Abstract
Repressor elements in the gp91(phox) promoter are necessary to restrict tissue-specific transcription to mature phagocytes. Deletion of these elements leads to significant promoter activity in cell lines such as HEL and K562 that do not normally express gp91(phox). The -100 to +12 base pair gp91(phox) promoter region is sufficient to direct maximal de-repressed transcription in these cells. However, promoter activity is dramatically decreased following a 16-base pair truncation that deletes an interferon-stimulated response element. This element interacts with IRF-1 and IRF-2, members of the interferon regulatory factor family of transcription factors. In addition, this promoter region is bound by a factor with properties similar to BID, a DNA-binding protein that also interacts with three upstream sites within the gp91(phox) promoter. Transient transfection studies using mutated promoters indicate that both the IRF and BID binding sites are required for maximal gp91(phox) promoter activity. Overexpression of IRF-1 or IRF-2 in K562 cells leads to transactivation of gp91(phox) promoter constructs, which is dependent on the presence of an intact IRF binding site. IRF-2 predominates in macrophages that express the gp91(phox) gene as well as in HEL and K562 cells. We conclude that IRF-2 and BID activate gp91(phox) promoter activity in the absence of transcriptional repression.
Highlights
The gp91phox gene encodes the cytochrome b558 heavy chain of the NADPH-dependent oxidase and is required for the generation of toxic free radicals and microbicidal activity by phagocytes [1]
Identification of a Cis Element Necessary for gp91phox Promoter Activity—Previously we demonstrated that the Ϫ100 to ϩ12 bp region of the gp91phox gene, which lacks multiple upstream repressor elements, exhibits significant promoter activity in cell lines such as K562 and HEL that do not normally express the gp91phox gene [9]
This study examines the cis elements and cognate DNAbinding proteins that are required for gp91phox promoter activity in the absence of CCAAT displacement protein (CDP)-mediated repression
Summary
The gp91phox gene encodes the cytochrome b558 heavy chain of the NADPH-dependent oxidase and is required for the generation of toxic free radicals and microbicidal activity by phagocytes [1]. Related if not identical DNA-binding proteins denoted BID (binding increased during differentiation) bind to three sites centered at Ϫ355 bp (DIST-BID), Ϫ225 bp (MID-BID), and Ϫ145 bp (PROX-BID) of the gp91phox promoter. These interactions are required for IFN-␥-induced transcription.. The restriction of gp91phox promoter activity to mature phagocytes requires regulated transcriptional repression mediated by the binding of CCAAT displacement protein (CDP). Transcriptional activating elements within this region of the endogenous gp91phox promoter are presumably interfered with by CDP binding in nonphagocytic cells and are possibly required for appropriate transcription in mature myeloid cells. We describe a cis element between Ϫ100 and Ϫ85 bp of the gp91phox promoter that is required for promoter activity in the absence of repression and which binds members of the IRF family and a factor similar to BID
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