Telomeric Silencing Research Articles

The epigenetic enzyme DOT1L orchestrates vascular smooth muscle cell-monocyte crosstalk and protects against atherosclerosis via the NF-κB pathway.

Histone H3 dimethylation at lysine 79 is a key epigenetic mark uniquely induced by methyltransferase disruptor of telomeric silencing 1-like (DOT1L). We aimed to determine whether DOT1L modulates vascular smooth muscle cell (VSMC) phenotype and how it might affect atherosclerosis in vitro and in vivo, unravelling the related mechanism. Gene expression screening of VSMCs stimulated with the BB isoform of platelet-derived growth factor led us to identify Dot1l as an early up-regulated epigenetic factor. Mouse and human atherosclerotic lesions were assessed for Dot1l expression, which resulted specifically localized in the VSMC compartment. The relevance of Dot1l to atherosclerosis pathogenesis was assessed through deletion of its gene in the VSMCs via an inducible, tissue-specific knock-out mouse model crossed with the ApoE-/- high-fat diet model of atherosclerosis. We found that the inactivation of Dot1l significantly reduced the progression of the disease. By combining RNA- and H3K79me2-chromatin immunoprecipitation-sequencing, we found that DOT1L and its induced H3K79me2 mark directly regulate the transcription of Nf-κB-1 and -2, master modulators of inflammation, which in turn induce the expression of CCL5 and CXCL10, cytokines fundamentally involved in atherosclerosis development. Finally, a correlation between coronary artery disease and genetic variations in the DOT1L gene was found because specific polymorphisms are associated with increased mRNA expression. DOT1L plays a key role in the epigenetic control of VSMC gene expression, leading to atherosclerosis development. Results identify DOT1L as a potential therapeutic target for vascular diseases.

DOT1L inhibition enhances pluripotency beyond acquisition of epithelial identity and without immediate suppression of the somatic transcriptome.

SummaryInhibiting the histone 3 lysine 79 (H3K79) methyltransferase, disruptor of telomeric silencing 1-like (DOT1L), increases the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs). Here, we find that, despite the enrichment of H3K79 methylation on thousands of actively transcribed genes in somatic cells, DOT1L inhibition (DOT1Li) does not immediately cause the shutdown of the somatic transcriptional profile to enable transition to pluripotency. Contrary to the prevalent view, DOT1Li promotes iPSC generation beyond the mesenchymal to epithelial transition and even from already epithelial cell types. DOT1Li is most potent at the midpoint of reprogramming in part by repressing Nfix that persists at late stages of reprogramming. Importantly, regulation of single genes cannot substitute for DOT1Li, demonstrating that H3K79 methylation has pleiotropic effects in maintaining cell identity.

The histone methyltransferase DOT1L is a new epigenetic regulator of pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with increasing occurrence, high death rates, and unfavorable treatment regimens. The pathogenesis underlying IPF is complex and the epigenetic contributions to IPF are largely unknown. Recent studies have shown that DOT1L (Disruptor of telomeric silencing-1 like), a histone H3K79 methyltransferase, contributes to fibrosis response, but its role in IPF remains unclear. DOT1L, H3K79me3, and the profibrotic proteins levels were upregulated in the pulmonary fibrosis models both in vivo and in vitro. Lentivirus-mediated DOT1L knockdown or DOT1L-specific inhibitor EPZ5676 alleviated the pathogenesis of bleomycin-induced mouse pulmonary fibrosis. Furthermore, heterozygous DOT1L-deficient mice (Dot1l+/−) showed less sensitive to pulmonary fibrosis, as shown by decreased lung fibrosis phenotypes in vivo. Mechanically, DOT1L regulated TGF-β1-induced fibroblasts fibrosis by increasing enrichments of H3K79me3 on the promoter of Jag1 gene (encoding the Notch ligand Jagged1), enhancing the expression of Jagged1, which in turn stimulated exuberant Notch signaling and actuated the fibrosis response. In conclusion, our study confirmed DOT1L to be an epigenetic modifier in the pathogenesis of lung fibrosis, revealed a counterbalancing mechanism governing Jag1 transcription by modulating H3K79 trimethylation at the Jag1 promoter, activating the Notch signaling, and affecting the expression of profibrotic proteins to accelerate the lung fibrosis.

Single-cell transcriptomics defines Dot1L interacting partners and downstream target genes in the mouse molar dental pulp.

