Cloning of the murine beta5 integrin subunit promoter. Identification of a novel sequence mediating granulocyte-macrophage colony-stimulating factor-dependent repression of beta5 integrin gene transcription.
We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin alphav beta5 and that granulocyte-macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the beta5 gene. We herein report cloning of the beta5 integrin gene promoter and identification of a GM-CSF-responsive sequence. A 13-kilobase (kb) genomic fragment containing part of the beta5 gene was isolated by screening a mouse genomic library with a probe derived from the most 5'-end of a murine beta5 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 beta5 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 beta5 mRNA levels. In summary, we have identified a 19-bp cis-element mediating GM-CSF-induced down-regulation of beta5 by a mechanism requiring protein synthesis.
Feng
X
X
Departments of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Teitelbaum
S L
SL
Quiroz
M E
ME
Towler
D A
DA
Ross
F P
FP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D039641:Integrin beta Chains; D016023:Integrins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012097:Repressor Proteins / Q000378:metabolism*; D014158:Transcription, Genetic
D039641:Integrin beta Chains; D016023:Integrins; D011500:Protein Synthesis Inhibitors; D012097:Repressor Proteins; C084344:integrin beta5; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003513:Cycloheximide