14-3-3 theta, a direct interactor of AF4, influences HOXA9 expression in RS4;11 leukemia cell line.

BACKGROUND: AF4 is the most common fusion partner of the MLL gene in t(4;11) infant acute lymphoblastic leukemia (ALL). The AF4 protein is a transcriptional activator with a central role in transcriptional elongation and chromatin remodeling. A hallmark of MLL leukemia is aberrant H3K79 hyper-methylation and over-expression of HOXA9, a target gene of the MLL-AF4 fusion protein. The MLL-AF4 chimera recruits AF4 and transactivates HOXA9. We recently demonstrated that AF4 interacts with the scaffold protein 14-3-3 theta. We studied the molecular features of such interaction in the context of the molecular pathogenesis of the t(4;11) ALL. METHODS: To evaluate the interaction between AF4 and 14-3-3 theta, we performed in vitro binding assay, western blot analysis and fluorescence resonance energy transfer (FRET) experiments. We transiently expressed 14-3-3 theta in RS4;11 leukemia cells and analyzed HOXA9 transcript levels by real time RT-PCR. RESULTS: Purified recombinant HA-14-3-3 theta and Flag-AF4 interact in vitro; FRET analysis showed that the recombinant proteins GFP-AF4 and Cherry-14-3-3 θ actually interact into the nucleus. Real time RT-PCR showed that transcript levels of HOXA9 increase in RS4;11 leukemia cells that ectopically express 14-3-3 theta. Notably, we found that HOXA9 expression level varies in direct proportion to the amount of ectopic 14-3-3 theta. CONCLUSIONS Our data indicate that 14-3-3 theta directly interacts with AF4 in the nucleus and probably contributes to trigger the epigenetic modifications that results in transcription of MLL-AF4 chimera target genes. Recently, small nonpeptidic molecules that inhibit 14-3-3 protein-protein interactions have been discovered. Therefore, 14-3-3 theta might be considered a promising molecular target in therapy of t(4;11) ALL.