PU.1 AS AN ESSENTIAL ACTIVATOR FOR THE EXPRESSION OF GP91(PHOX) GENE IN HUMAN PERIPHERAL NEUTROPHILS, MONOCYTES, AND B-LYMPHOCYTES

We have reported a deficiency of a 91-kDa glycoprotein component of th e phagocyte NADPH oxidase (gp91(phox)) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomat ous disease. Sequence analysis of his gp91(phox) gene revealed a singl e-base mutation (C --> T) at position -53. Electrophoresis mobility-sh ift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1. consensus sequence centered at po sition -53 of the gp91(phox) promoter, and the mutation at position -5 3 strongly inhibited the binding of both factors. It was also indicate d that a mutation at position -50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position -56 had an opposite binding specificity for these factors. In transient expressio n assay using HEL cells, which express PU.1 and HAF-1, the mutations a t positions -53 and -50 significantly reduced the gp91(phox) promoter activity; however, the mutation at position -56 did not affect the pro moter activity. In transient cotransfection study, PU.1 dramatically a ctivated the gp91(phox) promoter in Jurkat T cells, which originally c ontained HAF-1 but not PU.1. In addition, the single-base mutation (C --> T) at position -52 that was identified in a patient with chronic g ranulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expres sion of gp91(phox) gene in human neutrophils, monocytes, and B lymphoc ytes.