Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes including HOX A7 and HOX A9 and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. The mechanism by which this occurs and the relationship to normal MLL function is unknown. MLL and MLL fusion proteins bind with a similar distribution in hematopoietic cells at both promoters and coding sequences of target genes. Our studies suggest that a major mechanism of regulating MLL, which is expressed throughout hematopoiesis, is through modulating it’s binding to target promoters. MLL binds directly to the promoters and coding regions of HOX A7, HOX A9, and MEIS1 only in myeloblasts and not in neutrophils, indicating MLL is physically associated with genes only when they are actively transcribed. Expression of A cluster HOX loci and MEIS1 remains persistently elevated when MLL-ENL or dimerized MLL fusion proteins are expressed. Expression of either fusion protein is associated with increased binding of wild type MLL accompanied by increases in histone acetylation and histone H3 lysine 4, marks that are normally almost completely erased during myeloid differentiation. In addition MLL-ENL induces increased lysine 79 methylation. Both MLL and MLL fusion proteins interact with the tumor suppressor menin via sequences in the extreme amino terminus of MLL. In addition both proteins physically interact with RNA polymerase II, which shows abnormal pausing in the coding regions of HOX genes in Mll null cells. Genetic ablation of menin or expression of a dominant negative inhibitor of the MLL-menin interaction inhibits the growth of MLL fusion protein transformed cells. These findings suggest MLL fusion proteins act in concert with menin, MLL and other coactivators to deregulate HOX gene expression pivotal for transformation.