and five genes not previously known to be NF-κB-dependent (Gro-β and-γ, IκBε, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-κB regulated. Expression of constitutively active enhanced green fluorescent-NF-κB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-κB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IκBα/ε, Gro-β/γ and Naf-1 promoters directly bound NF-κ/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-κB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

AB - Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-κB (NF-κB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-κB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-κB inhibitor to systematically identify NF-κB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-κB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F) <0.001) and demonstrated a change in signal intensity of ± 3-fold relative to control. Of these, 28 were NF-κB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time FCR assays of eight characterized NF-κB-dependent genes and five genes not previously known to be NF-κB-dependent (Gro-β and-γ, IκBε, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-κB regulated. Expression of constitutively active enhanced green fluorescent-NF-κB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-κB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IκBα/ε, Gro-β/γ and Naf-1 promoters directly bound NF-κ/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-κB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

UR - http://www.scopus.com/inward/record.url?scp=20444478655&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=20444478655&partnerID=8YFLogxK

U2 - 10.1074/jbc.M500437200

DO - 10.1074/jbc.M500437200

M3 - Article

C2 - 15722553

AN - SCOPUS:20444478655

VL - 280

SP - 17435

EP - 17448

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -