The Experts below are selected from a list of 25221945 Experts worldwide ranked by ideXlab platform
Yi Zhang - One of the best experts on this subject based on the ideXlab platform.
-
DOT1L the h3k79 methyltransferase is required for mll af9 mediated leukemogenesis
Blood, 2011Co-Authors: Anh T Nguyen, Yi Zhang, Olena TaranovaAbstract: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.
-
DOT1L regulates dystrophin expression and is critical for cardiac function
Genes & Development, 2011Co-Authors: Anh T Nguyen, Bin Xiao, Ronald L Neppl, Eric M Kallin, Taiping Chen, Dazhi Wang, Xiao Xiao, Yi ZhangAbstract:Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of DOT1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of DOT1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin–glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease.
-
hDOT1L Links Histone Methylation to Leukemogenesis
Cell, 2005Co-Authors: Yuki Okada, Qin Feng, Yihui Lin, Qi Jiang, Vernon M. Coffield, Yi ZhangAbstract:Epigenetic modifications play an important role in human cancer. One such modification, histone methylation, contributes to human cancer through deregulation of cancer-relevant genes. The yeast Dot1 and its human counterpart, hDOT1L, methylate lysine 79 located within the globular domain of histone H3. Here we report that hDOT1L interacts with AF10, an MLL (mixed lineage leukemia) fusion partner involved in acute myeloid leukemia, through the OM-LZ region of AF10 required for MLL-AF10-mediated leukemogenesis. We demonstrate that direct fusion of hDOT1L to MLL results in leukemic transformation in an hDOT1L methyltransferase activity-dependent manner. Transformation by MLL-hDOT1L and MLL-AF10 results in upregulation of a number of leukemia-relevant genes, such as Hoxa9, concomitant with hypermethylation of H3-K79. Our studies thus establish that mistargeting of hDOT1L to Hoxa9 plays an important role in MLL-AF10-mediated leukemogenesis and suggests that the enzymatic activity of hDOT1L may provide a potential target for therapeutic intervention.
-
structure of the catalytic domain of human DOT1L a non set domain nucleosomal histone methyltransferase
Cell, 2003Co-Authors: Jinrong Min, Qin Feng, Yi ZhangAbstract:Abstract Dot1 is an evolutionarily conserved histone methyltransferase that methylates lysine-79 of histone H3 in the core domain. Unlike other histone methyltransferases, Dot1 does not contain a SET domain, and it specifically methylates nucleosomal histone H3. We have solved a 2.5 A resolution structure of the catalytic domain of human Dot1, hDOT1L, in complex with S-adenosyl-L-methionine (SAM). The structure reveals a unique organization of a mainly α-helical N-terminal domain and a central open α/β structure, an active site consisting of a SAM binding pocket, and a potential lysine binding channel. We also show that a flexible, positively charged region at the C terminus of the catalytic domain is critical for nucleosome binding and enzymatic activity. These structural and biochemical analyses, combined with molecular modeling, provide mechanistic insights into the catalytic mechanism and nucleosomal specificity of Dot1 proteins.
-
methylation of h3 lysine 79 is mediated by a new family of hmtases without a set domain
Current Biology, 2002Co-Authors: Qi Feng, Hengbi Wang, Hediye Erdjumentbromage, Paul Temps, Kevi Struhl, Yi ZhangAbstract:Abstract The N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation [1]. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity [2–4]. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family [3]. Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast to human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, we found that K79 methylation level is regulated throughout the cell cycle. Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase.
Zaneta Nikolovskacoleska - One of the best experts on this subject based on the ideXlab platform.
-
elucidating the importance of DOT1L recruitment in mll af9 leukemia and hematopoiesis
Cancers, 2021Co-Authors: Sierrah M Grigsby, James Ropa, Justin Serio, Jennifer Chase, Chenxi Shen, Andrew G Muntean, Ivan Maillard, Bridget Waas, Ann Friedman, Zaneta NikolovskacoleskaAbstract:MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute DOT1L inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9-DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.
-
peptidomimetics for targeting protein protein interactions between DOT1L and mll oncofusion proteins af9 and enl
ACS Medicinal Chemistry Letters, 2018Co-Authors: Sierrah M Grigsby, Aihong Yao, Yujie Chang, Garrett Johnson, Haiying Sun, Zaneta NikolovskacoleskaAbstract: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.
-
the af9 binding domain of DOT1L is critical for its recruitment by mll fusion proteins in leukemia and contributes to its functions in normal hematopoiesis
Blood, 2017Co-Authors: Sierrah M Grigsby, James Ropa, Justin Serio, Jennifer Chase, Chenxi Shen, Andrew G Muntean, Ivan Maillard, Zaneta NikolovskacoleskaAbstract:Abstract Mixed lineage leukemia (MLL) is a very aggressive therapy resistant leukemia. MLL-AF9 is one of the most common MLL-fusion proteins that recruits disruptor of telomeric silencing 1-like (DOT1L), to MLL-target genes HOXA9/MEIS1. DOT1L is a histone methyltransferase that catalyzes methylation of H3K79, a gene activating mark, that is essential for leukemogenesis and normal hematopoiesis. Our lab and others mapped the 10 amino acid (865 - 874) binding site on DOT1L, which binds to the ANC1 homology domain (AHD) domain of AF9. We also identified a point mutation, I867A, sufficient to disrupt the AF9-DOT1L interaction in vitro. We demonstrated that DOT1L lacking the 10 amino acids (Δ10) were unable to support transformation by ML-AF9. We hypothesize that by disrupting the AF9-DOT1L protein-protein interaction (PPI), we will be able to inhibit leukemogenesis and potentially circumvent the negative consequences of DOT1L loss in normal bone marrow. We used a genetic approach to explore the role of DOT1L recruitment in leukemogenesis and normal hematopoiesis. We generated cell lines derived from MLL-AF9-driven DOT1Lf/f CreER(T2)+ bone marrow by transducing DOT1L constructs, including wild type (WT-DOT1L), deletion of 10 amino acids (Δ10-DOT1L), point mutant (I867A-DOT1L), and an enzymatically inactive mutant (RCR-DOT1L). Both Δ10 and I867A mutants inhibited leukemogenesis similarly to the RCR mutant. This inhibited proliferation was coupled with a decrease in H3K79me2, a decrease in HOXA9/Meis1 gene expression, cellular differentiation, and induction of apoptosis demonstrating that disruption of the AF9-DOT1L PPI mimics the phenotype of as the enzymatic inhibition, deeming it as a potential therapeutic target for this cancer. To explore the impact of disrupting the AF9-DOT1L interaction in normal bone marrow, we transduced Mx-Cre+ DOT1Lf/f bone marrow with either WT-DOT1L, Δ10-DOT1L, or RCR-DOT1L constructs and injected these cells into lethally irradiated B6-CD45.1 recipients. After hematopoietic reconstitution, recipients were treated with poly(I:C) to inactivate endogenous DOT1L. In the absence of DOT1L's methyltransferase activity (RCR-DOT1L), transduced hematopoietic progenitors underwent rapid failure. Interestingly, expression of the Δ10-DOT1L mutant was sufficient to support short-term hematopoiesis but insufficient to sustain long-term hematopoietic stem cells (LT-HSCs). Together these results show that blocking DOT1L's recruitment eliminates the transformative potential of the MLL-AF9 oncogene and that while DOT1L's function is critical for all hematopoietic progenitor cell survival, removing the DOT1L AF9 binding domain allows for short term hematopoiesis. To further explore the role of AF9 role in recruiting DOT1L in MLL-AF9 leukemia and normal hematopoiesis, using high throughput screening approach, we identified small molecules that bind to the AF9 AHD domain. These compounds inhibit proliferation of cells harboring the MLL-AF9 fusion, decrease H3K79me2, and induce apoptosis similar to Δ10-DOT1L and I867A-DOT1L mutants. We are currently working to further characterize their on-target activity in cells, and test their effects on normal bone marrow, with the ultimate goal to validate additional avenues for therapeutic intervention for MLL-rearranged leukemia. Disclosures No relevant conflicts of interest to declare.
