Binding of Pleomorphic Adenoma Gene-like 2 to the Tumor Necrosis Factor (TNF)- alpha -responsive Region of the NCF2 Promoter Regulates p67 super(phox) Expression and NADPH Oxidase Activity

• Published in: The Journal of biological chemistry 2007-06, Vol.282 (24), p.17941-17952
• Main Authors: Ammons, Mary Cloud B, Siemsen, Daniel W, Nelson-Overton, Laura K, Quinn, Mark T, Gauss, Katherine A
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X

NCF2, the gene encoding the NADPH oxidase cytosolic component p67 super(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 super(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.