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


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.


CONCLUSIONS
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.
Hampson
L
L
CRC Department of Hematopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK. lynne.hampson@man.ac.uk
Hampson
I N
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Babichuk
C K
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Cotter
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Bleackley
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RC
Dexter
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Cross
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002874:Chromosome Mapping; D016384:Consensus Sequence; D003360:Cosmids; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D015843:Serpins / Q000096:biosynthesis / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; C490400:Serpina3k protein, mouse; D015843:Serpins