The interaction between two MLL fusion partner genes, AF4 and AF9

MLL is the gene at 11q23 whose rearrangement has been implicated in acute leukemia. MLL promiscuously pairs with over 30 cloned fusion partners and at least 20 more uncloned foci. The most common MLL fusion partners are AF4 and AF9, together accounting for over 60% of MLL acute leukemia.; We describe the cloning of the murine homologue of AF4. We investigated mAf4's localization using the EGFP reporter system. We show that mAf4 is targeted to subnuclear foci that we call “AF4 bodies.” We find that the AF4 body is excluded from the nucleolus and is not coincidental with either PODs or Cajal bodies. The AF4 body does not colocalize with DNA transcription or repair (PCNA), or with RNA synthesis (RNA polymerase II). We find that nascent RNA transcripts are excluded from the AF4 body.; We demonstrate the interaction of mAF4 and AF9 in the yeast 2-hybrid system, GST pull-downs, with endogenous proteins using antibodies and in the EGFP/DsRed reporter systems. We show that the carboxy terminal 100 amino acids of AF9 are required for mAf4 interaction and can disrupt mAf4 subnuclear localization. The portion of mAf4 necessary for AF9 interaction is narrowed down to a 51 amino acid area in a portion of AF4 included in MLL fusions. These 51 residues are coincidental with mAf4's subcellular targeting domain. Using an amino terminal MLL construct and an MLL-AF4 fusion construct, we show that the localization of MLL-AF4 is largely directed by MLL sequence. MLL-AF4 retains AF4's ability to interact with AF9 and redirect AF9 expression to the nucleolus, where it is normally not found. In a small percentage of cells, the AF9 interaction directs MLL-AF4 expression to the AF4 body.; A survey of the MLL partner literature, reveals that a number of seemingly unrelated MLL partner proteins interact, either directly or through intermediary proteins. We hypothesize that the MLL partner proteins are all globally related as components of a cellular process that is perverted by the inclusion of MLL sequence. The link we provide between AF9 and AF4 is key to this hypothesis.