Although histone methyltransferases are implicated in many key developmental processes, the contribution of individual chromatin modifiers in dental tissues is not well understood. Using single-cell RNA sequencing, we examined the expression profiles of the disruptor of telomeric silencing 1-like (Dot1L) gene in the postnatal day 5 mouse molar dental pulp. Dot1L is the only known enzyme that methylates histone 3 on lysine 79, a modification associated with gene expression. Our research revealed 15 distinct clusters representing different populations of mesenchymal stromal cells (MSCs), immune cells, pericytes, ameloblasts and endothelial cells. We documented heterogeneity in gene expression across different subpopulations of MSCs, a good indicator that these stromal progenitors undergo different phases of osteogenic differentiation. Interestingly, although Dot1L was broadly expressed across all cell clusters within the molar dental pulp, our analyses indicated specific enrichment of Dot1L within two clusters of MSCs, as well as cell clusters characterized as ameloblasts and endothelial cells. Moreover, we detected Dot1L co-expression with protein interactors involved in epigenetic activation such as Setd2, Sirt1, Brd4, Isw1, Bptf and Suv39h1. In addition, Dot1L was co-expressed with Eed2, Cbx3 and Dnmt1, which encode epigenetic factors associated with gene silencing and heterochromatin formation. Dot1l was co-expressed with downstream targets of the insulin growth factor and WNT signaling pathways, as well as genes involved in cell cycle progression. Collectively, our results suggest that Dot1L may play key roles in orchestrating lineage-specific gene expression during MSC differentiation.

Targeting of histone methyltransferase DOT1L plays a dual role in chemosensitization of retinoblastoma cells and enhances the efficacy of chemotherapy

Aberrant and exclusive expression of chromatin regulators in retinoblastoma (RB) in contrast to terminally differentiated normal retina presents a unique opportunity of selective targeting for RB. However, precise roles of these chromatin regulators in RB development and their potential as therapeutic targets have not been defined thoroughly. Here, we report that targeting of disruptor of telomeric silencing 1-like (DOT1L), a histone H3K79 methyltransferase, sensitizes RB cells to chemotherapeutic drugs by impairing the DNA damage response and thereby potentiating apoptosis while it is largely inefficacious as a single-agent therapy. Moreover, we identified high mobility group AT-hook 2 (HMGA2) as a novel DOT1L target gene in RB cells and found that its aberrant expression is dependent on DOT1L. As HMGA2 depletion reduced CHK1 phosphorylation during DNA damage response and augmented the drug sensitivity in RB cells, our results suggested that DOT1L targeting has a dual role in chemosensitization of RB cells by directly interfering with the immediate involvement of DOT1L in early DNA damage response upon genotoxic insults and also by downregulating the expression of HMGA2 as a rather late effect of DOT1L inhibition. Furthermore, we provide the first preclinical evidence demonstrating that combined therapy with a DOT1L inhibitor significantly improves the therapeutic efficacy of etoposide in murine orthotopic xenografts of RB by rendering the response to etoposide more potent and stable. Taken together, these results support the therapeutic benefits of DOT1L targeting in combination with other chemotherapeutic agents in RB, with mechanistic insights into how DOT1L targeting can improve the current chemotherapy in an RB cell-selective manner.

Histone Methyltransferase Dot1L Contributes to RIPK1 Kinase-Dependent Apoptosis in Cerebral Ischemia/Reperfusion.

BackgroundNeuron apoptosis is a pivotal process for brain damage in cerebral ischemia. Dot1L (disruptor of telomeric silencing 1‐like) is only known histone H3K79 methyltransferase. It is not clear whether the role and mechanism of Dot1L on cerebral ischemia is related to regulate neuron apoptosis.Methods and ResultsWe use a combination of mice middle cerebral artery occlusion stroke and neurons exposed to oxygen‐glucose deprivation followed by reoxygenation to investigate the role and mechanism of Dot1L on cerebral ischemia. We find knockdown or inhibition of Dot1L reversed ischemia‐induced neuronal apoptosis and attenuated the neurons injury treated by oxygen‐glucose deprivation followed by reoxygenation. Further, blockade of Dot1L prevents RIPK1 (receptor‐interacting protein kinase 1)‐dependent apoptosis through increased RIPK1 K63‐ubiquitylation and decreased formation of RIPK1/Caspase 8 complexes. In line with this, H3K79me3 enrichment in the promoter region of deubiquitin‐modifying enzyme A20 and deubiquitinase cylindromatosis gene promotes the increasing expression in oxygen‐glucose deprivation followed by reoxygenation ‐induced neuronal cells, on the contrary, oxygen‐glucose deprivation followed by reoxygenation decreases H3K79me3 level in the promoter region of ubiquitin‐modifying enzyme cIAP1 (cellular inhibitors of apoptosis proteins), and both these factors ultimately cause K63‐deubiquitination of RIPK1. Importantly, knockdown or inhibition of Dot1L in vivo attenuates apoptosis in middle cerebral artery occlusion mice and reduces the extent of middle cerebral artery occlusion ‐induced brain injury.ConclusionsThese data support for the first time, to our knowledge, that Dot1L regulating RIPK1 to the apoptotic death trigger contributes to cerebral ischemia injury. Therefore, targeting Dot1L serves as a new therapeutic strategy for ischemia stroke.

