Identification and characterization of a novel transcription factor that regulates NCF2 expression via the TNF-alpha responsive region

Abstract

The multicomponent NADPH oxidase is an essential enzyme complex found in professional phagocytic cells that mediates innate immune defence against multiple pathogens through the production of reactive oxygen species. The vital and functionally limiting cytosolic component of the NADPH oxidase, p67 phox, is transcriptionally regulated by TNF-alpha at the TNF-alpha Responsive Region (TRR) in the intragenic region of Neutrophil Cytosolic Factor 2 (NCF2), the gene which codes for p67 phox. The aim of this dissertation is to identify and charaterize the factor(s) that binds the TRR and regulates NCF2 expression in response to TNF-alpha. Using the TRR as bait in a yeast one-hybrid screen, we identified Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. In vitro and in vivo analysis confirmed that PLAGL2 binds to the TRR in NCF2 and that endogenous PLAGL2 binding is enhanced in the presence of TNF-alpha. Knock-down of endogenous PLAGL2 expression inhibited TNF-alpha-mediated NCF2 expression, p67 phox expression, and subsequent superoxide production. Characterization of PLAGL2 binding to the NCF2 TRR identified the essential core sequence which is recognized by zinc fingers six and seven of PLAGL2. Using PLAGL2 as bait in a yeast two-hybrid screen, we identified ubiquitin protein ligase (E2I), positive cofactor 2 (PC2,) four and a half LIM domain 3 (FHL3), and chromodomain helicase DNA binding protein 3 (CHD3) as putative PLAGL2 binding partners. In vitro and in vivo analysis confirmed that PLAGL2 binds PC2 434-748, FHL3, and CHD3 via interactions mediated through unique and overlapping domains of PLAGL2. In conclusion, we have identified a novel regulator of NCF2 transcription and have further characterized the features of PLAGL2 binding and recognition of the TRR sequence in the intragenic region of NCF2. Finally, through identification of PLAGL2 binding partners, we have begun to determine a model by which PLAGL2 regulates transcription in a context dependent manner.