SET Domain Methyltransferases Research Articles

S-adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3

Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases.

Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor.

Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

Sulfur-Oxygen Chalcogen Bonding Mediates AdoMet Recognition in the Lysine Methyltransferase SET7/9.

Recent studies have demonstrated that carbon-oxygen (CH···O) hydrogen bonds have important roles in S-adenosylmethionine (AdoMet) recognition and catalysis in methyltransferases. Here, we investigate noncovalent interactions that occur between the AdoMet sulfur cation and oxygen atoms in methyltransferase active sites. These interactions represent sulfur-oxygen (S···O) chalcogen bonds in which the oxygen atom donates a lone pair of electrons to the σ antibonding orbital of the AdoMet sulfur atom. Structural, biochemical, and computational analyses of an asparagine mutation in the lysine methyltransferase SET7/9 that abolishes AdoMet S···O chalcogen bonding reveal that this interaction enhances substrate binding affinity relative to the product S-adenosylhomocysteine. Corroborative quantum mechanical calculations demonstrate that sulfonium systems form strong S···O chalcogen bonds relative to their neutral thioether counterparts. An inspection of high-resolution crystal structures reveals the presence of AdoMet S···O chalcogen bonding in different classes of methyltransferases, illustrating that these interactions are not limited to SET domain methyltransferases. Together, these results demonstrate that S···O chalcogen bonds contribute to AdoMet recognition and can enable methyltransferases to distinguish between substrate and product.

Allele-Specific Crispr Targeting Reveals Epigenetic and Phenotypic Effects of a MMSET Gain of Function Mutation Found in Relapsed Acute Lymphoblastic Leukemia

Chromatin regulators including methyltransferases are frequently disrupted by inactivating mutations in a wide spectrum of cancers. In contrast gain of function mutations that increase enzyme activity are much more rare and found in only two enzymes within the family of approximately 60 SET domain methyltransferases, MMSET and EZH2. Specific recurrent mutations in either MMSET or EZH2 are detected in hematological malignancies in conjunction with increased methylation of their histone substrates, H3K36 and H3K27, respectively. We and others characterized the MMSET E1099K mutant, which generates a clear molecular hallmark of increased dimethylation of H3K36 (H3K36me2). This gain of function also induces a corresponding decrease in trimethylation at H3K27 (H3K27me3). The altered balance between H3K36me2 and H3K27me3 mimics global chromatin effects that we previously reported in multiple myeloma lines that overexpress wild type MMSET due to the t(4:14) translocation. Research led by the Pediatric Cancer Genome Project has shown that MMSET E1099K is detected in ~10% of B-cell acute lymphoblastic leukemia (ALL) and particularly enriched in samples following disease relapse. Therefore, we hypothesize that this mutation is a driver of ALL progression and mediates resistance to therapy. To address this question, we have rescued or disrupted the endogenous E1099K mutation in cell lines from relapsed ALL and characterized the resulting molecular and phenotypic effects.The sequence context of the E1099K mutation allows allele-specific CRISPR targeting, which we have exploited to create isogenic lines that differ by MMSET E1099K status. We isolated subclones from three parental lines and sequencing analysis revealed distinct outcomes: indel mutations that disrupt the E1099K allele or rescue to WT by interchromosomal gene conversion. Comparing these subclones to non-targeted controls showed that loss of E1099K causes a 3-fold reduction in H3K36me2 levels and 5-fold increase in H3K27me3. Mass spectrometry-based measurement of methylation kinetics revealed that E1099K accelerates the rate constant of conversion from unmodified H3K36 to monomethylation. This corresponds to an increased ability of the mutant enzyme to turnover substrate in vitro. Because H3K36 and H3K27 methylation both contribute to transcriptional regulation, we compared expression profiles and identified a common set of genes overexpressed in cells lines harboring the mutant allele. These overexpressed genes included components of the SLIT/ROBO, WNT/Beta-Catenin, and cell adhesion pathways. Correlating with these expression changes, several phenotypic changes resulted from loss of E1099K, such as reduction in proliferation, colony-forming ability, and adhesive properties. Loss of E1099K also increased sensitivity to chemotherapeutic agents used to treat ALL, such as doxorubicin and dexamethasone. One gene consistently upregulated in the presence of E1099K was the transcription factor ETV1. In support of ETV1 being a key mediator of E1099K-driven phenotypes we found that parental lines treated with an ETV1 inhibitor displayed reduced viability and adhesion. To further our phenotypic analysis, subclones and parental lines have been tagged with luciferase or fluorescent markers to assess invasion in models of metastasis and allow in vivo monitoring of tumors. Collectively, we have developed gene-targeting reagents specific for the MMSET E1099K mutation and used these tools to show its impact on global chromatin environment and cell phenotypes. DisclosuresNo relevant conflicts of interest to declare.