-
targeting recruitment of disruptor of telomeric silencing 1 like DOT1L characterizing the interactions between DOT1L and mixed lineage leukemia mll fusion proteins
Journal of Biological Chemistry, 2013Co-Authors: Chenxi Shen, Jay L Hess, Chenzhong Liao, Zaneta NikolovskacoleskaAbstract:The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865–874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia. Background: MLL fusion proteins use similar strategy for leukemic transformation through DOT1L recruitment. Results: Ten amino acids in DOT1L were identified as essential for binding and transformation by MLL-AF9. Conclusion: Biochemical and functional results indicate that blocking DOT1L recruitment represents a promising therapeutic strategy for mixed lineage leukemia. Significance: Identified DOT1L peptide will lay a foundation toward discovery of chemical tools able to block DOT1L recruitment.
Scott A Armstrong - One of the best experts on this subject based on the ideXlab platform.
-
targeting myc driven b cell lymphoma by inhibition of the histone methyltransferase DOT1L
Blood, 2018Co-Authors: Anagha Deshpande, Scott A Armstrong, Benson Chen, Parham Ramezanirad, Alessandro Pastore, Luyi Zhao, Oliver Weigert, Robert C Rickert, Bing Ren, Aniruddha J DeshpandeAbstract:Abstract Aberrant activation of the MYC proto-oncogene is a recurrent feature in human B-cell lymphomas of diverse sub-types, correlating with adverse prognosis and therapy resistance. Direct pharmacological MYC-targeting has proved difficult, but recent studies have shown that targeting chromatin regulators critical for MYC-driven oncogenesis may provide alternative avenues for therapeutic intervention. Recently, it has been demonstrated that MYC-driven oncogenesis in certain solid tumors is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L. We hypothesized that B-cell lymphomas with hyperactive MYC-signaling might be responsive to DOT1L inhibition. In order to test this hypothesis, we tested the effect of the DOT1L inhibitor Pinometostat (EPZ-5676) on a panel of human B-cell lymphoma cell lines featuring elevated MYC. Pinometostat treatment reduced global H3K79 methylation levels, accompanied by a time and dose-dependent decrease in proliferation of several Burkitt's lymphoma cell lines including P493-6, Daudi and Raji. We observed that key MYC-target genes including CDK4, PPAT and NPM1 were significantly downregulated upon Pinometostat treatment, suggesting that DOT1L is required for the transcriptional activation of MYC-target genes in these cells. Pinometostat-treated B-lymphoma cells showed a significant decrease of cells in S-phase compared to controls as assessed by BrdU-labeling assays. Similar results were also obtained in a panel of B-cell lymphoma cell lines with MYC-rearrangements including mantle cell lymphoma (MCL) cell lines Jeko-1, JVM2, Mino-1 and Maver-1 and the diffused large B-cell lymphoma (DLBCL) cell line Karpas 422. Next, we sought to investigate whether the DOT1L-dependence of MYC-driven B-cell lymphoma could be reproduced in a well-defined model of MYC-driven B-cell lymphoma. Towards this end, we utilized a mouse model in which expression of the Cre recombinase from a B cell specific promoter leads to ectopic expression of a transgenic human MYC allele and concomitant deletion of the tumor suppressor Pten in B cells. Similar to our in vitro studies, Pinometostat treatment led to a significant reduction in proliferation of B-cell lymphoma cells from these mice with an IC50 of 0.5 µM. Furthermore, we sought to ascertain whether these findings reflected on-target effects related to DOT1L inhibition. Therefore, we deleted DOT1L using CRISPR/Cas9 in B-cell lymphoma cell lines and assessed the effect on proliferation using competitive-proliferation assays. We observed that DOT1L-deletion progressively diminished the relative growth of anti-DOT1L sgRNA-expressing P493-6 and Jeko1 cells compared to non-targeted cells in vitro. In order to test the requirement for DOT1L in lymphoma propagation in vivo, we