Decoding Cinnabarinic Acid-Specific Stanniocalcin 2 Induction by Aryl Hydrocarbon Receptor.

Aryl hydrocarbon receptor (AhR) is a ligand-mediated transcription factor known for regulating response to xenobiotics, including prototypical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the activation of CYP1A1 expression. Upon ligand-binding, AhR translocates to the nucleus, interacts with the AhR nuclear translocator, and binds to xenobiotic response elements (XREs; GCGTG) present in the promoter region of AhR-regulated genes. Recently, we identified a novel tryptophan catabolite, cinnabarinic acid (CA), as an endogenous AhR agonist capable of activating expression of AhR target gene stanniocalcin 2 (stc2). The CA-driven stc2 induction bestowed cytoprotection against hepatotoxicity in an AhR-dependent manner. Interestingly, only CA but not TCDD was able to induce stc2 expression in liver, and CA was unable to upregulate the TCDD responsive cyp1a1 gene. In this report, we identified CA-specific histone H4 lysine 5 acetylation and H3 lysine 79 methylation at the AhR-bound stc2 promoter. Moreover, histone H4 lysine 5 acetylation writer, activating transcription factor 2 (Atf2), and H3 lysine 79 methylation writer, disruptor of telomeric silencing 1-like histone lysine methyltransferase (Dot1l), were interacting with the AhR complex at the stc2 promoter exclusively in response to CA treatment concurrent with the histone epigenetic marks. Suppressing Atf2 and Dot1l expression using RNA interference confirmed their role in stc2 expression. CRISPR/Cas9-assisted replacement of cyp1a1 promoter-encompassing XREs with stc2 promoter XREs resulted in CA-dependent induction of cyp1a1, underlining a fundamental role of quaternary structure of XRE sequence in agonist-specific gene regulation. In conclusion, CA-driven recruitment of specific chromatin regulators to the AhR complex and resulting histone epigenetic modifications may serve as a molecular basis for agonist-specific stc2 regulation by AhR. SIGNIFICANCE STATEMENT: Results reported here provide a mechanistic explanation for the agonist-specific differential gene regulation by identifying interaction of aryl hydrogen receptor with specific chromatin regulators concomitant with unique histone epigenetic marks. This study also demonstrated that the agonist-specific target-gene expression can be transferred with the gene-specific promoter xenobiotic response element-sequence in the context of chromatin architecture.

Establishing the Role of EPS15, DVL2 and Cortactin in CALM-AF10 Leukemogenesis

Background:Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoblastic leukemia (T-ALL)as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXA genes. To identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS).Methods:We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with BioID2-CALM-AF10 and grown in the presence or absence of biotin. MS was performed to identify candidate interacting proteins. We validated direct interactions of candidate proteins with CALM-AF10 using co-immunoprecipitation experiments in HEK293 cells transfected with a CALM-AF10 plasmid. We confirmed that candidate proteins are present in murine CALM-AF10 leukemia cells via Western blotting. In order to efficiently knockout (KO) candidate proteins, we have generated a human U937 cell line (which harbors a t(10;11) CALM-AF10 translocation) with a stable incorporated Cas9. To assess whether KO of EPS15, DVL2 or CTTN affects HOXA5 expression, we performed RT-qPCR in U937-Cas9 cells lines with confirmed KO.Results:We carried out three independent transfections/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 12 candidates were common to all three experiments . Importantly, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that interacts with CRM1 and that is involved in leukemogenic translocations. We chose EPS15, DVL2 and CTTN for further study, as each of these proteins plays a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that all three proteins co-precipitate with CALM-AF10. Western blotting showed that all three proteins are expressed in a murine CALM-AF10 leukemia cell line. We effectively knocked out EPS15 protein expression in U937 cells, and showed that HOXA5 expression is reduced in the setting of EPS15 knockout.Conclusion:We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate three candidate proteins - EPS15, DVL2 and CTTN - all of which are involved in leukemogenic transformation. We have shown that EPS15, DVL2 and CTTN are expressed in murine CALM-AF10 leukemia cells and directly interact with the CALM-AF10 fusion protein. Knockout of EPS15 in U937 cells results in decreased HOXA5 expression, suggesting the importance of EPS15 in CALM-AF10 leukemogenesis. Evaluation of the roles of these proteins in leukemogenesis may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Histone methyltransferase DOT1L mediates the TGF-β1/Smad3 signaling pathway through epigenetic modification of SYK in myocardial infarction.