SETDB2 Mediates Metabolic Stress in Liver

SET‐domain containing methyltransferases are important regulators of gene expression and metabolic homeostasis. SETDB2 is part of the Suv3‐9 group of SET domain methyltransferases, but little is known about its physiological role. Using adenoviral constructs for hepatic SETDB2 knockdown and overexpression, our studies reveal a role for SETDB2 in the glucocorticoid receptor (GR)‐mediated stress response of liver during starvation and tissue injury. SETDB2 is the most highly induced SET‐domain methyltransferase in livers of starved mice and its expression is robustly increased by glucocorticoids, both in vivo and in vitro. Furthermore, SETDB2 expression increases acutely with drug‐induced liver injury. Gene expression profiling of mouse primary hepatocytes deficient in SETDB2 shows blunted expression of stress‐related GR‐dependent genes. In vivo, hepatic SETDB2 knockdown also blunts expression of GR targets stimulated by the synthetic glucocorticoid dexamethasone (DEX). Glucocorticoids are known to exacerbate liver injury induced by CCl4; this injury is attenuated in mice with SETDB2 knockdown. SETDB2 overexpression induces stress‐related GR‐targets and causes liver damage, a phenotype not observed with a mutated SETDB2 construct predicted to lack methylation activity. Co‐IP studies show SETDB2 and GR interact in the nucleus following DEX treatment in hepatocytes. DEX also increases SETDB2 enrichment at liver GRE sites as shown by SETDB2‐ChIP experiments and studies are in progress to determine how SETDB2 alters GR activity. In summary, SETDB2 plays a role in mediating glucocorticoid‐mediated stress responses in liver likely through interaction with GR, thus influencing gene expression and metabolic homeostasis in stressed conditions.

Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN

Phosphatase and tensin homologue (PTEN), one of the well-characterized tumor suppressor proteins, counteracts the phosphatidylinositol 3-kinase-AKT pathway through its unique lipid phosphatase activity. The functions of PTEN are regulated by a variety of posttranslational modifications such as acetylation, oxidation, ubiquitylation, phosphorylation, and SUMOylation. However, methylation of PTEN has not been reported so far. In this study, we demonstrated that the oncogenic protein lysine methyltransferase SET and MYND domain containing 2 (SMYD2) methylates PTEN at lysine 313 in vitro and in vivo. Knockdown of SMYD2 suppressed the cell growth of breast cancer cells and attenuated phosphorylation levels of AKT, indicating that SMYD2-mediated methylation negatively regulates PTEN tumor suppressor activity and results in activation of the phosphatidylinositol 3-kinase-AKT pathway. Furthermore, PTEN protein with lysine 313 substitution diminished phosphorylation of PTEN at serine 380, which is known to inactivate tumor suppressor functions of PTEN. Taken together, our findings unveil a novel mechanism of PTEN dysregulation regulated by lysine methylation in human cancer.

The trxG family histone methyltransferase SET DOMAIN GROUP 26 promotes flowering via a distinctive genetic pathway.

Histone methylation is a major component in numerous processes such as determination of flowering time, which is fine-tuned by multiple genetic pathways that integrate both endogenous and environmental signals. Previous studies identified SET DOMAIN GROUP 26 (SDG26) as a histone methyltransferase involved in the activation of flowering, as loss of function of SDG26 caused a late-flowering phenotype in Arabidopsis thaliana. However, the SDG26 function and the underlying molecular mechanism remain largely unknown. In this study, we undertook a genetic analysis by combining the sdg26 mutant with mutants of other histone methylation enzymes, including the methyltransferase mutants Arabidopsis trithorax1 (atx1), sdg25 and curly leaf (clf), as well as the demethylase double mutant lsd1-like1 lsd1-like2 (ldl1 ldl2). We found that the early-flowering mutants sdg25, atx1 and clf interact antagonistically with the late-flowering mutant sdg26, whereas the late-flowering mutant ldl1 ldl2 interacts synergistically with sdg26. Based on microarray analysis, we observed weak overlaps in the genes that were differentially expressed between sdg26 and the other mutants. Our analyses of the chromatin of flowering genes revealed that the SDG26 protein binds at the key flowering integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/AGAMOUS-LIKE 20 (SOC1/AGL20), and is required for histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at this locus. Together, our results indicate that SDG26 promotes flowering time through a distinctive genetic pathway, and that loss of function of SDG26 causes a decrease in H3K4me3 and H3K36me3 at its target gene SOC1, leading to repression of this gene and the late-flowering phenotype.

PRMT1 Activates Leukemic Stem Cell Program in MLL-Rearranged Leukemia

In MLL-rearranged leukemia, the Mixed Lineage Leukemia (MLL) gene undergoes chromosomal translocation that results in the loss of C-terminal histone methyltransferase SET domain, whereas the N-terminal of MLL gene fuses in-frame with one of the 60 identified partner genes. The resultant MLL fusion proteins lead to a characteristic aberrant gene expression pattern in human acute myeloid and lymphoblastic leukemia. Epigenetic dysregulation mediated by MLL fusion proteins has been suggested to be a key event in MLL-rearranged leukemia. It has been demonstrated that MLL-EEN/PRMT1 oncogenic complex induces transformation of primary myeloid progenitors via introduction of aberrant H4R3me2 at target Hoxloci. PRMT1 is the predominant protein arginine methyltransferase in mammals and is responsible for over 85% of arginine methylation activity in mammalian cells. Dysregulation of PRMT1 has been implicated in different cancers such as leukemia, suggesting the expression of PRMT1 is positively correlated with cancer progression and clinical parameters. Nevertheless, the leukemogenic role of PRMT1 in the establishment of leukemic stem cell (LSC) remains unclear.Previously we have demonstrated that a MLL fusion protein, MLL-EEN, can strongly enhance the self-renewal ability of murine primary hematopoietic cells through multiple rounds of replating assays. We have created a conditional Mll-Een invertor mouse model (MllEen/+) in which the expression of fusion protein is restricted to hematopoietic progenitors. Immunophenotypic analysis demonstrated a significant increase in the immature myeloid cell population (c-kit+Mac-1+) in bone marrow of MllEen/+ mice, suggesting that the expression of Mll-Een induces the development of acute myeloid leukemia. We have also established an Mll-Een expressing cell line from the bone marrow of MllEen/+ mouse. These leukemic cells can persistently form colonies and they also demonstrated deregulation of Hox genes, which is frequently observed in human leukemia cases. The leukemogenicity of Mll-Een is closely associated with Prmt1, which was demonstrated through knockdown of Prmt1. Strikingly, we discovered a subpopulation of CD41+Mll-Een expressing cells, which showed enhanced self-renewal ability in the serial colony forming assays. The percentage of CD41+ leukemic cells is reduced once Prmt1 was knocked down, suggesting that Prmt1 is crucial in the maintenance of this subpopulation of cells. In addition, the CD41+ cells showed enhanced expression of genes associated with hematopoietic stem cell (HSC) activities (Bmi-1, Runx1, Tal-1 and Lmo2), implying that part of the HSC transcriptional program has been re-activated in these cells. We therefore speculate that the CD41+ cells may represent a group of MLL leukemic cells that harbors strong stem cell features, and presumably functions as LSCs. The CD41+ leukemic cells will be further characterized by their LSC functions and CD41 can potentially serve as a novel LSC marker in MLL-rearranged leukemia. Taken together, studies on the role of PRMT1 can provide novel insights on the establishment of LSC and the development of effective clinical treatment for MLL-rearranged leukemia. DisclosuresNo relevant conflicts of interest to declare.

The identification and structure of an N-terminal PR domain show that FOG1 is a member of the PRDM family of proteins.