performed intravenous injections of equal number of Jeko-1 cells with either anti-DOT1L or anti-Renilla control sgRNAs into sub-lethally irradiated non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID) mice. Mice injected with control anti-Renilla sgRNAs succumbed to disease with a median latency of 34 days while the latency of disease in the anti-DOT1L sgRNA cohort was 45 days. In summary, DOT1L depletion significantly delayed disease latency in this in vivo disseminated model of B-cell lymphoma (P=0.02). We then performed transcriptomic analyses of Pinometostat-treated B-cell lymphoma cell lines compared to DMSO-treated counterparts using RNA-seq. Gene-set enrichment analysis (GSEA) of RNA-seq data from three different B-cell lymphoma cell lines demonstrated that Pinometostat treatment significantly decreased the expression of MYC-target genes. In order to investigate the intriguing role of DOT1L in regulating MYC-target gene expression, we used ChIP-seq to assess the genome-wide occupancy of MYC following DOT1L inhibitor treatment. Strikingly, our studies demonstrated that DOT1L inhibition significantly reduced the chromatin occupancy of MYC. Taken together, our experiments demonstrate the role of DOT1L in MYC-driven B-cell lymphoma pathogenesis in vitro and in vivo. Furthermore, our genome-wide studies demonstrate the importance of DOT1L for genomic MYC occupancy. Based on these findings, we propose that therapeutic DOT1L targeting may be a viable strategy in MYC-driven B-cell lymphoma. Disclosures Weigert: Roche: Research Funding; Novartis: Research Funding. Rickert: Pfizer: Employment. Ren: Elli Lilly: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deshpande: Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.
-
the role of DOT1L in the maintenance of leukemia gene expression
Current Opinion in Genetics & Development, 2016Co-Authors: Xi Wang, Chunwei Chen, Scott A ArmstrongAbstract:Chromatin based (Epigenetic) mechanisms have been shown to play important roles in the regulation of gene expression during tumorigenesis and development. Mouse modeling suggests the methyltransferase DOT1L as a potential therapeutic target for MLL-rearranged leukemia. Epigenomic profiling indicates an abnormal H3K79me2 pattern on MLL-fusion targeted genes, but the molecular mechanism underlying this epigenetic dependency is not well understood. In this review, we will discuss recent advances in understanding the epigenetic mechanisms governed by DOT1L in the maintenance of gene expression. We will highlight the structural basis of chromatin targeting of DOT1L through its cofactors and the role of DOT1L in repelling transcription repressive complexes during leukemia development.
-
the pzp domain of af10 senses unmodified h3k27 to regulate DOT1L mediated methylation of h3k79
Molecular Cell, 2015Co-Authors: Shoudeng Chen, Scott A Armstrong, Aniruddha J Deshpande, Ze Yang, Alex W Wilkinson, Simone Sidoli, Krzysztof Krajewski, Brian D Strahl, Benjamin A Garcia, Dinshaw J PatelAbstract:AF10, a DOT1L cofactor, is required for H3K79 methylation and cooperates with DOT1L in leukemogenesis. However, the molecular mechanism by which AF10 regulates DOT1L-mediated H3K79 methylation is not clear. Here we report that AF10 contains a "reader" domain that couples unmodified H3K27 recognition to H3K79 methylation. An AF10 region consisting of a PHD finger-Zn knuckle-PHD finger (PZP) folds into a single module that recognizes amino acids 22-27 of H3, and this interaction is abrogated by H3K27 modification. Structural studies reveal that H3 binding triggers rearrangement of the PZP module to form an H3(22-27)-accommodating channel and that the unmodified H3K27 side chain is encased in a compact hydrogen-bond acceptor-lined cage. In cells, PZP recognition of H3 is required for H3K79 dimethylation, expression of DOT1L-target genes, and proliferation of DOT1L-addicted leukemic cells. Together, our results uncover a pivotal role for H3K27-via readout by the AF10 PZP domain-in regulating the cancer-associated enzyme DOT1L.
