title: Binding of pleomorphic adenoma gene-like 2 to the tumor necrosis factor (TNF)-alpha-responsive region of the NCF2 promoter regulates p67(phox) expression and NADPH oxidase activity.
abstract: NCF2, the gene encoding the NADPH oxidase cytosolic component p67(phox), is up-regulated by TNF-alpha, and we recently mapped a region in the NCF2 promoter that was required for this TNF-alpha-dependent response. Because this TNF-alpha-responsive region (TRR) lacked recognizable transcription factor binding elements, we performed studies to identify factors involved in regulating NCF2 via the TRR. Using the TRR sequence as bait in a yeast one-hybrid screen, we identified the zinc finger transcription factor Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. PLAGL2-specific antibodies were generated that detected the native and SUMO1-modified forms of endogenous PLAGL2. EMSA and DNA-binding protein affinity purification analyses demonstrated specific binding of in vitro-translated as well as endogenously expressed PLAGL2 to the TRR, and chromatin immunoprecipitation assays demonstrated enhanced binding of endogenous PLAGL2 to the TRR in vivo with TNF-alpha treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67(phox) protein expression, and subsequent superoxide production in response to TNF-alpha. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-alpha treatment. These data clearly identify PLAGL2 as a novel regulator of NCF2 gene expression as well as NADPH oxidase activity and contribute to a greater understanding of the transcriptional regulation of NCF2.
authors.affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: Mary Cloud B
authors.initials: MC
authors.lastname: Ammons
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Siemsen
authors.forename: Laura K
authors.initials: LK
authors.lastname: Nelson-Overton
authors.forename: Mark T
authors.initials: MT
authors.lastname: Quinn
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Gauss
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D025842:SUMO-1 Protein; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D014018:Tissue Distribution; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D004268:DNA-Binding Proteins; D016376:Oligonucleotides, Antisense; C114598:PLAGL2 protein, human; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases