Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes.
ABSTRACT: Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target.
Project description:The stem cell factor spalt-like transcription factor 4 (SALL4) plays important roles in normal hematopoiesis and also in leukemogenesis. We previously reported that SALL4 exerts its effect by recruiting important epigenetic factors such as DNA methyltransferases DNMT1 and lysine-specific demethylase 1 (LSD1/KDM1A). Both of these proteins are critically involved in mixed lineage leukemia (MLL)-rearranged (MLL-r) leukemia, which has a very poor clinical prognosis. Recently, SALL4 has been further linked to the functions of MLL and its target gene homeobox A9 (HOXA9). However, it remains unclear whether SALL4 is indeed a key player in MLL-r leukemia pathogenesis.Using a mouse bone marrow retroviral transduction/ transplantation approach combined with tamoxifen-inducible, CreERT2-mediated Sall4 gene deletion, we studied SALL4 functions in leukemic transformation that was induced by MLL-AF9-one of the most common MLL-r oncoproteins found in patients. In addition, the underlying transcriptional and epigenetic mechanisms were explored using chromatin immunoprecipitation (ChIP) sequencing (ChIP-Seq), mRNA microarray, qRT-PCR, histone modification, co-immunoprecipitation (co-IP), cell cycle, and apoptosis assays. The effects of SALL4 loss on normal hematopoiesis in mice were also investigated.In vitro and in vivo studies revealed that SALL4 expression is critically required for MLL-AF9-induced leukemic transformation and disease progression in mice. Loss of SALL4 in MLL-AF9-transformed cells induced apoptosis and cell cycle arrest at G1. ChIP-Seq assay identified that Sall4 binds to key MLL-AF9 target genes and important MLL-r or non-MLL-r leukemia-related genes. ChIP-PCR assays indicated that SALL4 affects the levels of the histone modification markers H3K79me2/3 and H3K4me3 at MLL-AF9 target gene promoters by physically interacting with DOT1-like histone H3K79 methyltransferase (DOT1l) and LSD1/KDM1A, and thereby regulates transcript expression. Surprisingly, normal Sall4 f/f /CreERT2 mice treated with tamoxifen or vav-Cre-mediated (hematopoietic-specific) Sall4 -/- mice were healthy and displayed no significant hematopoietic defects.Our findings indicate that SALL4 critically contributes to MLL-AF9-induced leukemia, unraveling the underlying transcriptional and epigenetic mechanisms in this disease and suggesting that selectively targeting the SALL4 pathway may be a promising approach for managing human MLL-r leukemia.
Project description:DOT1 (disruptor of telomeric silencing; also called Kmt4) was initially discovered in budding yeast in a genetic screen for genes whose deletion confers defects in telomeric silencing. Since the discovery ∼10 years ago that Dot1 and its mammalian homolog, DOT1L (DOT1-Like), possess histone methyltransferase activity toward histone H3 Lys 79, great progress has been made in characterizing their enzymatic activities and the role of Dot1/DOT1L-mediated H3K79 methylation in transcriptional regulation, cell cycle regulation, and the DNA damage response. In addition, gene disruption in mice has revealed that mouse DOT1L plays an essential role in embryonic development, hematopoiesis, cardiac function, and the development of leukemia. The involvement of DOT1L enzymatic activity in leukemogenesis driven by a subset of MLL (mixed-lineage leukemia) fusion proteins raises the possibility of targeting DOT1L for therapeutic intervention.
Project description:A number of acute leukemias arise from fusion of the mixed lineage leukemia 1 protein (MLL) N terminus to a variety of fusion partners that have been reported to reside in one or more poorly defined complexes linked to transcription elongation through interactions with the histone H3-K79 methyltransferase DOT1 and positive transcription elongation factor b (P-TEFb). Here we first identify natural complexes (purified through fusion partners AF9, AF4, and ELL) with overlapping components, different elongation activities, and different cofactor associations that suggest dynamic interactions. Then, through reconstitution of defined, functionally active minimal complexes, we identify stable subcomplexes that, through newly defined protein-protein interactions, form distinct higher order complexes. These definitive analyses show, for example, that (i) through direct interactions with AF9 and cyclinT1, family members AF4 and AFF4 independently mediate association of P-TEFb with AF9, (ii) P-TEFb, through direct interactions, provides the link for association of ELL and ELL-associated factors 1 and 2 (EAF1 and EAF2) with AF4, and (iii) in the absence of other factors, DOT1 forms a stable complex with AF9 and does not interact with AF9•AF4•P-TEFb complexes. Finally, we show the importance of defined higher order complex formation in MLL-AF9-mediated transcriptional up-regulation and cell immortalization potential in vivo. Thus, our study provides direct mechanistic insight into the role of fusion partners in MLL fusion-mediated leukemogenesis.
Project description:The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.
