A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription.
ABSTRACT: AF4 and ENL family proteins are frequently fused with MLL, and they comprise a higher order complex (designated AEP) containing the P-TEFb transcription elongation factor. Here, we show that AEP is normally recruited to MLL-target chromatin to facilitate transcription. In contrast, MLL oncoproteins fused with AEP components constitutively form MLL/AEP hybrid complexes to cause sustained target gene expression, which leads to transformation of hematopoietic progenitors. Furthermore, MLL-AF6, an MLL fusion with a cytoplasmic protein, does not form such hybrid complexes, but nevertheless constitutively recruits AEP to target chromatin via unknown alternative mechanisms. Thus, AEP recruitment is an integral part of both physiological and pathological MLL-dependent transcriptional pathways. Bypass of its normal recruitment mechanisms is the strategy most frequently used by MLL oncoproteins.
Project description:Gene rearrangements generate MLL fusion genes, which can lead to aggressive leukemia. In most cases, MLL fuses with a gene encoding a component of the AEP (AF4 family/ENL family/P-TEFb) coactivator complex. MLL-AEP fusion proteins constitutively activate their target genes to immortalize haematopoietic progenitors. Here we show that AEP and MLL-AEP fusion proteins activate transcription through selectivity factor 1 (SL1), a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). The pSER domain of AF4 family proteins associates with SL1 on chromatin and loads TATA-binding protein (TBP) onto the promoter to initiate RNA polymerase II (RNAP2)-dependent transcription. These results reveal a previously unknown transcription initiation mechanism involving AEP and a role for SL1 as a TBP-loading factor in RNAP2-dependent gene activation.
Project description:The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of 3 transcriptional modulators: AF4-ENL-P-TEFb complex (AEP), DOT1L-AF10-ENL complex (referred to as the DOT1L complex) and polycomb-repressive complex 1 (PRC1). Each complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (MLL) gene often fuses with ENL and AF10 family genes in leukemia. However, the functional interrelationship among those 3 complexes in leukemic transformation remains largely elusive. Here, we have shown that MLL-ENL and MLL-AF10 constitutively activate transcription by aberrantly inducing both AEP-dependent transcriptional activation and DOT1L-dependent transcriptional maintenance, mostly in the absence of PRC1, to fully transform hematopoietic progenitors. These results reveal a cooperative transcriptional activation mechanism of AEP and DOT1L and suggest a molecular rationale for the simultaneous inhibition of the MLL fusion-AF4 complex and DOT1L for more effective treatment of MLL-rearranged leukemia.
Project description:Gene rearrangement of the mixed lineage leukemia (MLL) gene causes leukemia by inducing the constitutive expression of a gene subset normally expressed only in the immature haematopoietic progenitor cells. MLL gene rearrangements often generate fusion products of MLL and a component of the AF4 family/ENL family/P-TEFb (AEP) complex. MLL-AEP fusion proteins have the potential of constitutively recruiting the P-TEFb elongation complex. Thus, it is hypothesized that relieving the promoter proximal pausing of RNA polymerase II is the rate-limiting step of MLL fusion-dependent transcription. AEP also has the potential to recruit the mediator complex via MED26. We recently showed that AEP activates transcription initiation by facilitating TBP loading to the TATA element through the SL1 complex. In the present study, we show that the key activity responsible for the oncogenic property of MLL-AEP fusion proteins is the TBP loading activity, and not the mediator recruitment or transcriptional elongation activities. Thus, we propose that TBP loading by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription.
Project description:MLL is a common target for chromosomal translocations associated with acute leukemia resulting in its fusion with a large variety of nuclear or cytoplasmic proteins that may activate its oncogenic properties by distinct but poorly understood mechanisms. The MLL-AF6 fusion gene represents the most common leukemogenic fusion of mixed lineage leukemia (MLL) to a cytoplasmic partner protein. Here, we identified a highly conserved Ras association (RA1) domain at the amino-terminus of AF6 as the minimal region sufficient for MLL-AF6 mediated myeloid progenitor immortalization in vitro and short latency leukemogenesis in vivo. Moreover, the ability of RA1 to activate MLL oncogenesis is conserved with its Drosophila ortholog, Canoe. Although the AF6 RA1 domain has previously been defined as an interaction surface for guanosine triphosphate-bound Ras, single amino acid substitutions known to abolish the AF6-Ras interaction did not abrogate MLL-AF6-mediated oncogenesis. Furthermore, fusion of MLL to heterologous RA domains of c-Raf1 or RalGDS, or direct fusion of MLL to constitutively active K-RAS, H-RAS, or RAP1 was not sufficient for oncogenic activation of MLL. Rather, the AF6 RA1 domain efficiently mediated self-association, suggesting that constitutive MLL self-association is a more common pathogenic mechanism for MLL oncogenesis than indicated by previous studies of rare MLL fusion partners.