-
structure guided design of DOT1L methyltransferase inhibitors by a novel label free assay platform
Blood, 2014Co-Authors: Alexander J Federation, Scott A Armstrong, Sirano Dhepaganon, Michael Hadler, Anthony Varca, Roodolph St Pierre, James E BradnerAbstract:Cooperation between several epigenetic modulators defines MLL-rearranged leukemia as an epigenomic-driven cancer. Wild type MLL catalyzes trimethylation of lysine 4 on histone 3 from the methyl donor S-adenosylmethionine (SAM) at homeobox and other genes important for hematopoiesis, promoting their expression during development. However, in MLL-rearrangements, its methyltransferase domain is ubiquitously lost and replaced with >70 known fusion partners. Many of these fusion partners recruit DOT1L, the only known SAM-dependent lysine methyltransferase responsible for the methylation of lysine 79 of histone 3 (H3K79)—a mark associated with most actively transcribed genes. Therefore, the recruitment of DOT1L by MLL fusion partners to MLL-target genes leads to aberrant H3K79 hypermethylation at these loci, resulting in inappropriate gene expression and leukemogenesis. DOT1L as a therapeutic target in MLL has been genetically validated by several groups, leading to the development of SAM-competitive small molecule inhibitors of DOT1L. These inhibitors exhibit excellent biochemical activity and selectivity, yet have delayed cellular activity and needing relatively high doses, with viability effects requiring 7-10 days and EC50s for H3K79 methylation depletion of 1-3 μM in cell lines. In animal studies, this translates to a modest survival benefit while requiring high doses through continuous osmotic subcutaneous infusion. Further optimization of DOT1L inhibitors is therefore needed. To date, development of DOT1L inhibitors has been slow, perhaps related to inadequacy of discovery chemistry assay technologies. All biochemical assays are radioactivity-based and are not miniaturizeable; low-throughput and delayed cellular effects of DOT1L inhibition all hamper the discovery of improved inhibitors. Therefore a pressing need towards improved DOT1L inhibitor discovery is a robust, accessible, and rapid profiling platform. Toward this goal, we synthesized both FITC- and biotin-tagged DOT1L probe ligands. We confirmed by structural studies that binding of the probes were similar to our previously published inhibitor, depleted H3K79 methylation, and had antiproliferative effects in MLL-rearranged cell lines. We then utilized the probes to devise two non-radioactive, orthogonal biochemical assays to competitively profile putative inhibitors: one employing bead-based, proxmity fluorescence technology and the second using fluorescence polarization technology. These assays are robust and adaptable to high-throughput screening. We also designed a miniaturizable high-content imaging, immunofluorescence-based assay to assess the effect of DOT1L inhibitors on H3K79 methylation, reporting cellular IC50s after just four days of treatment. These three assays were validated against three known DOT1L inhibitors of different potencies, accurately differentiating between the compounds. Together, these orthogonal assays define an accessible platform capability to discover and optimize DOT1L inhibitors. Our platform rank-ordered a library of SAM derivatives that we synthesized, indicating that large substituents off the SAM base does not affect DOT1L binding. We also explored other features of the SAM core structure, identifying several chlorinated probes that had increased cellular potency (IC50 values ~10nM) relative to the initial compounds published, without losing specificity for DOT1L. The inhibitory effect on MLL-target gene expression correlated to the H3K79me2 decrease reported in high content assay, validating that our high-content assay accurately reports on downstream biology seen later in treatment. And as expected, the high-content potencies of our chlorinated DOT1L probes also correlated to increased anti-proliferative effect in MLL cells. Overall, we utilized chemistry, biology, and chemical biology tools to develop this profiling platform capability for more rapid discovery and optimization of small molecule DOT1L inhibitors. These assays can additionally be used to screen for non-SAM competitive inhibitors in high-throughput fashion. Furthermore, the DOT1L inhibitors and probes synthesized here (available as open-source tools) are useful in deeper mechanistic studies of the DOT1L complex and its role in MLL. Disclosures Armstrong:Epizyme: Consultancy.
