Transcriptional control of Pactolus: evidence of a negative control region and comparison with its evolutionary paralogue, CD18 (β2 integrin)

The mouse Pactolus and CD18 genes are highly conserved paralogues. The expression patterns of these genes are diverse in that most cells of hematopoietic lineage express CD18, but Pactolus is only expressed by maturing neutrophils. The minimal promoters of these two genes are homologous, including t...

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Veröffentlicht in:Journal of leukocyte biology 2006-08, Vol.80 (2), p.383-398
Hauptverfasser: Hale, J. Scott, Dahlem, Timothy J., Margraf, Rebecca L., Debnath, Irina, Weis, Janis J., Weis, John H.
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container_end_page 398
container_issue 2
container_start_page 383
container_title Journal of leukocyte biology
container_volume 80
creator Hale, J. Scott
Dahlem, Timothy J.
Margraf, Rebecca L.
Debnath, Irina
Weis, Janis J.
Weis, John H.
description The mouse Pactolus and CD18 genes are highly conserved paralogues. The expression patterns of these genes are diverse in that most cells of hematopoietic lineage express CD18, but Pactolus is only expressed by maturing neutrophils. The minimal promoters of these two genes are homologous, including the conservation of two tandem PU.1‐binding sites upstream of the transcriptional start site. To define the means by which these two structurally similar but functionally distinct promoters operate, a series of reporter assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation analyses, were performed. Transfection of Pactolus constructs into mouse macrophages, which do not express Pactolus, defined a negative control element within the first 100 base pairs. The presence of this negative regulatory site, distinct from the PU.1‐binding site, was confirmed by EMSA oligonucleotide competition and gene reporter assays of Pactolus/CD18 chimeric constructs. Although PU.1 binding can be detected on Pactolus and CD18 minimal promoter segments with EMSA, only the CD18 promoter shows PU.1 binding in vivo, suggesting that the negative regulatory protein may block PU.1 from binding to the Pactolus promoter, thus inhibiting transcription of the gene. Sequence analysis of the negative control region in the Pactolus promoter suggested potential control by Snail and/or Smad families of transcription regulators. EMSA supershift analysis with antibodies against these proteins, using extracts from macrophages and mucosal mast cells, identified specific binding of Smuc to the promoter element, including a Smuc/PU.1/DNA trimeric complex. These data implicate Smuc as blocking Pactolus transcription in cells expressing PU.1 (and CD18) but not Pactolus.
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Transfection of Pactolus constructs into mouse macrophages, which do not express Pactolus, defined a negative control element within the first 100 base pairs. The presence of this negative regulatory site, distinct from the PU.1‐binding site, was confirmed by EMSA oligonucleotide competition and gene reporter assays of Pactolus/CD18 chimeric constructs. Although PU.1 binding can be detected on Pactolus and CD18 minimal promoter segments with EMSA, only the CD18 promoter shows PU.1 binding in vivo, suggesting that the negative regulatory protein may block PU.1 from binding to the Pactolus promoter, thus inhibiting transcription of the gene. Sequence analysis of the negative control region in the Pactolus promoter suggested potential control by Snail and/or Smad families of transcription regulators. EMSA supershift analysis with antibodies against these proteins, using extracts from macrophages and mucosal mast cells, identified specific binding of Smuc to the promoter element, including a Smuc/PU.1/DNA trimeric complex. 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To define the means by which these two structurally similar but functionally distinct promoters operate, a series of reporter assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation analyses, were performed. Transfection of Pactolus constructs into mouse macrophages, which do not express Pactolus, defined a negative control element within the first 100 base pairs. The presence of this negative regulatory site, distinct from the PU.1‐binding site, was confirmed by EMSA oligonucleotide competition and gene reporter assays of Pactolus/CD18 chimeric constructs. Although PU.1 binding can be detected on Pactolus and CD18 minimal promoter segments with EMSA, only the CD18 promoter shows PU.1 binding in vivo, suggesting that the negative regulatory protein may block PU.1 from binding to the Pactolus promoter, thus inhibiting transcription of the gene. Sequence analysis of the negative control region in the Pactolus promoter suggested potential control by Snail and/or Smad families of transcription regulators. EMSA supershift analysis with antibodies against these proteins, using extracts from macrophages and mucosal mast cells, identified specific binding of Smuc to the promoter element, including a Smuc/PU.1/DNA trimeric complex. 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Scott</creatorcontrib><creatorcontrib>Dahlem, Timothy J.</creatorcontrib><creatorcontrib>Margraf, Rebecca L.</creatorcontrib><creatorcontrib>Debnath, Irina</creatorcontrib><creatorcontrib>Weis, Janis J.</creatorcontrib><creatorcontrib>Weis, John H.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of leukocyte biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hale, J. 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subjects Animals
Base Sequence
Binding Sites
CD18 Antigens - genetics
Cell Line
Down-Regulation
Evolution, Molecular
Gene Expression Regulation
granulocytes
Integrin beta1 - genetics
macrophages
Mice
Molecular Sequence Data
neutrophils
Promoter Regions, Genetic
Proto-Oncogene Proteins - genetics
Snail Family Transcription Factors
Trans-Activators - genetics
Transcription Factors - metabolism
Transcription, Genetic
Transfection
title Transcriptional control of Pactolus: evidence of a negative control region and comparison with its evolutionary paralogue, CD18 (β2 integrin)
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