Myocardial infarction (MI) represents the most critical condition in coronary artery disease, and the fibrotic process, detrimental to optimal recovery, often sustains. In the present work, we assessed whether suppression of disruptor of telomeric silencing 1-like (DOT1L) could alleviate fibrosis in vivo and cardiac fibroblast (CFS) proliferation in vitro, and elucidated the possible mechanism involved in these events. After left coronary artery ligation, we found that the MI mice exhibited a decrease in cardiac function, along with evident MI and myocardial fibrosis. In addition, AngII increased CFS viability and migration, and enhanced the expression of fibrotic proteins. Inhibition of DOT1L ameliorated proliferation and fibrosis in CFS. Furthermore, DOT1L promoted the expression of spleen tyrosine kinase (SYK) by increasing the H3K79me2 modification of the SYK promoter. SYK upregulation reversed the inhibitory effect of DOT1L knockdown on CFS proliferation and fibrosis by activating the TGF-β1/Smad3 signaling. SYK also mitigated the ameliorative effect of DOT1L knockdown on myocardial injury and fibrosis caused by MI in vivo. In conclusion, these data indicated that DOT1L depletion might be a promising therapeutic target for fibrosis in MI.

Loss of telomere silencing is accompanied by dysfunction of Polo kinase and centrosomes during Drosophila oogenesis and early development.

Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. We studied the mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. We found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction.

Computational modeling reveals key molecular properties and dynamic behavior of disruptor of telomeric silencing 1-like (DOT1L) and partnering complexes involved in leukemogenesis.

Disruptor of telomeric silencing 1‐like (DOT1L) is the only non‐SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time‐dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1‐AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L–AF9 complex are less extensive and highly dynamic, resembling a swivel‐like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.

IGluK-Deep: computational identification of lysine glutarylation sites using deep neural networks with general pseudo amino acid compositions

Lysine glutarylation is a post-translation modification which plays an important regulatory role in a variety of physiological and enzymatic processes including mitochondrial functions and metabolic processes both in eukaryotic and prokaryotic cells. This post-translational modification influences chromatin structure and thereby results in global regulation of transcription, defects in cell-cycle progression, DNA damage repair, and telomere silencing. To better understand the mechanism of lysine glutarylation, its identification in a protein is necessary, however, experimental methods are time-consuming and labor-intensive. Herein, we propose a new computational prediction approach to supplement experimental methods for identification of lysine glutarylation site prediction by deep neural networks and Chou’s Pseudo Amino Acid Composition (PseAAC). We employed well-known deep neural networks for feature representation learning and classification of peptide sequences. Our approach opts raw pseudo amino acid compositions and obsoletes the need to separately perform costly and cumbersome feature extraction and selection. Among the developed deep learning-based predictors, the standard neural network-based predictor demonstrated highest scores in terms of accuracy and all other performance evaluation measures and outperforms majority of previously reported predictors without requiring expensive feature extraction process. iGluK-Deep:Computational Identification of lysine glutarylationsites using deep neural networks with general Pseudo Amino Acid Compositions Sheraz Naseer, Rao Faizan Ali, Yaser Daanial Khan, P.D.D Dominic Communicated by Ramaswamy H. Sarma

Disruptor of telomeric silencing 1-like promotes ovarian cancer tumor growth by stimulating pro-tumorigenic metabolic pathways and blocking apoptosis

