Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling.
Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB 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-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/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-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.
Tian
Bing
B
Department of Medicine, The University of Texas Medical Branch, Galveston, Texas 77555-1060, USA.
Nowak
David E
DE
Jamaluddin
Mohammad
M
Wang
Shaofei
S
Brasier
Allan R
AR
D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers / Q000737:chemistry; D004318:Doxycycline / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007249:Inflammation; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000737:chemistry; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D051996:Transcription Factor RelA; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D017931:DNA Primers; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009841:Oligonucleotides; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D012313:RNA; D013752:Tetracycline; D004318:Doxycycline