FOG1 is a transcriptional regulator that acts in concert with the hematopoietic master regulator GATA1 to coordinate the differentiation of platelets and erythrocytes. Despite considerable effort, however, the mechanisms through which FOG1 regulates gene expression are only partially understood. Here we report the discovery of a previously unrecognized domain in FOG1: a PR (PRD-BF1 and RIZ) domain that is distantly related in sequence to the SET domains that are found in many histone methyltransferases. We have used NMR spectroscopy to determine the solution structure of this domain, revealing that the domain shares close structural similarity with SET domains. Titration with S-adenosyl-L-homocysteine, the cofactor product synonymous with SET domain methyltransferase activity, indicated that the FOG PR domain is not, however, likely to function as a methyltransferase in the same fashion. We also sought to define the function of this domain using both pulldown experiments and gel shift assays. However, neither pulldowns from mammalian nuclear extracts nor yeast two-hybrid assays reproducibly revealed binding partners, and we were unable to detect nucleic-acid-binding activity in this domain using our high-diversity Pentaprobe oligonucleotides. Overall, our data demonstrate that FOG1 is a member of the PRDM (PR domain containing proteins, with zinc fingers) family of transcriptional regulators. The function of many PR domains, however, remains somewhat enigmatic for the time being.

Manipulating unconventional CH-based hydrogen bonding in a methyltransferase via noncanonical amino acid mutagenesis.

Recent studies have demonstrated that the active sites of S-adenosylmethionine (AdoMet)-dependent methyltransferases form strong carbon-oxygen (CH···O) hydrogen bonds with the substrate's sulfonium group that are important in AdoMet binding and catalysis. To probe these interactions, we substituted the noncanonical amino acid p-aminophenylalanine (pAF) for the active site tyrosine in the lysine methyltransferase SET7/9, which forms multiple CH···O hydrogen bonds to AdoMet and is invariant in SET domain enzymes. Using quantum chemistry calculations to predict the mutation's effects, coupled with biochemical and structural studies, we observed that pAF forms a strong CH···N hydrogen bond to AdoMet that is offset by an energetically unfavorable amine group rotamer within the SET7/9 active site that hinders AdoMet binding and activity. Together, these results illustrate that the invariant tyrosine in SET domain methyltransferases functions as an essential hydrogen bonding hub and cannot be readily substituted by residues bearing other hydrogen bond acceptors.

The lysine methyltransferase SMYD3 interacts with hepatitis C virus NS5A and is a negative regulator of viral particle production

Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner.

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

WDR5, ASH2L, and RBBP5 control the efficiency of FOS transcript processing

H3K4 trimethylation is strongly associated with active transcription. The deposition of this mark is catalyzed by SET-domain methyltransferases, which consist of a subcomplex containing WDR5, ASH2L, and RBBP5 (the WAR subcomplex); a catalytic SET-domain protein; and additional complexspecific subunits. The ERK MAPK pathway also plays an important role in gene regulation via phosphorylation of transcription factors, co-regulators, or histone modifier complexes. However, the potential interactions between these two pathways remain largely unexplored. We investigated their potential interplay in terms of the regulation of the immediate early gene (IEG) regulatory network. We found that depletion of components of the WAR subcomplex led to increased levels of unspliced transcripts of IEGs that did not necessarily reflect changes in their mature transcripts. This occurs in a manner independent from changes in the H3K4me3 levels at the promoter region. We focused on FOS and found that the depletion of WAR subcomplex components affected the efficiency of FOS transcript processing. Our findings show a new aspect of WAR subcomplex function in coordinating active transcription with efficient pre-mRNA processing.Electronic Supplementary MaterialSupplementary material is available for this article at 10.2478/s11658-014-0190-8 and is accessible for authorized users.

Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.

Structural Context of Disease-Associated Mutations and Putative Mechanism of Autoinhibition Revealed by X-Ray Crystallographic Analysis of the EZH2-SET Domain

The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.

