Two-way inhibition of PAX5 transcriptional activity by PAX5::CBFA2T3.

Abstract

PAX5 promotes B-cell differentiation by transcriptional activation of B-lineage-specific genes. Chromosomal rearrangements in PAX5 account for 2-3% of B-ALL cases, and most lead to the expression of in-frame fusion transcripts. These fusions can encode chimeric proteins composed of the N-terminal portion of PAX5 and the C-terminal region of a variety of heterogeneous fusion partners. We analyzed the function of PAX5::CBFA2T3 (PAX5-C), a fusion protein found in B-cell acute lymphoblastic leukemia. PAX5-C strongly repressed PAX5 transcriptional activity in luciferase assays. In co-immunoprecipitation assays, PAX5-C bound to PAX5 and HDAC1/3. However, neither HDAC knockdown nor treatment with a HDAC inhibitor showed any effect on the repression of PAX5 transactivity by PAX5-C. In addition, PAX5-C with DNA binding-defective mutations (PAX5 M-C) could still repress PAX5 transactivity; however, the repression of PAX5 transactivity by PAX5 M-C was abolished by inhibition or knockdown of HDAC. These findings indicate that PAX5-C exhibits two mechanisms of repression: a DNA binding-dependent and a HDAC-dependent mechanism, with either being sufficient for the repression of PAX5 transactivity by PAX5-C. We performed ChIP-qPCR under conditions of the luciferase assay and inferred that these two mechanisms involved the inhibition of direct binding of PAX5 to the promoter due to promoter occupancy by PAX5-C, and recruitment of HDAC1/3 to the PAX5 transcription complex by the binding of PAX5-C to PAX5 on the promoter. The present results provide novel insight into the mechanisms of how PAX5-fusion proteins inhibit PAX5 function.