A minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells.

2.50
Hdl Handle:
http://hdl.handle.net/10541/85585
Title:
A minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells.
Authors:
Hampson, Lynne; Hampson, Ian N; Babichuk, Charolyn K; Cotter, Laura A; Bleackley, R Chris; Dexter, T Michael; Cross, Michael A
Abstract:
INTRODUCTION: The serine protease inhibitor Serpin 2A is highly expressed in ex vivo bipotent granulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The gene undergoes rapid down-regulation as these cells are induced to differentiate, and constitutive expression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in T cells as a consequence of activation. We now report analysis of the upstream regulatory elements that control Serpin 2A transcription. MATERIALS AND METHODS: Using primer extension and rapid amplification of cDNA ends the transcription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstream fragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoretic mobility shift assays. RESULTS: A minimal promoter fragment was identified with high activity dependent on NF-kappa and Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cells and activated T cells. NF-kappa was shown to be the main DNA binding protein in T cells, whereas that in haematopoietic stem cells appears to be novel. CONCLUSION: Serpin 2A promoter activity in T cells is due predominantly to NF-kappa binding to its consensus site. Activity in haematopoietic stem cells appears to be mediated by a novel protein, which recognises the NF-kappa consensus only in the context of flanking sequences. This concise regulatory element may be of potential value in gene therapeutic applications.
Affiliation:
CRC Department of Hematopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK. lynne.hampson@man.ac.uk
Citation:
A minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells. 2001, 2 (3):150-60 Hematol. J.
Journal:
The Hematology Journal
Issue Date:
2001
URI:
http://hdl.handle.net/10541/85585
DOI:
10.1038/sj/thj/6200102
PubMed ID:
11920240
Type:
Article
Language:
en
ISSN:
1466-4860
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorHampson, Lynneen
dc.contributor.authorHampson, Ian Nen
dc.contributor.authorBabichuk, Charolyn Ken
dc.contributor.authorCotter, Laura Aen
dc.contributor.authorBleackley, R Chrisen
dc.contributor.authorDexter, T Michaelen
dc.contributor.authorCross, Michael Aen
dc.date.accessioned2009-11-06T16:16:43Z-
dc.date.available2009-11-06T16:16:43Z-
dc.date.issued2001-
dc.identifier.citationA minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells. 2001, 2 (3):150-60 Hematol. J.en
dc.identifier.issn1466-4860-
dc.identifier.pmid11920240-
dc.identifier.doi10.1038/sj/thj/6200102-
dc.identifier.urihttp://hdl.handle.net/10541/85585-
dc.description.abstractINTRODUCTION: The serine protease inhibitor Serpin 2A is highly expressed in ex vivo bipotent granulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The gene undergoes rapid down-regulation as these cells are induced to differentiate, and constitutive expression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in T cells as a consequence of activation. We now report analysis of the upstream regulatory elements that control Serpin 2A transcription. MATERIALS AND METHODS: Using primer extension and rapid amplification of cDNA ends the transcription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstream fragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoretic mobility shift assays. RESULTS: A minimal promoter fragment was identified with high activity dependent on NF-kappa and Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cells and activated T cells. NF-kappa was shown to be the main DNA binding protein in T cells, whereas that in haematopoietic stem cells appears to be novel. CONCLUSION: Serpin 2A promoter activity in T cells is due predominantly to NF-kappa binding to its consensus site. Activity in haematopoietic stem cells appears to be mediated by a novel protein, which recognises the NF-kappa consensus only in the context of flanking sequences. This concise regulatory element may be of potential value in gene therapeutic applications.en
dc.language.isoenen
dc.subjectHaematopoietic Stem Cellsen
dc.subject.meshAnimals-
dc.subject.meshBase Sequence-
dc.subject.meshBinding Sites-
dc.subject.meshCells, Cultured-
dc.subject.meshChromosome Mapping-
dc.subject.meshConsensus Sequence-
dc.subject.meshCosmids-
dc.subject.meshDNA-Binding Proteins-
dc.subject.meshElectrophoretic Mobility Shift Assay-
dc.subject.meshExons-
dc.subject.meshGene Expression Regulation-
dc.subject.meshGenes, Reporter-
dc.subject.meshHematopoietic Stem Cells-
dc.subject.meshLymphocyte Activation-
dc.subject.meshMice-
dc.subject.meshMice, Inbred BALB C-
dc.subject.meshMice, Inbred CBA-
dc.subject.meshMolecular Sequence Data-
dc.subject.meshMutagenesis, Site-Directed-
dc.subject.meshNF-kappa B-
dc.subject.meshOrgan Specificity-
dc.subject.meshPromoter Regions, Genetic-
dc.subject.meshRNA, Messenger-
dc.subject.meshReverse Transcriptase Polymerase Chain Reaction-
dc.subject.meshSequence Deletion-
dc.subject.meshSerpins-
dc.subject.meshT-Lymphocytes-
dc.subject.meshTranscription, Genetic-
dc.subject.meshTransfection-
dc.titleA minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells.en
dc.typeArticleen
dc.contributor.departmentCRC Department of Hematopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK. lynne.hampson@man.ac.uken
dc.identifier.journalThe Hematology Journalen

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