Leukemogenic MLL Fusion Proteins Promote MLL Association with HOX Loci Resulting in Persistent Expression.

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. To address this we performed a detailed study of where MLL and MLL fusion proteins bind at target genes in hematopoietic cells using quantitative chromatin immunoprecipitation (ChIP). In addition we characterized the histone modifications at the HOX A9 locus that occur with normal down modulation of HOX A9 expression during hematopoietic differentiation and how this is perturbed by induction of conditionally transforming MLL fusion proteins. These 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 is transiently increased by the addition of IL-3 but 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 methylation, marks that are normally almost completely erased during myeloid differentiation. In addition MLL-ENL induces increased lysine 79 methylation. MLL extensively colocalizes with RNA polymerase II, which shows abnormal pausing in the coding regions of HOX genes in Mll null cells. These findings suggest MLL fusion proteins maintain expression of HOX genes critical for leukemogenesis through increased MLL binding resulting in both MLL dependent histone modifications and lysine 79 methylation and ultimately promotion of transcriptional elongation.