-
structure guided DOT1L probe optimization by label free ligand displacement
ACS Chemical Biology, 2014Co-Authors: Alexander J Federation, Sirano Dhepaganon, Michael Hadler, Roodolph St Pierre, Anthony C Varca, Jason J Marineau, William B Smith, Amanda Souza, Emma J Chory, Scott A ArmstrongAbstract:The DOT1L lysine methyltransferase has emerged as a validated therapeutic target in MLL-rearranged (MLLr) acute leukemias. Although S-adenosylmethionine competitive inhibitors have demonstrated pharmacological proof-of-principle in MLLr-leukemia, these compounds require further optimization to improve cellular potency and pharmacokinetic stability. Limiting DOT1L inhibitor discovery and ligand optimization have been complex biochemical methods often using radionucleotides and cellular methods requiring prolonged culture. We therefore developed a new suite of assay technologies that allows comparative assessment of chemical tools for DOT1L in a miniaturized format. Coupling these assays with structural information, we developed new insights into DOT1L ligand binding and identified several functionalized probes with increased cellular potency (IC50 values ∼10 nM) and excellent selectivity for DOT1L. Together these assay technologies define a platform capability for discovery and optimization of small-molecu...
Robert G Roeder - One of the best experts on this subject based on the ideXlab platform.
-
DOT1L complex regulates transcriptional initiation in human erythroleukemic cells
Proceedings of the National Academy of Sciences of the United States of America, 2021Co-Authors: Junhong Zhi, Tom W Muir, Tian Tian, Ali Cihan, Murat A Cevher, Ziling Liu, Yael David, Robert G RoederAbstract:DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17, and ENL or AF9, is dysregulated in most cases of mixed-lineage leukemia (MLLr), and has been believed to regulate transcriptional elongation on the basis of its colocalization with RNA polymerase II (Pol II), the sharing of subunits (AF9 and ENL) between the DOT1L and super elongation complexes, and the distribution of H3K79 methylation on both promoters and transcribed regions of active genes. Here we show that DOT1L depletion in erythroleukemic cells reduces its global occupancy without affecting the traveling ratio or the elongation rate (assessed by 4sUDRB-seq) of Pol II, suggesting that DOT1L does not play a major role in elongation in these cells. In contrast, analyses of transcription initiation factor binding reveal that DOT1L and ENL depletions each result in reduced TATA binding protein (TBP) occupancies on thousands of genes. More importantly, DOT1L and ENL depletions concomitantly reduce TBP and Pol II occupancies on a significant fraction of direct (DOT1L-bound) target genes, indicating a role for the DOT1L complex in transcription initiation. Mechanistically, proteomic and biochemical studies suggest that the DOT1L complex may regulate transcriptional initiation by facilitating the recruitment or stabilization of transcription factor IID, likely in a monoubiquitinated H2B (H2Bub1)-enhanced manner. Additional studies show that DOT1L enhances H2Bub1 levels by limiting recruitment of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. These results advance our understanding of roles of the DOT1L complex in transcriptional regulation and have important implications for MLLr leukemias.
