A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification

Abstract

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...

The heterogeneity of pediatric MLL-rearranged acute myeloid leukemia

“…For example, direct binding interactions have already been described between AFF1, MLLT3 and MLLT1, and AFF1/ MLLT10, which have a functional role in leukemogenesis. 54,55 The complex of these proteins, which is called the MLLT1/ENLassociated protein complex (EAP), 55 was also linked to DOT1L, which methylates H3K79, 56 and pTEFb, which is necessary to convert 'promotor-arrested' RNA polymerase-II into an 'elongating' RNA polymerase-II. 57 In addition, it has been shown that MLLT10 binds to DOT1L and that binding of DOT1L was necessary for the transforming activity of MLL-MLLT10.…”

Chromatin maps, histone modifications and leukemia

“…83,84 DOT1L binds the leukemogenic MLL-AF10 and MLL-ENL fusion proteins and indirect evidence suggests that DOT1L catalytic activity is required for transformation by these fusions. 85,86 Furthermore, DOT1L is found in nuclear complexes with AF10, ENL, and other MLL translocation partners AF4 and AF9. 85,[87][88][89][90] Ectopically expressed MLL-AF10, or a synthetic, not naturally occurring MLL-DOT1 fusion, was shown to localize to the HOXA9 locus and increase K79 methylation.…”

“…85,86 Furthermore, DOT1L is found in nuclear complexes with AF10, ENL, and other MLL translocation partners AF4 and AF9. 85,[87][88][89][90] Ectopically expressed MLL-AF10, or a synthetic, not naturally occurring MLL-DOT1 fusion, was shown to localize to the HOXA9 locus and increase K79 methylation. This corresponded to increased HOXA9 expression, consistent with the notion that H3K79 methylation is an activating chromatin modification.…”

DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs and Hypomethylating Agents inMLL-Rearranged Leukemia Cells

“…In the context of the MLL-r-associated fusion proteins, MLL loses its catalytic domain. However, a unifying feature of many of the common MLL fusion partner proteins is the ability to bind to another HMT known as DOT1L (Okada et al, 2005;Bitoun et al, 2007;Mueller et al, 2007;Mohan et al, 2010;Park et al, 2010;Yokoyama et al, 2010;Biswas et al, 2011). In this manner, DOT1L is recruited to gene locations normally under the control of MLL (Okada et al, 2005;Mueller et al, 2007;Monroe et al, 2011).…”

“…However, a unifying feature of many of the common MLL fusion partner proteins is the ability to bind to another HMT known as DOT1L (Okada et al, 2005;Bitoun et al, 2007;Mueller et al, 2007;Mohan et al, 2010;Park et al, 2010;Yokoyama et al, 2010;Biswas et al, 2011). In this manner, DOT1L is recruited to gene locations normally under the control of MLL (Okada et al, 2005;Mueller et al, 2007;Monroe et al, 2011). DOT1L catalyzes the specific methylation of histone H3 at lysine 79 (H3K79), and this site-specific marking of histone H3 leads to transcriptional activation (Milne et al, 2005;Okada et al, 2005;Guenther et al, 2008;Krivtsov et al, 2008;Mueller et al, 2009;Thiel et al, 2010;Monroe et al, 2011;Nguyen et al, 2011).…”