ABSTRACTOvarian cancer is the leading cause of gynecological malignancy-related deaths. Current therapies for ovarian cancer do not provide meaningful and sustainable clinical benefits, highlighting the need for new therapies. We show that the histone H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is overexpressed in ovarian cancer and that a higher level of DOT1L expression correlates with shorter progression-free and overall survival (OS). Pharmacological inhibition of DOT1L (EPZ-5676, EPZ004777, and SGC0946) or genetic inhibition of DOT1L attenuates the growth of ovarian cancer cells in cell culture and in a mouse xenograft model of ovarian cancer. Transcriptome-wide mRNA expression profiling shows that DOT1L inhibition results in the downregulation of genes involved in cellular biosynthesis pathways and the upregulation of proapoptotic genes. Consistent with the results of transcriptome analysis, the unbiased large-scale metabolomic analysis showed reduced levels of several metabolites of the amino acid and nucleotide biosynthesis pathways after DOT1L inhibition. DOT1L inhibition also resulted in the upregulation of the NKG2D ligand ULBP1 and subsequent increase in natural killer (NK) cell-mediated ovarian cancer eradication. Collectively, our results demonstrate that DOT1L promotes ovarian cancer tumor growth by regulating apoptotic and metabolic pathways as well as NK cell-mediated eradication of ovarian cancer and identifies DOT1L as a new pharmacological target for ovarian cancer therapy.

The SESAME complex regulates cell senescence through the generation of acetyl-CoA.

Acetyl-CoA is a central node in carbon metabolism and plays critical roles in regulatory and biosynthetic processes. The acetyl-CoA synthetase Acs2, which catalyses acetyl-CoA production from acetate, is an integral subunit of the serine-responsive SAM-containing metabolic enzyme (SESAME) complex, but the precise function of Acs2 within the SESAME complex remains unclear. Here, using budding yeast, we show that Acs2 within the SESAME complex is required for the regulation of telomere silencing and cellular senescence. Mechanistically, the SESAME complex interacts with the histone acetyltransferase SAS protein complex to promote histone H4K16 acetylation (H4K16ac) enrichment and the occupancy of bromodomain-containing protein, Bdf1, at subtelomeric regions. This interaction maintains telomere silencing by antagonizing the spreading of Sir2 along the telomeres, which is enhanced by acetate. Consequently, dissociation of Sir2 from telomeres by acetate leads to compromised telomere silencing and accelerated chronological ageing. In human endothelial cells, ACSS2, the ortholog of yeast Acs2, also interacts with H4K16 acetyltransferase hMOF and are required for acetate to increase H4K16ac, reduce telomere silencing and induce cell senescence. Altogether, our results reveal a conserved mechanism to connect cell metabolism with telomere silencing and cellular senescence.

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

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. DisclosuresNo relevant conflicts of interest to declare.

DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A.

Oncogene-induced senescence (OIS) is a stable cell cycle arrest that occurs in normal cells upon oncogene activation. Cells undergoing OIS express a wide variety of secreted factors that affect the senescent microenvironment termed the senescence-associated secretory phenotype (SASP), which is beneficial or detrimental in a context-dependent manner. OIS cells are also characterized by marked epigenetic changes. We globally assessed histone modifications of OIS cells and discovered an increase in the active histone marks H3K79me2/3. The H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) was necessary and sufficient for increased H3K79me2/3 occupancy at the IL1A gene locus, but not other SASP genes, and was downstream of STING. Modulating DOT1L expression did not affect the cell cycle arrest. Together, our studies establish DOT1L as an epigenetic regulator of the SASP, whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.

DOT1L Regulates Thermogenic Adipocyte Differentiation and Function via Modulating H3K79 Methylation.

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.

Inhibition of histone demethylases as an approach to restore deficient DOT1L activity in osteoarthritis