-
DOT1L complex regulates transcriptional initiation 1 in human cells
bioRxiv, 2020Co-Authors: Robert G Roeder, Junhong Zhi, Tian Tian, Lixue ChenAbstract:SUMMARY DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17 and ENL/AF9, is dysregulated in most of the cases of mixed lineage leukemia (MLL) and is believed to regulate transcriptional elongation without much evidence. Here we show that the depletion of DOT1L reduced the global occupancy without affecting the traveling ratio or the elongation rate of Pol II, suggesting it not a major elongation factor. An examination of general transcription factors (GTFs) binding revealed globally reduced TBP and TFIIA occupancies near promoters after DOT1L loss, pointing to a role in transcriptional initiation. Proteomic studies uncovered that DOT1L regulates transcriptional initiation likely by facilitating the recruitment of TFIID. Moreover, ENL, a DOT1L complex subunit with a known role in DOT1L recruitment, also regulates transcriptional initiation. Furthermore, DOT1L stimulates H2B monoubiquitination by limiting the recruitment of human SAGA complex, and the connection between Dot1/DOT1L and SAGA complex is conserved between yeast and human. These results advanced current understanding of roles of DOT1L complex in transcriptional regulation and MLL.
-
chemically ubiquitylated histone h2b stimulates hDOT1L mediated intranucleosomal methylation
Nature, 2008Co-Authors: Robert K. Mcginty, Robert G Roeder, Jaehoon Kim, Champak Chatterjee, Tom W MuirAbstract:Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDOT1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDOT1L. This effect is mediated through the catalytic domain of hDOT1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.
Lin Yang - One of the best experts on this subject based on the ideXlab platform.
-
DOT1L regulates thermogenic adipocyte differentiation and function via modulating h3k79 methylation
Diabetes, 2021Co-Authors: Lin Shuai, Haowen Jiang, Lin YangAbstract:Brown and beige adipocytes are characterized as thermogenic adipocytes and have great potential for treating obesity and associated metabolic diseases. In this article, we identify a conserved mammalian lysine 79 of histone H3 (H3K79) methyltransferase, disruptor of telomeric silencing-1 like (DOT1L), as a new epigenetic regulator that controls thermogenic adipocyte differentiation and function. We show that deletion of DOT1L in thermogenic adipocytes potently protects mice from diet-induced obesity, improves glucose homeostasis, alleviates hepatic steatosis, and facilitates adaptive thermogenesis in vivo. Loss of DOT1L in primary preadipocytes significantly promotes brown and beige adipogenesis and thermogenesis in vitro. Mechanistically, DOT1L epigenetically regulates the brown adipose tissue-selective gene program by modulating H3K79 methylation, in particular H3K79me2 modification. Thus, our study demonstrates that DOT1L exerts an important role in energy homeostasis by regulating thermogenic adipocyte differentiation and function.
-
DOT1L mediated h3k79 methylation regulates thermogenic adipocyte differentiation and function
Social Science Research Network, 2020Co-Authors: Lin Shuai, Haowen Jiang, Yanan Duan, Lin YangAbstract:Brown and beige adipocytes are characterized as thermogenic adipocytes and have great potential for treating obesity and associated metabolic diseases. Here, we identify a conserved mammalian lysine 79 of histone H3 (H3K79) methyltransferase, disruptor of telomeric silencing -1 like (DOT1L), as a new epigenetic regulator that controls thermogenic adipocyte development and function. We show that loss of DOT1L in primary preadipocytes significantly promotes brown and beige adipogenesis and thermogenesis in vitro. Notably, deletion of DOT1L in thermogenic adipocytes potently protects mice from diet-induced obesity, improves glucose homeostasis, alleviates hepatic steatosis, and facilitates adaptive thermogenesis in vivo. Mechanistically, DOT1L negatively regulates the BAT-selective gene program through H3K79 methylation. Thus, our study demonstrates that DOT1L exerts an important role in energy homeostasis by regulating thermogenic adipocyte function in mice.