Myeloid hematopoiesis involves the transcriptional regulation of a number of tissue specific genes. The gp91-phox gene is a marker of terminal differentiation in phagocytic leukocytes. This gene codes for the 91-kilodalton subunit of a unique b cytochrome involved in the NADPH- dependent respiratory burst oxidase. Respiratory burst activity is characteristic of mature phagocytes and is a marker for terminal hematopoieses in this lineage. The proximal promoter region of the gp91- phox gene is adequate to target reporter gene expression to a subset of monocyte/macrophages in transgenic mice. In the proposed experiments, the cis-elements of the gp91-phox promoter that target expression to monocyte/macrophages will be identified. This will be done employing site directed mutagenesis to regions of the proximal gp91-phox promoter that have sequence specific in vitro protein binding to ablate the protein binding site. In vitro protein binding assays will be done using nuclear extracts from terminally differentiated myeloid cell lines. Function of the cis-elements will be assayed by detection of reporter gene transcription in transgenic mice and terminally differentiated myeloid cell lines. DNA-binding proteins that interact with the identified cis-elements will be cloned using lambda gt 11 expression library screening or protein purification employing DNA-affinity chromatography. Full length proteins will be expressed in E coli to assay function in vitro.
| Eklund, E A; Skalnik, D G (1995) Characterization of a gp91-phox promoter element that is required for interferon gamma-induced transcription. J Biol Chem 270:8267-73 |
