H3K4 Methyltransferase Activity Research Articles

Overexpression of Solanum lycopersicum DDB1 interacting protein 4 (SlDDI4) extends tomato shelf life by methylation-mediated suppression of softening-related genes expression

Prolonging the fruit storage period is strategically important due to the limitation of postharvest life determined by excessive softening. Efforts have been made to alleviate the issue of fruit softening in transgenic fruit by suppressing genes that encode cell wall-degrading proteins. However, individual genes related to the degradation of cell walls have limited ability, resulting in minimal success. SET domain-containing protein (SET protein) is widely expressed in most plant organs and crucial in plant growth and development. A tomato SET protein was previously identified as SlDDI4 (Solanum lycopersicum DDB1 interacting protein 4) with a H3K9 methyltransferase activity involving cell proliferation and organ size regulation. In this study, we demonstrated that SlDDI4 overexpression (SlDDI4-OE) promotes fruit firmness in addition to increased fruit size. Further experimental investigations revealed that the increased expression of SlDDI4 inhibited cellulase and pectinase activities, consequently increasing the proportion of cellulose and insoluble pectin. RNA-seq profiling indicated the genes involved in cell wall metabolism in SlDDI4-OE fruit were significantly down-regulated. DNA methylation and McrBC-PCR analysis at the SlCEL2, SlPG2, and SlEXP12 loci revealed that their down-regulation was associated with the elevated methylation level in the individual promoter regions. Furthermore, up-regulation of SlDDI4 generated fruit with increased lycopene content and prolonging shelf-life by approximately 20 d without adverse impacts on the flavor indicators. Thus, our results suggest that manipulation of SlDDI4 expression enables broad favorable effects on postharvest life as well as fruit size and quality through chromatin remodeling.

Methylation of histone H3 lysine 4 is required for maintenance of beta cell function in adult mice

Aims/hypothesisBeta cells control glucose homeostasis via regulated production and secretion of insulin. This function arises from a highly specialised gene expression programme that is established during development and then sustained, with limited flexibility, in terminally differentiated cells. Dysregulation of this programme is seen in type 2 diabetes but mechanisms that preserve gene expression or underlie its dysregulation in mature cells are not well resolved. This study investigated whether methylation of histone H3 lysine 4 (H3K4), a marker of gene promoters with unresolved functional importance, is necessary for the maintenance of mature beta cell function.MethodsBeta cell function, gene expression and chromatin modifications were analysed in conditional Dpy30 knockout mice, in which H3K4 methyltransferase activity is impaired, and in a mouse model of diabetes.ResultsH3K4 methylation maintains expression of genes that are important for insulin biosynthesis and glucose responsiveness. Deficient methylation of H3K4 leads to a less active and more repressed epigenome profile that locally correlates with gene expression deficits but does not globally reduce gene expression. Instead, developmentally regulated genes and genes in weakly active or suppressed states particularly rely on H3K4 methylation. We further show that H3K4 trimethylation (H3K4me3) is reorganised in islets from the Leprdb/db mouse model of diabetes in favour of weakly active and disallowed genes at the expense of terminal beta cell markers with broad H3K4me3 peaks.Conclusions/interpretationSustained methylation of H3K4 is critical for the maintenance of beta cell function. Redistribution of H3K4me3 is linked to gene expression changes that are implicated in diabetes pathology.Graphical abstract

Abstract A021: Lysine methylation in EHMT1/GLP acts as a molecular switch to reprogram transcription networks to drive prostate cancer progression

