J Biol Chem, Vol. 274, Issue 3, 1366-1374, January 15, 1999

Cloning of the Murine ₅ Integrin Subunit Promoter
IDENTIFICATION OF A NOVEL SEQUENCE MEDIATING GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR-DEPENDENT REPRESSION OF ₅ INTEGRIN GENE TRANSCRIPTION

Xu Feng, Steven L. Teitelbaum, Marisol E. Quiroz, Dwight A. Towler, and F. Patrick Ross

From the Departments of Pathology and  Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin _v5 and that granulocyte-macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the ₅ gene. We herein report cloning of the ₅ integrin gene promoter and identification of a GM-CSF-responsive sequence. A 13-kilobase (kb) genomic fragment containing part of the ₅ gene was isolated by screening a mouse genomic library with a probe derived from the most 5'-end of a murine ₅ cDNA. A combination of primer extension and S1 nuclease studies identifies two transcriptional start sites, with the major one designated +1. A 1-kb subclone containing sequence 875 to + 110 is transcriptionally active in a murine myeloid cell line. This 1-kb fragment contains consensus binding sequences for basal (Sp1), lineage-specific (PU.1), and regulatable (signal transducer and activator of transcription) transcription factors. Reflecting our earlier findings, promoter activity is repressed in transfected myeloid cells treated with GM-CSF. Using deletion mutants, we localized a 109-base pair (bp) promoter region responsible for GM-CSF-inhibited ₅ transcription. We further identified a 19-bp sequence within the 109-bp region that binds GM-CSF-induced nuclear proteins by gel shift/competition assays. Mutation of the 19-bp sequence not only ablates its capacity to bind nuclear proteins from GM-CSF-treated cells, in vitro, but the same mutation, when introduced in the 1-kb promoter, abolishes its ability to respond to GM-CSF treatment. Northern analysis demonstrates that cycloheximide treatment abrogates the capacity of GM-CSF to decrease ₅ mRNA levels. In summary, we have identified a 19-bp cis-element mediating GM-CSF-induced down-regulation of ₅ by a mechanism requiring protein synthesis.