Dominant-negative mechanism of leukemogenic PAX5 fusions

Abstract

PAX5 encodes a master regulator of B-cell development. It fuses to other genes associated with acute lymphoblastoid leukemia (ALL). These fusion products are potent dominant-negative (DN) inhibitors of wild-type PAX5 resulting in a blockade of B-cell differentiation. Here, we show that multimerization of PAX5 DNA-binding domain (DBD) is necessary and sufficient to cause extremely stable chromatin binding and DN-activity. ALL-associated PAX5-C20S results from fusion of the N-terminal region of PAX5 including its paired DBD, to the C-terminus of C20orf112, a protein of unknown function. We report that PAX5-C20S is a tetramer which interacts extraordinarily stably with chromatin as determined by fluorescence recovery after photobleaching (FRAP) in living cells. Tetramerization, stable chromatin-binding and DN-activity all require a putative five-turn amphipathic α-helix at the C-terminus of C20orf112, and does not require potential co-repressor binding peptides elsewhere in the sequence. In vitro, the monomeric PAX5 DBD and PAX5-C20S binds a PAX5-binding site with equal affinity when it is at the center of an oligonucleotide too short to bind to more than one PAX5 DBD. But PAX5-C20S binds the same sequence with tenfold higher affinity than the monomeric PAX5 DBD when it is in a long DNA molecule. We suggest that the increased affinity results from interactions of one or more of the additional DBDs with neighboring non-specific sites in a long DNA molecule, and that this can account for the increased stability of PAX5-C20S chromatin binding compared to wt PAX5, resulting in DN-activity by competition for binding to PAX5-target sites. Consistent with this model, the ALL-associated PAX5 fused to ETV6 or the multimerization domain of ETV6 SAM results in stable chromatin binding and DN-activity. In addition, PAX5 DBD fused to artificial dimerization, trimerization, and tetramerization domains result in parallel increases in the stability of chromatin binding and DN-activity. Our studies suggest that oncogenic fusion proteins that retain the DBD of the transcription factor and the multimerization sequence of the partner protein can act in a DN fashion by multimerizing and binding avidly to gene targets preventing the normal transcription factor from binding and inducing expression of its target genes. Inhibition of this multimeriztion may provide a novel therapeutic approach for cancers with this or similar fusion proteins.