Abstract Background: Prostate Cancer (PCa) progresses to a metastatic form of cancer called Castration-Resistant Prostate Cancer (CRPC) after treatment with androgen deprivation therapies (ADTs). Epigenetic reprogramming through altered expression and activity of histone modifier proteins is one major mechanism of tumor resistance. In particular, histone lysine methyltransferases (KMTs) and demethylases (KDMs) are important epigenetic targets in CRPC. In this study, we have focused on studying the function and regulation of Euchromatic Histone Methyltransferase 1 protein (EHMT1/GLP), which is a well-known H3K9 methyltransferase and functions in repressing gene transcription. EHMT family genes are altered in ~2-3% of primary PCa and ~10% of CRPC (primarily gene amplification) and their expression levels are significantly increased in CRPC, suggesting these proteins may have oncogenic activities in driving CRPC progression. Methods: We conducted an integrated analysis of ChIP-seq and RNA-seq analysis in LNCaP PCa cells to characterize the transcription program of EHMT1. We then performed proteomics analyses in VCaP and LNCaP PCa cells to identify post-translational modifications of EHMT1 and functionally validated the findings by generating loss-of-function mutations. We performed a histone demethylase assay to discover if EHMT1 is a substrate of LSD1 (a member of the KDM family). Moreover, we also assessed the effects of EHMT1 silencing or inhibition (in multiple PCa cell lines) on cell proliferation and metastasis in vitro using cell cycle analysis and transwell migration assay, and tumor growth and metastasis in vivo using mouse subcutaneous injection and zebrafish embryo injection approaches. Results: Our data showed that EHMT1 can transcriptionally activate multiple oncogenic pathways, including E2F and MYC signaling. Proteomic analyses revealed that EHMT1 is methylated in PCa cell lines, with dual-lysine methylation occurring at the K450/K451 sites. The histone demethylase assay showed that methylated K450, but not K451 is a potential substrate of LSD1. By generating the K450R, K451R, and K450/451R mutants, we showed that the K450/451R mutant, but not any single lysine mutants, can greatly expand EHMT1 chromatin binding independent of its H3K9 methyltransferase activity. This mutant also significantly induced EHMT1 oncogenic activity by activating E2F and MYC pathways. Moreover, EHMT1 silencing, or inhibition can significantly suppress tumor growth and metastasis. Conclusion: We have demonstrated that EHMT1 can function to activate oncogenic transcriptional programs in PCa by an H3K9-independent mechanism, possibly mediated through dual-lysine demethylation at K450/451 sites. Our data also suggest that targeting EHMT1 in CRPC may be a potential therapeutic strategy to suppress tumor growth and metastasis. Citation Format: Anna Besschetnova, Wanting Han, Mingyu Liu, Yanfei Gao, Muqing Li, Zifeng Wang, Maryam Labaf, Susan Patalano, Kavita Venkataramani, Rachel Muriph, Jill Macoska, Kellee Siegfried-Harris, Jason Evans, Steven Balk, Shuai Gao, Dong Han, Changmeng Cai. Lysine methylation in EHMT1/GLP acts as a molecular switch to reprogram transcription networks to drive prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A021.

IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors invitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML invivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

Abstract 2957: Inhibition of epigenetic regulator menin is a novel therapeutic approach for high-risk neuroblastoma

Abstract Controlling epigenetic factors such as histone methyltransferases and demethylases has recently emerged as an attractive therapeutic approach for different cancers. KMT2 family protein KMT2A or MLL1 is a H3K4 methyltransferase, which is known to play an essential role in cancer development. MLL1 forms a COMPASS complex to function as an epigenetic regulator and Menin is one of the specific and required binding partner for the activation and function of this epigenetic enzyme. Menin acts as a tumor suppressor protein, and inhibition of Menin-MLL1 interaction has been shown to destabilize the MLL1 epigenetic complex, inhibits H3K4 methyltransferase activity, and hinder the oncogenic potential of various cancers. High-risk neuroblastoma (NB) is the most common extracranial solid pediatric tumor and accounts for almost 15% of all pediatric cancer-related deaths. Recent reports indicate that aberrant activation of epigenetic regulators may drive NB progression and relapse. Therefore, in the present study, we used a specific small-molecule inhibitor, MI-503 to disrupt Menin-MLL1 interaction, and to further understand the role of Menin in pediatric NB. We used different NB cell lines including MYCN-non-amplified (SH-SY5Y, SK-N-AS, CHLA-255) and MYCN-amplified (NGP, LAN-5, IMR-32), and performed cytotoxicity and Colonogenic assays to determine the effect of Menin inhibition on NB proliferation. Results showed that MI-503 significantly inhibits NB proliferation and colony formation capacity in a dose-dependent manner in contrast to controls. Further, we performed apoptosis and cell cycle assays and observed that MI-503 significantly induces apoptosis as detected by the increase of Annexin-V positive early apoptotic cells, and blocks cell cycle progression at the S phase in all NB cell lines tested. Additionally, Western blot assays demonstrated an overall inhibition of H3K3me3 levels in NB cells in response to MI-503. To further determine the effects of Menin inhibition on NB tumor growth, we developed a NB 3D spheroid tumor model that recapitulate in vivo tumor growth for solid tumors. Treatment with MI-503 significantly inhibits spheroidal tumor growth by inducing tumor cell death in a dose-dependent manner. Overall, these data highlight the role of epigenetic regulation, MLL1 complex, and of Menin in NB growth and tumorigenicity. In our future efforts, we will further elucidate the effects of MI-503 and the role of Menin in NB by using in vivo tumor models. Epigenetic inhibitor such as MI-503 holds significant potential for further clinical development and as a novel therapeutic approach for NB. Citation Format: Rameswari Chilamakuri, Saurabh Agarwal. Inhibition of epigenetic regulator menin is a novel therapeutic approach for high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2957.

