Transcription factors regulating the Btk promoter

• Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase-related to the Src family of kinases. Mutations in various parts of the gene have been shown to lead to X-linked agammaglobulinemia, an immunodeficiency disease which is characterized by a defect in B-cell development. Although the disease manifests itself only in the B-cell compartment, Btk was found to be expressed in all hematopoietic cells but mature T cells and plasma cells. In an attempt to investigate the regulation of the Btk gene the genomic organization of mouse and human Btk genes were determined. Both loci were found to be very similarly organized and consisted of 19 exons. The transcriptional start sites in both genes were found to be a cluster of sites surrounding a putative initiator sequence. Sequences upstream were able to drive a reporter gene in an orientation dependent manner and therefore fulfill the criteria for a promoter. No obvious TATA-box was found. The280 bp upstream contained the elements necessary for the cell restricted expression of 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 transactivation domains A and B nor the D domain of Sp 1 could be replaced by the corresponding domain of Sp3. The transcription factors PU. 1 and Spi-B activated the Btk promoter in cotransfection experiments. However, analysis of Btk expression in fetal liver of PU. 1-/- mice, which lack lymphoid and myeloid cells revealed that transcription of Btk also occurred in the absence of PU. 1, albeit at a reduced level. Further analysis in Drosophila Schneider cells, which are devoid of Sp- or PU.l-like activity, demonstrated that PU.l and Spl/3 synergistically activated the Btk promoter.This synergism is mediated through adjacent binding sites implicating a possible direct interaction between the proteins. Understanding the regulation of the Btk gene is a first step towards a somatic gene therapy approach for X-linked agammaglobulinerma using endogenous regulatory elements.

Nyckelord

Btk, X-linked agammaglobulinemia, transcription factors, repression,synergism, Sp 1, Sp3, PU.l, Spi-B, genetherapy.

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