122 SUPPRESSION OF EPIGENETIC MODIFIERS ALTERS THE BOVINE EMBRYONIC DEVELOPMENTAL PROGRAM DURING IN VITRO CULTURE

During early bovine embryogenesis, the regular establishment of DNA methylation and histone modification patterns is essential for proper gene expression and continuation of embryonic development. Epigenome patterns established during this period, if improperly maintained, can lead to developmental anomalies and may partially explain the lower pregnancy rates of in vitro-produced embryos. We hypothesised that the suppression of translation of the genes euchromatic histone-lysine N-methyltransferase 2 (EHMT2), DNA methyltransferase 3A (DNMT3A), absent, small, or homeotic-like (ASH2L), and SET domain, bifurcated 1 (SETDB1) would provide insightful information on the importance of these genes during early embryonic development in an in vitro setting. In order to define the roles of these genes, small interfering RNA (siRNA) targeting the gene of interest were synthesised and target verified in bovine cell culture using quantitative real-time RT-PCR (RT-qPCR). We acquired matured bovine oocytes from commercial suppliers, followed by IVF by standard laboratory procedures. Eighteen hours post IVF, cumulus cells were removed and zygotes separated into 3 different treatment groups: non-injected controls (CNTL), non-targeting siRNA injected controls (siNULL), and injection with siRNA targeting the gene of interest (si “gene target”). Each siRNA was mixed with a green fluorescent dextran at a concentration of 20 μM and ~100 pL injected cytoplasmically. The green fluorescent dextran was used to give visual confirmation that zygotes were indeed injected. Post-injection, fluorescent embryos were separated and cultured in Bovine Evolve (Zentih Biotech) medium supplemented with 4 mg mL–1 of BSA (Probumin, Millipore). Cleavage rates were monitored on Day 2, and only cleaved embryos were cultured further. On Day 8 post-IVF, embryos were morphologically examined and numbers of blastocysts recorded. Mean development rates between siNULL and targeting siRNA were compared using a t-test statistic. Over the course of these experiments the mean blastocyst rate for CNTL zygotes was 34.5% ± 2.6 s.e.m. (n = 1647). None of the zygotes injected with siEHMT2 (n = 1184) or siSETDB1 (n = 361) reached the blastocyst stage and these rates differed from the siNULL rate (21.0% ± 2.5 s.e.m., n = 1587; P < 0.05). Morphologically, embryos from both groups developed to the morula stage before they exhibited fragmentation. Injection of siDNMT3A also resulted in significant loss of viability at the 8-cell stage and few zygotes injected (n = 1057) developed to blastocyst (2.1% ± 0.5 s.e.m.; P < 0.001). Inhibiting gene expression of ASH2L showed little variation in blastocyst rate from our siNULL embryos (31.3% ± 2.0 s.e.m., n = 466 v. 34.8% ± 1.9 s.e.m., n = 418, respectively, P > 0.2). It is unknown at this time if inhibition of ASH2L translation will have effects later in development. Ongoing experiments analysing DNA methylation and histone modifications through immunocytochemistry and global gene expression via RT-qPCR will further explore the establishment and maintenance of these genes in the embryonic epigenome.

Horizontal gene transfer of epigenetic machinery and evolution of parasitism in the malaria parasite Plasmodium falciparum and other apicomplexans

BackgroundThe acquisition of complex transcriptional regulatory abilities and epigenetic machinery facilitated the transition of the ancestor of apicomplexans from a free-living organism to an obligate parasite. The ability to control sophisticated gene expression patterns enabled these ancient organisms to evolve several differentiated forms, invade multiple hosts and evade host immunity. How these abilities were acquired remains an outstanding question in protistan biology.ResultsIn this work, we study SET domain bearing genes that are implicated in mediating immune evasion, invasion and cytoadhesion pathways of modern apicomplexans, including malaria parasites. We provide the first conclusive evidence of a horizontal gene transfer of a Histone H4 Lysine 20 (H4K20) modifier, Set8, from an animal host to the ancestor of apicomplexans. Set8 is known to contribute to the coordinated expression of genes involved in immune evasion in modern apicomplexans. We also show the likely transfer of a H3K36 methyltransferase (Ashr3 from plants), possibly derived from algal endosymbionts. These transfers appear to date to the transition from free-living organisms to parasitism and coincide with the proposed horizontal acquisition of cytoadhesion domains, the O-glycosyltransferase that modifies these domains, and the primary family of transcription factors found in apicomplexan parasites. Notably, phylogenetic support for these conclusions is robust and the genes clearly are dissimilar to SET sequences found in the closely related parasite Perkinsus marinus, and in ciliates, the nearest free-living organisms with complete genome sequences available.ConclusionsAnimal and plant sources of epigenetic machinery provide new insights into the evolution of parasitism in apicomplexans. Along with the horizontal transfer of cytoadhesive domains, O-linked glycosylation and key transcription factors, the acquisition of SET domain methyltransferases marks a key transitional event in the evolution to parasitism in this important protozoan lineage.