Direct targeting of epigenetic regulator Menin inhibits neuroblastoma growth

Histone modifiers such as methyltransferases and demethylases have recently emerged as an attractive target to treat different cancers. Menin is a tumor suppressor protein and an essential oncogenic co‐factor of MLL1 COMPASS complex. Menin is a highly specific binding partner of the N‐terminus of MLL1 complex that have H3K4 methyltransferase activity. Menin‐MLL1 as an epigenetic regulator regulates the expression of target oncogenes, including HOXA, MEIS1, and AF9. Blocking menin‐MLL1 interaction destabilizes the COMPASS complex and hinders the oncogenic potential of cancer cells and cancer stem cells (CSCs). Neuroblastoma (NB) is an aggressive pediatric solid tumor develops during the early embryonic stage from extracranial sympathetic nervous system, and accounts for 10‐15% of all pediatric cancer‐related deaths. In the present study, we hypothesized that disrupting menin‐MLL1 interaction by using a specific small molecule inhibitor could disrupt COMPASS complex and overall inhibit NB growth, metastasis, and relapse. In the present study, we analyzed 1235 NB patients from different NB patient datasets and found that the upregulation of menin inversely correlate with overall survival of NB patients. We used six NB cell lines, including MYCN‐ amplified and ‐non‐amplified cell lines and performed cytotoxic assays. Results showed that inhibition of menin‐MLL1 interaction by MI‐503 significantly inhibits NB proliferation and colony formation capacity in contract to control treatments. Furthermore, MI‐503 significantly and in a dose‐dependent manner blocked NB cell cycle progression at S phase and enhanced apoptosis in treatment groups compared to controls. Additionally, we developed a 3D spheroidal tumor model for NB to mimic in vivo tumor conditions and tested the ability of MI‐503 in inhibiting tumor growth. Results showed significant inhibition of spheroidal tumor growth by enhancing tumor cell death in response to MI‐503 treatment. To further determine the effect of MI‐503 on inhibition of H3K4me3 levels, a key function of menin‐MLL1 COMPASS complex, we performed Western blot assays and observed a significant and dose dependent inhibition of H3K4me3 levels in response to MI‐503 treatment. Overall, our study highlights the role of menin‐MLL1 interaction in NB. Inhibition of menin‐MLL1 interaction by a specific small‐molecule inhibitor MI‐503 leads to the inhibition of epigenetic regulator functions and inhibits NB growth. Our future efforts will focus on further elucidating the role of menin in NB and to develop effective therapeutic strategies incorporating epigenetic inhibitors for NB patients.

Discovery of a potent MLL1 and WDR5 protein-protein interaction inhibitor with invivo antitumor activity.

