Role of BSAP in the regulation of human epsilon germ line transcription

Ig isotype switching in B lymphocytes is directed by cytokines, which activate transcription of the target gene in germ line configuration. Germ line transcripts (GLT) contain short I exons spliced to the heavy chain codons. Expression of GLT is regulated by nuclear factors that bind the I exon promoter and adjacent regions. IL-4 induces epsilon GLT in murine and human B cells. A major IL-4 responsive element (IL-4RE) has been identified upstream of the human I epsilon exon. A Xmal-BclI fragment downstream of the IL-4RE is highly conserved, and contains a site that has been shown to bind the B cell-specific factor BSAP (Pax5) in the mouse. BSAP has been implicated in the regulation of murine epsilon GLT, and in the negative regulation of the Ig 3′ enhancer We have identified a human EBV-negative B cell line, BJAB, that expresses epsilon GLT upon IL-4 stimulation. EMSA analysis with nuclear extracts from unstimulated and IL-4-stimulated BJAB cells has shown that: a) The human epsilon GLT promoter binds BSAP. b) BSAP activity is constitutively expressed in BJAB cells, and in normal peripheral blood B cells. IL-4 does not seem to affect human BSAP binding activity, c) The invariant C in the BSAP binding site is essential for DNA/protein interactions. In order to test the role of BSAP in the IL-4-dependent induction of epsilon GLT, we are currently testing the effects of deletions and/or point mutations in the BSAP binding site in reporter assays.