Identification and comparative analysis of Pax5 C-terminal isoforms expressed in human cord blood-derived B cell progenitors

We identified three Pax5 isoforms due to alternative splicing of the C-terminal exons of its gene in cord blood (CB)-derived B cell progenitors cultivated on the murine bone marrow stromal (HESS-5) cells. Apart from wild type (wt), one isoform skips exon 9 without subsequent frameshift (del9), while the other has a frameshift insert between exons 8 and 9, resulting in novel C-terminal sequences (ins8′). Quantitative reverse transcription-polymerase chain reaction analysis revealed that wt mRNA could be detected in CB CD34+ cells, but that del9 and ins8′ isoforms only appeared after 1 or 3 weeks of co-culture, respectively. Expression of each isoform mRNA was markedly upregulated during B cell differentiation in vitro, and wild type continued to be the most abundant isoform. In a luciferase reporter assay using a synthetic CD19 enhancer, del9 isoform revealed slightly lower activity and ins8′ isoform showed much lower activity, compared with Pax5-wt. Furthermore, retroviral expression of each Pax5 isoform in CB CD34+ cells induced aberrant CD19 expression in a fraction of immature myeloid cells after 1 week of culture, although del9 and ins8′ isoforms showed much less potent activity than Pax5-wt. These results suggest that Pax5-wt is quantitatively and qualitatively dominant over other C-terminal isoforms during human B cell differentiation.