MLL1-WDR5 interaction is essential for the formation of MLL core complex and its H3K4 methyltransferase activity. Disrupting MLL1-WDR5 interaction has been proposed as a potential therapeutic approach in the treatment of leukemia. A "toolkit" of well-characterized chemical probe will allow exploring animal studies. Based on a specific MLL1-WDR5 PPI inhibitor (DDO-2117), which was previously reported by our group, we conducted a bioisosterism approach by click chemistry to discover novel phenyltriazole scaffold MLL1-WDR5 interaction blockers. Here, our efforts resulted in the best inhibitor 24 (DDO-2093) with high binding affinity (Kd=11.6nM) and with improved drug-like properties. Both invitro and invivo assays revealed 24 could efficiently block the MLL1-WDR5 interaction. Furthermore, 24 significantly suppressed tumor growth in the MV4-11 xenograft mouse model and showed a favorable safety profile. We propose 24 as a chemical probe that is suitable for invivo pharmacodynamic and biological studies of MLL1-WDR5 interaction.

P53 inactivation unmasks histone methylation-independent WDR5 functions that drive self-renewal and differentiation of pluripotent stem cells.

Summaryp53 alterations occur during culture of pluripotent stem cells (PSCs), but the significance of these events on epigenetic control of PSC fate determination remains poorly understood. Wdr5 deletion in p53-null (DKO) mouse ESCs (mESCs) leads to impaired self-renewal, defective retinal neuroectoderm differentiation, and de-repression of germ cell/meiosis (GCM)-specific genes. Re-introduction of a WDR5 mutant with defective H3K4 methylation activity into DKO ESCs restored self-renewal and suppressed GCM gene expression but failed to induce retinal neuroectoderm differentiation. Mechanistically, mutant WDR5 targets chromatin that is largely devoid of H3K4me3 and regulates gene expression in p53-null mESCs. Furthermore, MAX and WDR5 co-target lineage-specifying chromatin and regulate chromatin accessibility of GCM-related genes. Importantly, MAX and WDR5 are core subunits of a non-canonical polycomb repressor complex 1 responsible for gene silencing. This function, together with canonical, pro-transcriptional WDR5-dependent MLL complex H3K4 methyltransferase activity, highlight how WDR5 mediates crosstalk between transcription and repression during mESC fate choice.

Differential effects of two catalytic mutations on full-length PRDM9 and its isolated PR/SET domain reveal a case of pseudomodularity.

PRDM9 is a DNA-binding histone methyltransferase that designates and activates recombination hotspots in mammals by locally trimethylating lysines 4 and 36 of histone H3. In mice, we recently reported two independently produced point mutations at the same residue, Glu360Pro (Prdm9EP) and Glu360Lys (Prdm9EK), which severely reduce its H3K4 and H3K36 methyltransferase activities in vivo. Prdm9EP is slightly less hypomorphic than Prdm9EK, but both mutations reduce both the number and amplitude of PRDM9-dependent H3K4me3 and H3K36me3 peaks in spermatocytes. While both mutations cause infertility with complete meiotic arrest in males, Prdm9EP, but not Prdm9EK, is compatible with some female fertility. When we tested the effects of these mutations in vitro, both Prdm9EP and Prdm9EK abolished H3K4 and H3K36 methyltransferase activity in full-length PRDM9. However, in the isolated PRDM9 PR/SET domain, these mutations selectively compromised H3K36 methyltransferase activity, while leaving H3K4 methyltransferase activity intact. The difference in these effects on the PR/SET domain vs the full-length protein shows that PRDM9 is not an intrinsically modular enzyme; its catalytic domain is influenced by its tertiary structure and possibly by its interactions with DNA and other proteins in vivo. These two informative mutations illuminate the enzymatic chemistry of PRDM9, and potentially of PR/SET domains in general, reveal the minimal threshold of PRDM9-dependent catalytic activity for female fertility, and potentially have some practical utility for genetic mapping and genomics.

SET DOMAIN GROUP 721 protein functions in saline-alkaline stress tolerance in the model rice variety Kitaake.