Project description:Acute Myeloid Leukemia (AML) with MLL gene rearrangements demonstrate unique gene expression profiles driven by MLL-fusion proteins. Here, we identify the circadian clock transcription factor SHARP1 as a novel oncogenic target in MLL-AF6 AML, which has the worst prognosis among all subtypes of MLL-rearranged AMLs. SHARP1 is expressed solely in MLL-AF6 AML, and its expression is regulated directly by MLL-AF6/DOT1L. Suppression of SHARP1 induces robust apoptosis of human MLL-AF6 AML cells. Genetic deletion in mice delays the development of leukemia and attenuated leukemia-initiating potential, while sparing normal hematopoiesis. Mechanistically, SHARP1 binds to transcriptionally active chromatin across the genome and activates genes critical for cell survival as well as key oncogenic targets of MLL-AF6. Our findings demonstrate the unique oncogenic role for SHARP1 in MLL-AF6 AML.
Project description:The t(6;11)(q27;q23) is a recurrent chromosomal rearrangement that encodes the MLLAF6 fusion oncoprotein and is observed in patients with diverse hematologic malignancies. The presence of the t(6;11)(q27;q23) has been linked to poor overall survival in patients with AML. In this study, we demonstrate that MLL-AF6 requires continued activity of the histone-methyltransferase DOT1L to maintain expression of the MLL-AF6-driven oncogenic gene-expression program. Using gene-expression analysis and genome-wide chromatin immunoprecipitation studies followed by next generation sequencing, we found that MLL-fusion target genes display markedly high levels of histone 3 at lysine 79 (H3K79) dimethylation in murine MLL-AF6 leukemias as well as in ML2, a human myelomonocytic leukemia cell line bearing the t(6;11)(q27;q23) translocation. Targeted disruption of Dot1l using a conditional knockout mouse model inhibited leukemogenesis mediated by the MLL-AF6 fusion oncogene. Moreover, both murine MLL-AF6-transformed cells as well as the human MLL-AF6-positive ML2 leukemia cell line displayed specific sensitivity to EPZ0004777, a recently described, selective, small-molecule inhibitor of Dot1l. Dot1l inhibition resulted in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6-transformed cells. These results indicate that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.
Project description:Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.
Project description:Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79-specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.
Project description:Cancer cells transcribe RNAs in a characteristic manner in order to maintain their oncogenic potentials. In eukaryotes, RNA is polymerized by three distinct RNA polymerases, RNA polymerase I, II, and III (RNAP1, RNAP2, and RNAP3, respectively). The transcriptional machinery that initiates each transcription reaction has been purified and characterized. Selectivity factor 1 (SL1) is the complex responsible for RNAP1 pre-initiation complex formation. However, whether it plays any role in RNAP2-dependent transcription remains unclear. Our group previously found that SL1 specifically associates with AF4 family proteins. AF4 family proteins form the AEP complex with ENL family proteins and the P-TEFb elongation factor. Similar complexes have been independently characterized by several different laboratories and are often referred to as super elongation complex. The involvement of AEP in RNAP2-dependent transcription indicates that SL1 must play an important role in RNAP2-dependent transcription. To date, this role of SL1 has not been appreciated. In leukemia, AF4 and ENL family genes are frequently rearranged to form chimeric fusion genes with MLL. The resultant MLL fusion genes produce chimeric MLL fusion proteins comprising MLL and AEP components. The MLL portion functions as a targeting module, which specifically binds chromatin containing di-/tri-methylated histone H3 lysine 36 and non-methylated CpGs. This type of chromatin is enriched at the promoters of transcriptionally active genes which allows MLL fusion proteins to selectively bind to transcriptionally-active/CpG-rich gene promoters. The fusion partner portion, which recruits other AEP components and SL1, is responsible for activation of RNAP2-dependent transcription. Consequently, MLL fusion proteins constitutively activate the transcription of previously-transcribed MLL target genes. Structure/function analysis has shown that the ability of MLL fusion proteins to transform hematopoietic progenitors depends on the recruitment of AEP and SL1. Thus, the AEP/SL1-mediated gene activation pathway appears to be the central mechanism of MLL fusion-mediated transcriptional activation. However, the molecular mechanism by which SL1 activates RNAP2-dependent transcription remains largely unclear. This review aims to cover recent discoveries of the mechanism of transcriptional activation by MLL fusion proteins and to introduce novel roles of SL1 in RNAP2-dependent transcription by discussing how the RNAP1 machinery may be involved in RNAP2-dependent gene regulation.
Project description:RNA-Seq of 1) human AML samples; 2) sorted, uncultured distinct population from human cord blood (CB); 3) short-term (ST) cultured sorted CB cells transduced with MLL-ENL, MLL-AF6 or untransduced; and 4) cultured (LT) sorted CB cells transformed with MLL-ENL or MLL-AF6. Cells from MLL-fusion AML patients are bulk. Several cords were used for the sorting (CB1, CB2, CB3, 135, 141...) and these represent biological replicates. Several samples were sequenced several times in different lanes and results were merged together for the analysis (rep1,rep2...). Samples were used to determine the different effect of MLL-fusions in different celltypes just after the transduction, and after a longer time period when cells were transformed. Sorted CB samples, uncultured as well as transformed by MLL-fusions, were used in machine learning approach to predict which of the patients originated from which cell-type of origin.