Transcription factors regulating the Btk promoter
Sammanfattning: Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinaserelated to the Src family of kinases. Mutations in various parts of the gene havebeen shown to lead to X-linked agammaglobulinemia, an immunodeficiency disease whichis characterized by a defect in B-cell development. Although the disease manifestsitself only in the B-cell compartment, Btk was found to be expressed in all hematopoieticcells but mature T cells and plasma cells. In an atternpt to investigate the regulation of the Btk gene the genomic organizationof mouse and human Btk genes were determined. Both loci were found to be very similarlyorganized and consisted of 19 exons. The transcriptional start sites in both geneswere found to be a cluster of sites surrounding a putative initiator sequence. Sequencesupstream were able to drive a reporter gene in an orientation dependent manner andtherefore fulfill the criteria for a promoter. No obvious TATA-box was found. The280 bp upstream contained the elements necessary for the cell restricted expressionof a reporter gene which was mediated through the Sp-family members Sp I and Sp3and the Ets family members PU. 1 and Spi-B. The transcription factor Sp3 was shown to possess a dual function as activator,in the Btk promoter, and repressor of Sp 1 mediated activation in several other promoters.Despite the conserved structure between Sp 1 and Sp3 neither the glutamine-rich transactivationdomains A and B nor the D domain of Sp 1 could be replaced by the corresponding domainof Sp3. The transcription factors PU. I and Spi-B activated the Btk promoter in cotransfectionexperiments. However, analysis of Btk expression in fetal liver of PU. 1-/- mice,which lack Iymphoid and myeloid cells revealed that transcription of Btk also occurredin the absence of PU. 1, albeit at a reduced level. Further analysis in Drosophila Schneider cells, which are devoid of Sp- or PU.l-likeactivity, demonstrated that PU.l and Spl/3 synergistically activated the Btk promoter.This synergism is mediated through adjacent binding sites implicating a possibledirect interaction between the proteins. Understanding the regulation of the Btk gene is a first step towards a somaticgene therapy approach for X-linked agammaglobulinerma using endogenous regulatoryelements. Keywords: Btk, X-linked agammaglobulinemia, transcription factors, repression,synergism, Sp 1, Sp3, PU.l, Spi-B, genetherapy. ISBN 91-628-2717-0
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