Arabidopsis histone methyltransferase SET DOMAIN GROUP2 is required for regulation of various hormone responsive genes

Histone modifications are known to play important roles in plant development through epigenetic regulation of gene expression. How these modifications regulate downstream targets in response to various environmental cues and developmental stimuli is still largely unknown. Here, we provide evidence that Arabidopsis histone H3K4 methyltransferase SET DOMAIN GROUP2 (SDG2) is required for full activation of hormone responsive genes upon hormone treatment. The pleiotropic phenotypes of sdg2 were closely related to those of auxin deficient mutants and RNA analysis revealed that expression of early hormone responsive genes was significantly reduced in sdg2-5. By ChIP analyses we found that H3K4 tri-methylations on chromatin region of hormone responsive genes such as SAUR27, KIN1 and GASA6 were enriched in WT upon hormone treatments whereas these enrichments were largely abolished in sdg2-5. After hormone treatment, chromatin regions of responsive genes that accumulated H3K4me3 in WT overlapped with those displaying decreased H3K4me3 levels in sdg2-5. Histone H3K4 di-methylation levels on tested genes were increased rather than decreased in sdg2-5, suggesting that SDG2 mediates transition of H3K4me2 to H3K4me3. Taken together, we conclude that the SDG2 activity is required to regulate the expression of hormone responsive genes via histone H3K4 tri-methylation.

Ordered changes in histone modifications at the core of the Arabidopsis circadian clock

Circadian clock function in Arabidopsis thaliana relies on a complex network of reciprocal regulations among oscillator components. Here, we demonstrate that chromatin remodeling is a prevalent regulatory mechanism at the core of the clock. The peak-to-trough circadian oscillation is paralleled by the sequential accumulation of H3 acetylation (H3K56ac, K9ac), H3K4 trimethylation (H3K4me3), and H3K4me2. Inhibition of acetylation and H3K4me3 abolishes oscillator gene expression, indicating that both marks are essential for gene activation. Mechanistically, blocking H3K4me3 leads to increased clock-repressor binding, suggesting that H3K4me3 functions as a transition mark modulating the progression from activation to repression. The histone methyltransferase SET DOMAIN GROUP 2/ARABIDOPSIS TRITHORAX RELATED 3 (SDG2/ATXR3) might contribute directly or indirectly to this regulation because oscillator gene expression, H3K4me3 accumulation, and repressor binding are altered in plants misexpressing SDG2/ATXR3. Despite divergences in oscillator components, a chromatin-dependent mechanism of clock gene activation appears to be common to both plant and mammal circadian systems.

Notch1 counteracts WNT/β-catenin signaling through chromatin modification in colorectal cancer

Crosstalk between the Notch and wingless-type MMTV integration site (WNT) signaling pathways has been investigated for many developmental processes. However, this negative correlation between Notch and WNT/β-catenin signaling activity has been studied primarily in normal developmental and physiological processes in which negative feedback loops for both signaling pathways are intact. We found that Notch1 signaling retained the capability of suppressing the expression of WNT target genes in colorectal cancers even when β-catenin destruction by the adenomatous polyposis coli (APC) complex was disabled. Activation of Notch1 converted high-grade adenoma into low-grade adenoma in an Apcmin mouse colon cancer model and suppressed the expression of WNT target genes in human colorectal cancer cells through epigenetic modification recruiting histone methyltransferase SET domain bifurcated 1 (SETDB1). Extensive microarray analysis of human colorectal cancers also showed a negative correlation between the Notch1 target gene, Notch-regulated ankyrin repeat protein 1 (NRARP), and WNT target genes. Notch is known to be a strong promoter of tumor initiation, but here we uncovered an unexpected suppressive role of Notch1 on WNT/β-catenin target genes involved in colorectal cancer.