Identification of an oncogenic MLL fusion complex with histone methylation and acetylation activities

Abstract

The Mixed Lineage Leukemia gene (MLL) encoding a histone methyltransferase is rearranged to form chimeric fusion proteins with more than 50 different fusion partners in about 10% of adult, 50% of infant and secondary leukemia related to topoisomerase-II inhibitor treatment. We and others have proposed two alternative mechanisms for oncogenic activation of MLL by direct fusion with transcriptional activation domains or acquisition of homo-oligomerization domains, which will both convert MLL into constitutive transcriptional activators that aberrantly activate downstream targets such as Hox genes. While SH3 domain containing proteins constitute the biggest family of MLL fusion partners with defined-structural domains, very little is known about the transformation mechanisms. To gain further insights into MLL-leukemogenesis, the present study molecularly and functionally dissects the underlying mechanisms mediated by MLL-EEN fusion protein, the founding member of SH3 domain containing MLL fusions. Using a retroviral transduction and transplantation assay, we demonstrated the ability of MLL-EEN to enhance self-renewal of primary hematopoietic cells and induce AML in mice. Structure/function analysis revealed the SH3 domain as the minimal domain in EEN protein that was necessary and sufficient for MLL-mediated transformation. To further characterize the underlying mechanisms, we affinity purified the protein complex recruited by EEN-SH3 domain and identified the interacting components by mass spectrometry. While all the identified components consistently have putative roles in transcriptional regulation, the current study will focus on an interacting partner called Sam68. Sam68 is a member of Signal Transduction and Activation of RNA (STAR) family of RNA-binding proteins, which can recruit both histone acetyltransferase (CBP) and histone methyltransferase (PRMT1) that cooperatively encodes positive histone codes for gene expression. Immunoprecipitation and immunofluorescent staining have confirmed the in vivo interaction between Sam68 and MLL-EEN. Further deletion analysis mapped the interaction between the SH3 domain of EEN and the proline rich motif in Sam68, which are consistent with the binding consensus of SH3 domain. We also demonstrated the in vivo association of CBP and PRMT1 with both Sam68 and MLL-EEN by immunoprecipitation assay. Moreover, histone acetylation activity could be detected in protein complex associated with EEN-SH3 but not EEN SH3 point mutant (mSH3), which is incapable of interacting with Sam68. Consistently, MLL-EEN mSH3 lost its ability to transform primary hematopoietic cells, strongly indicating essential functions of Sam68 as a critical mediator for MLL-EEN mediated transformation. To this end, we constructed a synthetic MLL-Sam68 fusion and tested if direct fusion of Sam68 to MLL can replace EEN for transformation. As a result, MLL-Sam68 not only could recruit CBP, but also enhanced self-renewal of primary hematopoietic cells and transformation myeloid progenitors with phenotypes indistinguishable from MLL-EEN transformed cells in spite of a reduction of colony number. Taken together, these results not only establish transformation mechanisms mediated by an MLL-SH3 domain containing fusion protein but also identify a novel MLL-fusion complex with both histone acetylation and methylation activities, which may provide potential avenues for molecular targeting. ©2006, The American Society of Hematology