IntroductionHematopoietic development is controlled by an intricate network of finely tuned transcriptional programs. Consequently, a perturbation of the transcription factors involved can block differentiation. This developmental roadblock cooperates with mutations in pathways that signal growth and/or survival to cause acute leukemias. 1 A prime example for such a mechanism is mixed lineage leukemia. In this disease, the gene for the histone methyltransferase MLL participates in chromosomal translocations that eventually create maturation-blocking and therefore leukemogenic MLL fusion proteins. 2,3 These protein chimeras consist of N-terminal portions of MLL joined to a variety of mostly unrelated fusion partners that replace the original MLL C-terminus, including the methyltransferase domain (http://atlasgeneticsoncology.org/Genes/ MLL.html). MLL fusion proteins are aberrant transcription factors that ectopically activate genes important for hematopoietic development like the abdominal-type Hox genes Hoxa7 and Hoxa9 and their dimerization partner Meis1. [4][5][6][7] Despite intensive study, little is known about the biological function of MLL fusion partners in normal cells, and it is mostly unclear how these proteins activate the oncogenic potential of MLL. In the rare cases where MLL is joined to a cytoplasmatic protein, domains introduced by the partner force a dimerization of the fusion that is crucial for oncogenic activity. 8,9 The overwhelming majority of leukemias with MLL rearrangement, however, involves nuclear proteins as translocation partners. The data available seem to indicate a role of some nuclear MLL partners in transcriptional control and histone modification. Support for this speculation comes from the detection of direct protein-protein interactions of the homologous MLL fusion partners AF4 and AF5q31 that both bind to ENL and the closely related AF9. 10,11 ENL in turn interacts with histone H3. 11 In addition, another MLL fusion partner, AF10, recruits the histone H3 lysine 79-specific methyltransferase DOT1L that introduces a dimethyl mark during transcriptional elongation. 12 The same modification is also a hallmark of genes activated by MLL-ENL. 13 Finally, the proteins CBX8 (chromobox 8) and BCoR (BCL6 corepressor) that are involved in chromatin-dependent gene repression have also been found to associate with ENL and AF9. [14][15][16] In order to learn more about the biological function of a classical MLL fusion partner, we identified proteins associated with the "Eleven Nineteen Leukemia" protein (ENL) originally discovered as an MLL fusion partner in the recurrent translocation t(11;19). Here, we describe the purification and analysis of ENL-associated proteins (EAPs) by tandem immunoprecipitation of ENL. This protein assembly contains several other MLL fusion partners, positive transcription elongation factor b (pTEFb), DOT1L, and polycomb group proteins. The composition of EAP suggests that ENL works in a new unit of transcriptional regulation that coordinates transcriptional elo...

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Mixed lineage leukemia (MLL) fusion proteins are derived from translocations at 11q23 that occur in aggressive subtypes of leukemia. As a consequence, MLL is joined to different unrelated proteins to form oncogenic transcription factors. Here we demonstrate a direct interaction between several nuclear MLL fusion partners and present evidence for a role of these proteins in histone binding. In two-hybrid studies, ENL interacted with AF4 and AF5q31 as well as with a fragment of AF10. A structure-function analysis revealed that the AF4/ AF5q31/AF10 binding domain in ENL coincided with the C-terminus that is essential for transformation by MLL-ENL. The ENL/AF4 association was corroborated by GST-pulldown experiments and by mutual coprecipitation. Both proteins colocalized in vivo in a nuclear speckled pattern. Moreover, AF4 and ENL coeluted on sizing columns together with the known ENL binding partner Polycomb3, suggesting the presence of a multiprotein complex. The overexpression of ENL alone activated a reporter construct and a mutational screen indicated the conserved YEATS domain as essential for this function. Overlay and pulldown-assays finally showed a specific and YEATS domain-dependent association of ENL with histones H3 and H1. In summary, our studies support a common role for nuclear MLL fusion partners in chromatin biology.