To isolate the genetic locus responsible for saline–alkaline stress tolerance, we developed a high‐throughput activation tagging‐based T‐DNA insertion mutagenesis method using the model rice (Oryza sativa L.) variety Kitaake. One of the activation‐tagged insertion lines, activation tagging 7 (AC7), showed increased tolerance to saline–alkaline stress. This phenotype resulted from the overexpression of a gene that encodes a SET DOMAIN GROUP 721 protein with H3K4 methyltransferase activity. Transgenic plants overexpressing OsSDG721 showed saline–alkaline stress‐tolerant phenotypes, along with increased leaf angle, advanced heading and ripening dates. By contrast, ossdg721 loss‐of‐function mutants showed increased sensitivity to saline–alkaline stress characterized by decreased survival rates and reduction in plant height, grain size, grain weight and leaf angle. RNA sequencing (RNA‐seq) analysis of wild‐type Kitaake and ossdg721 mutants indicated that OsSDG721 positively regulates the expression level of HIGH‐AFFINITY POTASSIUM (K+ ) TRANSPORTER1;5 (OsHKT1;5), which encodes a Na+‐selective transporter that maintains K+/Na+ homeostasis under salt stress. Furthermore, we showed that OsSDG721 binds to and deposits the H3K4me3 mark in the promoter and coding region of OsHKT1;5, thereby upregulating OsHKT1;5 expression under saline–alkaline stress. Overall, by generating Kitaake activation‐tagging pools, we established that the H3K4 methyltransferase OsSDG721 enhances saline–alkaline stress tolerance in rice.

Preparation of the ubiquitination-triggered active form of SETDB1 in Escherichia coli for biochemical and structural analyses.

Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an Escherichia coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that co-expression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E.coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1. Graphical Abstract.

Distinct kinetic mechanisms of H3K4 methylation catalyzed by MLL3 and MLL4 core complexes

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi–bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi–bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.

CHD7 regulates cardiovascular development through ATP-dependent and -independent activities

CHD7 encodes an ATP-dependent chromatin remodeling factor. Mutation of this gene causes multiple developmental disorders, including CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth/development, Genital abnormalities, and Ear anomalies) syndrome, in which conotruncal anomalies are the most prevalent form of heart defects. How CHD7 regulates conotruncal development remains unclear. In this study, we establish that deletion of Chd7 in neural crest cells (NCCs) causes severe conotruncal defects and perinatal lethality, thus providing mouse genetic evidence demonstrating that CHD7 cell-autonomously regulates cardiac NCC development, thereby clarifying a long-standing controversy in the literature. Using transcriptomic analyses, we show that CHD7 fine-tunes the expression of a gene network that is critical for cardiac NCC development. To gain further molecular insights into gene regulation by CHD7, we performed a protein-protein interaction screen by incubating recombinant CHD7 on a protein array. We find that CHD7 directly interacts with several developmental disorder-mutated proteins including WDR5, a core component of H3K4 methyltransferase complexes. This direct interaction suggested that CHD7 may recruit histone-modifying enzymes to target loci independently of its remodeling functions. We therefore generated a mouse model that harbors an ATPase-deficient allele and demonstrates that mutant CHD7 retains the ability to recruit H3K4 methyltransferase activity to its targets. Thus, our data uncover that CHD7 regulates cardiovascular development through ATP-dependent and -independent activities, shedding light on the etiology of CHD7-related congenital disorders. Importantly, our data also imply that patients carrying a premature stop codon versus missense mutations will likely display different molecular alterations; these patients might therefore require personalized therapeutic interventions.

Suppression of Mll1-Complex by Stat3/Cebpβ-Induced miR-21a/21b/181b Maintains the Accumulation, Homeostasis, and Immunosuppressive Function of Polymorphonuclear Myeloid-Derived Suppressor Cells.

Mixed-lineage leukemia 1 (MLL1), which exerts its H3K4 methyltransferase activity by interacting with WDR5, ASH2L, and RBBP5, plays a pivotal role in regulating hematopoietic stem cell homeostasis. Disrupting the integrity of MLL1-complex has been reported to be associated with acute leukemia. However, the exact role of MLL1-complex in myeloid cells is unknown. In this study, microarray analysis revealed that the core components of the Mll1-complex, Wdr5, Ash2l, and Mll1, were concurrently downregulated by tumor-secreted factors as well as GM-CSF + IL-6 during the accumulation and activation of murine myeloid-derived suppressor cells (MDSCs). These changes were further validated by quantitative RT-PCR and Western blotting both in vitro and in vivo. The expression levels of WDR5 and ASH2L were also significantly decreased in bone marrow MDSCs of lung cancer patients compared with that of healthy controls. Functionally, ectopic expression of Wdr5, Ash2l, and Mll1 (C terminus) reversed the accumulation and function of GM-CSF + IL-6-induced as well as tumor-cocultured polymorphonuclear MDSCs (PMN-MDSCs) by promoting them to differentiate into mature neutrophil-like cells. Mechanistically, GM-CSF + IL-6-activated Stat3 and Cebpβ synergistically induced the expression of miR-21a, miR-21b, and miR-181b, and thus inhibited the expression of Wdr5, Ash2l, and Mll1 by targeting to their 3' untranslated regions, respectively. Furthermore, knockdown of these microRNAs also suppressed the expansion and function of GM-CSF + IL-6-induced PMN-MDSCs. Taken together, our findings indicate that the Stat3/Cebpβ-miR-21a/b/181b-Mll1-complex axis may play a critical role in PMN-MDSC expansion, activation, and differentiation, and this axis may provide an effectively immunological therapeutic approach for patients with cancer or other immunological diseases.

