Cloning of the murine β5 integrin subunit promoter. Identification of a novel sequence mediating granulocyte-macrophage colony-stimulating factor- dependent repression of β5 integrin gene transcription

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

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin α(v)β5 and that granulocyte- macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the β5 gene. We herein report cloning of the β5 integrin gene promoter and identification of a GM-CSF- responsive sequence. A 13-kilobase (kb) genomic fragment containing part of the β5 gene was isolated by screening a mouse genomic library with a probe derived from the most 5'-end of a murine β5 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 β5 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 β5 mRNA levels. In summary, we have identified a 19-bp cis-element mediating GM-CSF-induced down-regulation of β5 by a mechanism requiring protein synthesis.

Original languageEnglish (US)
Pages (from-to)1366-1374
Number of pages9
JournalJournal of Biological Chemistry
Volume274
Issue number3
DOIs
StatePublished - Jan 15 1999

Fingerprint

Cloning
Transcription
Granulocyte-Macrophage Colony-Stimulating Factor
Integrins
Organism Cloning
Genes
Base Pairing
Myeloid Cells
Nuclear Proteins
Cells
Mutation
Genomic Library
Consensus Sequence
Osteoclasts
Cycloheximide
Transducers
Genetic Promoter Regions
Assays
Screening
Transcription Factors

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cloning of the murine β5 integrin subunit promoter. Identification of a novel sequence mediating granulocyte-macrophage colony-stimulating factor- dependent repression of β5 integrin gene transcription. / Feng, Xu; Teitelbaum, Steven L.; Quiroz, Marisol E.; Towler, Dwight A.; Ross, F. Patrick.

In: Journal of Biological Chemistry, Vol. 274, No. 3, 15.01.1999, p. 1366-1374.

Research output: Contribution to journalArticle

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abstract = "We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin α(v)β5 and that granulocyte- macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the β5 gene. We herein report cloning of the β5 integrin gene promoter and identification of a GM-CSF- responsive sequence. A 13-kilobase (kb) genomic fragment containing part of the β5 gene was isolated by screening a mouse genomic library with a probe derived from the most 5'-end of a murine β5 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 β5 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 β5 mRNA levels. In summary, we have identified a 19-bp cis-element mediating GM-CSF-induced down-regulation of β5 by a mechanism requiring protein synthesis.",
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T1 - Cloning of the murine β5 integrin subunit promoter. Identification of a novel sequence mediating granulocyte-macrophage colony-stimulating factor- dependent repression of β5 integrin gene transcription

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AU - Teitelbaum, Steven L.

AU - Quiroz, Marisol E.

AU - Towler, Dwight A.

AU - Ross, F. Patrick

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AB - We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin α(v)β5 and that granulocyte- macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the β5 gene. We herein report cloning of the β5 integrin gene promoter and identification of a GM-CSF- responsive sequence. A 13-kilobase (kb) genomic fragment containing part of the β5 gene was isolated by screening a mouse genomic library with a probe derived from the most 5'-end of a murine β5 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 β5 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 β5 mRNA levels. In summary, we have identified a 19-bp cis-element mediating GM-CSF-induced down-regulation of β5 by a mechanism requiring protein synthesis.

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