Purpose: Osteoarthritis (OA), the most common chronic joint disease worldwide, is characterized by progressive damage to the articular cartilage, increased joint-associated bone remodelling, and synovial inflammation. Current OA treatments are limited to pain relief, physiotherapy, or replacement surgeries in severe cases, yet disease-modifying drugs are lacking. A genome wide association study (GWAS) revealed a genetic association between polymorphisms in the DOT1L gene and OA. The Disruptor of telomeric silencing 1-like (DOT1L) gene encodes a histone methyltransferase that methylates Lysine 79 of Histone H3 (H3K79). We identified DOT1L as a key protector of cartilage homeostasis, using both human articular chondrocytes and different Dot1l genetic mouse models. Furthermore, we reported that DOT1L activity, indicated by the levels of H3K79 methylation (H3K79me), is reduced in OA as compared to non-OA cartilage. Therefore, maintaining H3K79me seems to be critical to preserve joint health and prevent the development or progression of OA. Here, we hypothesized that H3K79me could be restored or maintained by inhibiting demethylation at the H3K79 site, via targeting specific histone demethylases. There are two main families of histone demethylases: the lysine-specific demethylases (LSD) and Jumonji C (JmjC) demethylases. The LSD family contains two members: LSD1 and LSD2. The JmjC family is further classified into 6 subfamilies: from KDM2 to KDM7. In this new study, we aimed to investigate which histone demethylases are responsible for H3K79 demethylation and whether their specific targeting can lead to protective effects in OA. Methods: We determined the baseline mRNA expression of 28 reported histone demethylases in primary human articular chondrocytes (hACs) from non-OA patients, using real-time qPCR. We also determined the changes in histone demethylase expression in vitro upon OA-mimicking stimuli, namely proinflammatory cytokine IL-1β and the Wnt signalling activator CHIR99021. To interrogate the role of JmjC demethylase family in H3K79me, human articular chondrocyte C28/I2 cells were treated with the JmjC pan inhibitor JIB-04. In parallel, the LSD family members LSD1 and LSD2 were pharmacologically inhibited using LSD1 inhibitor II or silenced using siRNA against LSD2, respectively. The role of the different JmjC subfamilies on H3K79me was studied using selective pharmacological inhibitors. We assessed the levels of H3K79me by Western blot and immunofluorescence analysis. The translational impact of individual JmjC demethylase targeting was evaluated using a siRNA knockdown strategy in hACs from OA patients. In this setup, gene expression of healthy cartilage markers Collagen2a1 (COL2A1) and Aggrecan, as well as of catabolic markers MMP13 and ADAMTS5, was assessed using real-time qPCR. Results: The baseline expression of histone demethylases was mapped in hACs, showing differences in absolute expression among the different enzymes. Treatment with IL1-β resulted in an increase in KDM6B and KDM7A mRNA expression. Conversely, Wnt signalling activation by CHIR99021 led to a downregulation in most of histone demethylases’ mRNA expression. Interestingly, pharmacological inhibition of the JmjC family using JIB-04 resulted in increased H3K79me levels. However, the blockade of LSD family members did not lead to H3K79me changes in articular chondrocytes. Moreover, inhibition of all JmjC demethylase subfamilies increased H3K79me levels. Individual silencing of specific JmjC demethylases increased the expression of chondrocyte healthy markers while reducing the expression of catabolic markers. Conclusions: The discovery approach performed in this study unravels that histone demethylases from the JmjC family control H3K79me in the articular cartilage. Silencing of individual demethylases from this family exerted protective effects in the articular chondrocyte. Therefore, targeting specific histone demethylases could restore the loss of DOT1L activity in OA and lead to therapeutic benefits in this chronic disease.

Synthesis and biological activity of selenopsammaplin A and its analogues as antitumor agents with DOT1L inhibitory activity

Disruptor of telomeric silencing-1 like (DOT1L) is a histone H3 methyltransferase which specifically catalyzes the methylation of histone H3 lysine-79 residue. Recent findings demonstrate that DOT1L is abnormally overexpressed and the upregulated DOT1L evokes the proliferation and metastasis in human breast cancer cells. Therefore, the DOT1L inhibitor is considered a promising strategy to treat breast cancers. Non-nucleoside DOT1L inhibitors, selenopsammaplin A and its analogues, were firstly reported in the present study. Selenopsammaplin A was newly designed and synthesized with 25% overall yield in 8 steps from 3-bromo-4-hydroxybenzaldahyde, and thirteen analogues of selenopsammaplin A were prepared for structure–activity relationship studies of their cytotoxicity against cancer cells and inhibitory activity toward DOT1L for antitumor potential. All synthetic selenopsammaplin A analogues exhibited the higher cytotoxicity compared to psammaplin A with up to 6 – 60 times depending on cancer cells, and most analogues showed significant inhibitory activities against DOT1L. Among the prepared analogues, the phenyl analogue (10) possessed the most potent activity with both cytotoxicity and inhibition of DOT1L. Compound 10 also exhibited the antitumor and antimetastatic activity in an orthotopic mouse metastasis model implanted with MDA-MB-231 human breast cancer cells. These biological findings suggest that analogue 10 is a promising candidate for development as a cancer chemotherapeutic agent in breast cancers.

Repression of a large number of genes requires interplay between homologous recombination and HIRA.

During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51–DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.