Chromosomal translocations at 11q23 are characteristic for a subset of particularly aggressive acute lymphoid and myeloid leukemias. This event fuses the mixed lineage leukemia (MLL) gene to a puzzling variety of seemingly unrelated fusion partners to create novel transcription factors with potent oncogenic activity. Despite the molecular heterogeneity, MLL−associated leukemias show similar clinical presentations and an almost identical gene expression pattern regardless of the translocation partner. We and others previously identified interactions between individual MLL translocation partners suggesting that these might share a common mechanism for transformation. To characterize these interactions in more detail we generated a HEK293−derived producer line expressing a FLAG−tagged derivative of the frequent fusion partner ENL. Double affinity purification of nuclear extracts on FLAG and ENL antibody resins reproducibly yielded a macromolecular complex of 10 proteins we term MPAC, for MLL Partner Associated Complex, which in addition to ENL includes the MLL fusion partners AF4, AF9 and AF5q31, and two components involved in transcriptional elongation: pTEFb and DOT1L. The pTEFb dimer consists of cyclin T1/2 and CDK9 and is responsible for serine−2 phosphorylation of the C−terminal repeat domain of elongating RNA Polymerase II. DOT1L is the only known histone methyltransferase that catalyzes dimethylation of H3K79, a modification introduced during elongation of actively transcribed transcription units. Surprisingly, PC3 and RING1b, members of the polycomb repressive complex I were also identified in the MPAC complex. Further examination demonstrated a direct interaction of ENL with DOT1L through C−terminal interaction domains by yeast 2−hybrid, pulldown and colocalization experiments. Knock−down of ENL diminished global H3K79 dimethylation and also impaired various promoters in transient reporter assays suggesting the MPAC complex is involved in transcriptional regulation mediated by DOT1L, even in the absence of MLL fusion proteins. In summary, our results indicate that the most common MLL fusion partners are associated in an hitherto undescribed complex involved in transcriptional elongation.

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