Proton pump inhibitors selectively suppress MLL rearranged leukemia cells via disrupting MLL1-WDR5 protein-protein interaction

Genetic rearrangements of the mixed lineage leukemia (MLL) leading to oncogenic MLL-fusion proteins (MLL-FPs). MLL-FPs occur in about 10% of acute leukemias and are associated with dismal prognosis and treatment outcomes which emphasized the need for new therapeutic strategies. In present study, by a cell-based screening in-house compound collection, we disclosed that Rabeprazole specially inhibited the proliferation of leukemia cells harboring MLL-FPs with little toxicity to non-MLL cells. Mechanism study showed Rabeprazole down-regulated the transcription of MLL-FPs related Hox and Meis1 genes and effectively inhibited MLL1 H3K4 methyltransferase (HMT) activity in MV4-11 cells bearing MLL-AF4 fusion protein. Displacement of MLL1 probe from WDR5 protein suggested that Rabeprazole may inhibit MLL1 HMT activity through disturbing MLL1-WDR5 protein-protein interaction. Moreover, other proton pump inhibitors (PPIs) also indicated the inhibition activity of MLL1-WDR5. Preliminary SARs showed the structural characteristics of PPIs were also essential for the activities of MLL1-WDR5 inhibition. Our results indicated the drug reposition of PPIs for MLL-rearranged leukemias and provided new insight for further optimization of targeting MLL1 methyltransferase activity, the MLL1-WDR5 interaction or WDR5.

288-OR: Novel Epigenetic Mechanism for Maternal Exercise Effects on Offspring Metabolic Health

Obesity and type 2 diabetes in mothers can initiate a vicious cycle of multi-generational metabolic dysfunction. Conversely, maternal exercise (MatEx) in mice improves offspring metabolic health. We previously found that the mechanism for this critical benefit of MatEx involves multiple epigenetic changes inducing increased expression of TCA and fatty acid oxidation (FAO) genes in offspring liver, leading to enhanced liver function. Here, we hypothesize that maternal high fat diet (MatHFD) and MatEx result in histone modifications, an important means of epigenetic regulation. C57BL/6 female mice were fed chow or HFD, housed with (trained) or without (sedentary) running wheels for 2 weeks before breeding and through gestation. Offspring were chow fed and sedentary and livers studied up to age 52 weeks. Trimethylation of lysine 4 on histone H3 (H3K4me3), a hallmark of active transcription, was decreased in promoters of TCA and FAO genes in offspring livers from MatHFD. Importantly, this decrease was fully reversed in offspring livers from MatEx. In fact, offspring livers from MatEx had increased H3K4me3 in these genes as early as embryonic day 13.5 and at all times after birth. Altered H3K4me3 was associated with changes in H3K4 methyltransferase (HKMT) activity, as MatHFD decreased, and MatEx increased HKMT activity in offspring livers. MatHFD induced protein carbonylation in offspring liver, and MatEx prevented this harmful effect on protein function. LC-MS/MS showed that WDR82, a regulatory component of HKMT, was robustly carbonylated in offspring liver of MatHFD, an effect reversed by MatEx. Overexpression of WDR82 in offspring hepatoblasts from MatHFD recovered H3K4me3 levels and hepatic gene expression. In sum, these data demonstrate that maternal exercise reverses the detrimental effects of maternal HFD on H3K4me3 function, likely via protection of WDR82 from carbonylation. We have identified a novel epigenetic mechanism by which maternal exercise improves offspring metabolic health. Disclosure J. Kusuyama: None. A.B. Wagner: None. M.F. Hirshman: Stock/Shareholder; Self; Abbott Laboratories, AbbVie Inc., Amgen Inc., Colgate-Palmolive Company, Medtronic. L.J. Goodyear: None. Funding National Institutes of Health (to L.J.G.); Sunstar Foundation (to J.K.)

