Different Isoforms of BSAP Protein Regulate the Expression of Activation-Induced Cytidine Deaminase (AID) in Normal and CLL B-Cells.

Background: In B-cells somatic hypermutation (SHM) and class switch recombination (CSR) depend of AID expression, a typical event in germinal centers upon CD40 ligand (CD40L) stimulation. We recently showed, that in contrast to normal B-cells, AID is constitutively expressed in a majority of unmutated CLL patients (Oppezzo et al, Blood, 2003). Pax-5 gene, encodes for the B-specific activator protein (BSAP) an essential protein in B-cell development. BSAP has been shown to up-regulate AID gene expression, through binding to its promoter region, whereas inhibitor of differentiation Id-2 down-regulates it. To study the molecular mechanisms underlying AID regulation, we analyzed expression of BSAP and Id-2 transcripts in normal and CLL B-cells and correlated these results to expression of AID and CSR process.

Results: B-cells from 6 healthy donors and 40 CLL patients were analyzed for Pax-5, Id-2, AID and CSR. Results show that CLL and normal B-cells not expressing AID exhibit diminished expression of BSAP and express a spliced variant of the Pax-5 gene (Pax-5 ΔEx8) displaying a deletion in the C-terminal-active domain. Upon CD40-L and IL-4 stimulation expression of AID transcripts and disappearance of the Pax-5 ΔEx8 transcripts was observed. Translation of complete Pax-5 and Pax-5/ΔEx8 isoforms and their binding to AID gene promoter have been demonstrated by Western Blot and EMSA assays, respectively. In contrast, CLL B-cells with constitutive AID expression and active CSR, constantly show increased levels of BSAP and absence of Pax-5ΔEx8 isoform. High expression of AID and BSAP is also correlated with a decrease of Id-2 transcripts.

Conclusion: These results suggest that the presence of BSAP isoform deleted in exon 8 may interfere with the binding of the complete BSAP protein to AID promoter and down-regulate the expression of Pax-5a. Thus, we show for the first time a subtle regulation mechanism controlling the expression of AID protein through the differential splicing of a transcription factor as BSAP.