Profiling of histone modification, transcription factor binding of MLL-AF9 of HSCs, GMPs and L-GMPs. The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations that involve the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in a murine MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3 and H3K36me3. Histone methylation patterns are highly abnormal on MLL-AF9 fusion target loci, defining a distinct epigenetic lesion involving H3K79. Conditional inactivation of Dot1l leads to specific down-regulation of direct MLL-AF9 targets and an MLL-translocation associated gene expression signature, while global transcription levels remain largely unaffected. This correlated with a greater sensitivity of leukemic blasts towards loss of Dot1l compared to normal hematopoietic cells. Development of in vivo leukemia was absolutely dependent on Dot1l.