The identification of novel small-molecule inhibitors targeting WDR5-MLL1 interaction through fluorescence polarization based high-throughput screening

The protein-protein interaction between WDR5 (WD40 repeat protein 5) and MLL1 (mixed-lineage leukemia 1) is important for maintaining optimal H3K4 methyltransferase activity of MLL1. Dysregulation of MLL1 catalytic function is relevant to mixed-lineage leukemia, and targeting WDR5-MLL1 interaction could be a promising therapeutic strategy for leukemia harboring MLL1 fusion proteins. To date, several peptidomimetic and non-peptidomimetic small-molecule inhibitors targeting WDR5-MLL1 interaction have been reported, yet the discovery walk of new drugs inhibiting MLL1 methytransferase activity is still in its infancy. It’s urgent to find other small-molecule WDR5-MLL1 inhibitors with novel scaffolds. In this study, through fluorescence polarization (FP)-based high throughput screening, several small-molecule inhibitors with potent inhibitory activities in vitro against WDR5-MLL1 interaction were discovered. Nuclear Magnetic Resonance (NMR) assays were carried out to confirm the direct binding between hit compounds and WDR5. Subsequent similarity-based analog searching of the 4 hits led to several inhibitors with better activity, among them, DC_M5_2 displayed highest inhibitory activity with IC50 values of 9.63 ± 1.46 µM. Furthermore, a molecular docking study was performed and disclosed the binding modes and interaction mechanisms between two most potent inhibitors and WDR5.

Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability.

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and regulation of gene expression1–4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H35-11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help limit inappropriate H3K9me spreading12–15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me leading to aberrant gene silencing and epigenetic instability is not fully understood. Here we reveal an autoinhibited conformation in the conserved fission yeast S. pombe H3K9 methyltransferase Clr4/Suv39h that plays a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits its catalytic activity by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances Clr4 H3K9 methylation activity. Mutations predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains, and growth inhibition, demonstrating the importance of Clr4 intrinsic inhibition and auto-activation in regulating H3K9me deposition and preventing epigenetic instability. Conservation of the Clr4 autoinhibitory loop in other H3K9 methyltransferases, and automethylation of a corresponding lysine in the human SUV39H2 homolog16, suggest that the mechanism described here is broadly conserved.

Dissecting KMT2D missense mutations in Kabuki syndrome patients.

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.

MKL1 mediates TNF-α induced pro-inflammatory transcription by bridging the crosstalk between BRG1 and WDR5.

Tumor necrosis factor alpha (TNF-α) is a cytokine that can potently stimulate the synthesis of a range of pro-inflammatory mediators in macrophages. The underlying epigenetic mechanism, however, is underexplored. Here we report that the transcriptional modulator megakaryocytic leukemia 1 (MKL1) is associated with a histone H3K4 methyltransferase activity. Re-ChIP assay suggests that MKL1 interacts with and recruits WDR5, a component of the COMPASS complex responsible for H3K4 methylation, to the promoter regions of pro-inflammatory genes in macrophages treated with TNF-α. WDR5 enhances the ability of MKL1 to stimulate the promoter activities of pro-inflammatory genes. In contrast, silencing of WDR5 attenuates TNF-α induced production of pro-inflammatory mediators and erases the H3K4 methylation from the gene promoters. Of interest, the chromatin remodeling protein BRG1 also plays an essential role in maintaining H3K4 methylation on MKL1 target promoters by interacting with WDR5. MKL1 knockdown disrupts the interaction between BRG1 and WDR5. Together, our data illustrate a role for MKL1 in moderating the crosstalk between BRG1 and WDR5 to activate TNF-α induced pro-inflammatory transcription in macrophages.