The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin

Abstract

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.

The leukemogenic AF4–MLL fusion protein causes P-TEFb kinase activation and altered epigenetic signatures

“…42 We have not identified AF6, AF17 or ELL1-3 in our purified complexes (data not shown), suggesting that cells are able to form different complexes by AF4 family members that exert, however, highly similar functions. 43,44 In conclusion, the purification and characterization of AF4 and AF4-MLL extends very much our current knowledge and explains the functional consequences of expressed AF4-MLL fusion protein. Further studies to dissect the protein interaction network within the AF4 and AF4-MLL complexes are currently underway.…”

New insights to the MLL recombinome of acute leukemias

“…Finally, the most frequent TPGs in MLL translocations encode nuclear proteins (AFF1/AF4, AFF3/LAF4, AFF4/AF5Q31, MLLT3/ AF9, MLLT1/ENL and MLLT10/AF10) that belong to a protein network that transmits DOT1L 38 and pTEF-B to promoterarrested RNA polymerase II, and thus, allows active transcription and elongation. 39,40 pTEF-B phosphorylates the C-terminal domain of RNA polymerase II, whereas DOT1L enables methylation of lysine 79 of histone H3 proteins, a prerequisite for the maintenance of RNA transcription. 41 In addition, expression of this MLL Á AF4 fusion protein confers a global increase of H3K79 methylation, a potentially novel oncogenic mechanism.…”

The MLL recombinome of acute leukemias

“…18 By contrast, the AF4 protein seems to be required for growth and differentiation of lymphocytic progenitors. 19 ENL was shown to interact directly with the AF4 and AF10 proteins, 20 and in addition, AF10 binds to hDOT1L. 21 The hDOT1L protein is a non-SETdomain protein that is able to mediate the methylation of lysine 79 of histone H3 proteins.…”