The transcription factor B-cell-specific activator protein is not involved in the IL-4-induced activation of the human IgE germline promoter.

Abstract

Transcriptional activity of the human IgE germline gene is a prerequisite for a subsequent deletional rearrangement of the Ig heavy-chain locus, the hallmark of isotype switching to IgE. The B-cell-specific transcription factor B cell-specific activator protein (BSAP) was described for being critically involved in the IL-4 up-regulation of the murine IgE germline gene. Our study was initiated to evaluate the regulatory role of BSAP in the human IgE germline promoter. It is shown that BSAP binds to a DNA element located immediately upstream of the most 5' transcriptional start site. The authenticity of BSAP was determined by electrophoretic mobility shift assays in which oligonucleotides corresponding to published BSAP binding sites efficiently competed for binding to the novel identified sequence. In addition, recombinant purified BSAP protein bound this motif and comigrated with the band seen with nuclear extracts. Finally, a polyclonal anti-BSAP antiserum specifically prevented interaction of the protein with its DNA recognition sequence. The affinity of BSAP for its recognition sequence was low compared with the sites identified in the CD19, the blk gene, and an LR1 transcription factor binding sequence located in the Ig gamma 1 switch region. Reporter gene constructs in which binding of BSAP was abolished by site-directed mutagenesis responded to IL-4 stimulation better than the wild-type construct in both cell lines tested. In addition, the basal activity of the mutated promoter did not change significantly despite the close proximity of the BSAP motif to the transcriptional start site. It is concluded that BSAP plays no direct regulatory role in the cytokine-induced response of the human IgE germline promoter.