Project description:Chromosomal translocations of the mixed lineage leukemia (MLL) gene are a common cause of acute leukemias. The oncogenic function of MLL fusion proteins is, in part, mediated through aberrant activation of Hoxa genes and Meis1, among others. Here we demonstrate using a tamoxifen-inducible Cre-mediated loss of function mouse model that DOT1L, an H3K79 methyltransferase, is required for both initiation and maintenance of MLL-AF9-induced leukemogenesis in vitro and in vivo. Through gene expression and chromatin immunoprecipitation analysis we demonstrate that mistargeting of DOT1L, subsequent H3K79 methylation, and up-regulation of Hoxa and Meis1 genes underlie the molecular mechanism of how DOT1L contributes to MLL-AF9-mediated leukemogenesis. Our study not only provides the first in vivo evidence for the function of DOT1L in leukemia, but also reveals the molecular mechanism for DOT1L in MLL-AF9 mediated leukemia. Thus, DOT1L may serve as a potential therapeutic target for the treatment of leukemia caused by MLL translocations.
Project description:BACKGROUND: Methylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome. RESULTS: Targeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1. CONCLUSIONS: Targeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.
Project description:The expression of genes residing near telomeres is attenuated through telomere position-effect variegation (TPEV). By using a URA3 reporter located at TEL-VII-L of Saccharomyces cerevisiae, it was proposed that the disruptor of telomeric silencing-1 (Dot1) regulates TPEV by catalyzing H3K79 methylation. URA3 reporter assays also indicated that H3K79 methylation is required for HM silencing. Surprisingly, a genome-wide expression analysis of H3K79 methylation-defective mutants identified only a few telomeric genes, such as COS12 at TEL-VII-L, to be subject to H3K79 methylation-dependent natural silencing. Consistently, loss of Dot1 did not globally alter Sir2 or Sir3 occupancy in subtelomeric regions, but only led to some telomere-specific changes. Furthermore, H3K79 methylation by Dot1 did not play a role in the maintenance of natural HML silencing. Therefore, commonly used URA3 reporter assays may not report on natural PEV, and therefore, studies concerning the epigenetic mechanism of silencing in yeast should also employ assays reporting on natural gene expression patterns.
Project description:Acute leukaemias express high levels of MYB which are required for the initiation and maintenance of the disease. Inhibition of MYB expression or activity has been shown to suppress MLL-fusion oncoprotein-induced acute myeloid leukaemias (AML), which are among the most aggressive forms of AML, and indeed MYB transcription has been reported to be regulated by the MLL-AF9 oncoprotein. This highlights the importance of understanding the mechanism of MYB transcriptional regulation in these leukaemias. Here we have demonstrated that the MLL-AF9 fusion protein regulates MYB transcription directly at the promoter region, in part by recruiting the transcriptional regulator kinase CDK9, and CDK9 inhibition effectively suppresses MYB expression as well as cell proliferation. However, MYB regulation by MLL-AF9 does not require H3K79 methylation mediated by the methyltransferase DOT1L, which has also been shown to be a key mediator of MLL-AF9 leukemogenicity. The identification of specific, essential and druggable transcriptional regulators may enable effective targeting of MYB expression, which in turn could potentially lead to new therapeutic approaches for acute myeloid leukaemia with MLL-AF9.
Project description:MLL-fusion proteins, AF9 and ENL, play an essential role in the recruitment of DOT1L and the H3K79 hypermethylation of MLL target genes, which is pivotal for leukemogenesis. Blocking these interactions may represent a novel therapeutic approach for MLL-rearranged leukemia. Based on the 7 mer DOT1L peptide, a class of peptidomimetics was designed. Compound 21 with modified middle residues, achieved significantly improved binding affinities to AF9 and ENL, with KD values of 15 nM and 57 nM, respectively. Importantly, 21 recognizes and binds to the cellular AF9 protein and effectively inhibits the AF9-DOT1L interactions in cells. Modifications of the N- and C-termini of 21 resulted in 28 with 2-fold improved binding affinity to AF9 and much decreased peptidic characteristics. Our study provides a proof-of-concept for development of nonpeptidic compounds to inhibit DOT1L activity by targeting its recruitment and the interactions between DOT1L and MLL-oncofusion proteins AF9 and ENL.
Project description:The conserved histone methyltransferase Dot1 establishes an H3K79 methylation pattern consisting of mono-, di- and trimethylation states on histone H3 via a distributive mechanism. This mechanism has been shown to be important for the regulation of the different H3K79 methylation states in yeast. Dot1 enzymes in yeast, Trypanosoma brucei (TbDot1A and TbDot1B, which methylate H3K76) and human (hDot1L) generate very divergent methylation patterns. To understand how these species-specific methylation patterns are generated, the methylation output of the Dot1 enzymes was compared by expressing them in yeast at various expression levels. Computational simulations based on these data showed that the Dot1 enzymes have highly distinct catalytic properties, but share a distributive mechanism. The mechanism of methylation and the distinct rate constants have implications for the regulation of H3K79/K76 methylation. A mathematical model of H3K76 methylation during the trypanosome cell cycle suggests that temporally-regulated consecutive action of TbDot1A and TbDot1B is required for the observed regulation of H3K76 methylation states.