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A combination of chemical and genetic approaches has established a proof of concept in mouse models—with strong mechanistic underpinning—indicating that targeting the aberrantly recruited histone methyltransferase activity of DOT1L has therapeutic potential in aggressive leukemias driven by MLL fusion genes.

We demonstrate that RING finger protein MSL2 in the MOF-MSL complex is a histone ubiquitin E3 ligase. MSL2, together with MSL1, has robust histone ubiquitylation activity that mainly targets nucleosomal H2B on lysine 34 (H2B K34ub), a site within a conserved basic patch on H2B tail. H2B K34ub by MSL1/2 directly regulates H3 K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells. The significance of MSL1/2 mediated histone H2B ubiquitylation is underscored by facts that MSL1/2 activity is important for transcription activation at HOXA9 and MEIS1 loci and that this activity is evolutionarily conserved in the Drosophila dosage compensation complex. Altogether, these results establish that the MOF-MSL complex possesses two distinct chromatin-modifying activities (i.e. H4 K16 acetylation and H2B K34 ubiquitylation) through MOF and MSL2 subunits. They also shed new lights on how intricate network of chromatin modifying enzymes functions coordinately in gene activation.

Epigenetic modifications have been reported in a number of non-germ-line tumor types. Epigenetic modifications to the genome, especially DNA methylation and histone modifications, affect gene expression causing increased risk for cancers and other diseases. We have summarized information about DNA methylation percentages in Glioblastoma multiforme (GBM) line HTB-12, alveolar cell carcinoma, and acute lymphocytic leukemia samples and determined H3 (K27) methyltransferase activity in GBM and leukemia cells and made comparisons to H3 (K27) methyltransferase activity in normal astrocyte, lung, and lymphocyte cells. GBM and alveolar cell carcinoma gDNA possessed lower gDNA methylation percentages compared to normal cells. Methyl-sensitive cut counting analysis (MSCC) showed fold decreases in GBM CpG methylation sites for genes PBK, KIF23, COL6A3, and LOX. There was no significant difference in CpG DNA methylation, but less histone methyltransferase activity in acute lymphocytic leukemia compared to normal cells. GBM possessed increased histone methyltransferase activity compared to normal samples. Challenges in the field in diagnosis and prognosis for cancer risk especially with regard to the results of this work are discussed.

The aim of the present study was to demonstrate that ribavirin, a known inhibitor of eIF4E and inosine 5'-phosphate dehydrogenase (IMPDH), also inhibits histone methyltransferase zeste homolog 2 (EZH2). A computational searching revealed that ribavirin has a high structural similarity to 3-deazaneplanocin A (DZNep). The growth inhibitory effects of ribavirin as well as its effects upon epigenetic enzymes were evaluated in various cancer cell lines. siRNA assays were used to downregulate eIF4E, EZH2 and IMPDH to determine the contribution of these targets to the growth inhibitory effects of ribavirin. Ribavirin decreased EZH2 expression, inhibited histone methyltransferase activity and decreased H3K27 trimethylation. Ribavirin induced variable growth inhibition in a number of cell lines and downregulation of the targets, EZH2, eIF4E and IMPDH1 and 2 by siRNA led to comparable growth inhibition while no significant further reduction in viability was observed when siRNA transfected cells were treated with ribavirin. The results showed that ribavirin inhibits these cancer targets and should thus be studied for cancer therapy.

Human cancer genome sequencing has recently revealed that genes that encode subunits of SWI/SNF chromatin remodeling complexes are frequently mutated across a wide variety of cancers, and several subunits of the complex have been shown to have bona fide tumor suppressor activity. However, whether mutations in SWI/SNF subunits result in shared dependencies is unknown. Here we show that EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is essential in all tested cancer cell lines and xenografts harboring mutations of the SWI/SNF subunits ARID1A, PBRM1, and SMARCA4, which are several of the most frequently mutated SWI/SNF subunits in human cancer, but that co-occurrence of a Ras pathway mutation is correlated with abrogation of this dependence. Notably, we demonstrate that SWI/SNF-mutant cancer cells are primarily dependent on a non-catalytic role of EZH2 in the stabilization of the PRC2 complex, and that they are only partially dependent on EZH2 histone methyltransferase activity. These results not only reveal a shared dependency of cancers with genetic alterations in SWI/SNF subunits, but also suggest that EZH2 enzymatic inhibitors now in clinical development may not fully suppress the oncogenic activity of EZH2.

The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. In this study, we report that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btd) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity. DOI: http://dx.doi.org/10.7554/eLife.03502.001, eLife digest Whereas the majority of cells in the brain are unable to divide to produce new cells, neural stem cells can divide numerous times and have the potential to become many different types of brain cells. However, between these two extremes there is another group of cells called neural progenitors. These cells can give rise to multiple types of neurons but, in contrast to stem cells, they can undergo only a limited number of divisions. Many of the molecular mechanisms by which stem cells give rise to progenitors are similar in mammals and in the fruit fly Drosophila. In the brains of fly larvae, a subset of neural stem cells called type II neuroblasts give rise to ‘intermediate neural progenitors’, each of which can divide between four and six times. Every division generates a replacement intermediate neural progenitor and a cell called a ganglion mother cell, which divides one last time to produce two brain cells. Thus, intermediate neural progenitors increase the overall output of cells derived from every division of a type II neuroblast. The ability of type II neuroblasts to generate intermediate neural progenitors is important for development. Loss of this ability will result in a shortage of cells, disrupting brain development, while the faulty generation of intermediate neural progenitors will result in the formation of tumors. Now, using Drosophila brain cells cultured in the laboratory, Komori et al. show that an evolutionarily conserved enzyme called Trithorax has an important role in maintaining this ability. Trithorax acts through a protein called Buttonhead. The role of Buttonhead in regulating intermediate neural progenitors has also been identified by Xie et al. Komori et al. show that type II neuroblasts that lack Trithorax activity lose their unique identity and behave as type I neuroblasts, which never generate intermediate neural progenitors. Trithorax maintains the cellular memory of a type II neuroblast by keeping regions of chromatin—a macromolecule made of DNA and proteins called histones—in an active state. These regions contain key genes, such as the gene for Buttonhead. Re-introducing Buttonhead in type II neuroblasts that lack Trithorax activity can reinstate their ability to produce intermediate neural progenitors. DOI: http://dx.doi.org/10.7554/eLife.03502.002

Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription.

In 2007, the Ubiquitously Transcribed Tetratricopeptide Repeat on chromosome X (UTX) was identified as a histone demethylase that specifically targets di- and tri-methyl groups on lysine 27 of histone H3 (H3K27me2/3). Since then, UTX has been proven essential during normal development, as it is critically required for correct reprogramming, embryonic development and tissue-specific differentiation. UTX is a member of the MLL2 H3K4 methyltransferase complex and its catalytic activity has been linked to regulation of HOX and RB transcriptional networks. In addition, an H3K27me2/3 demethylase independent function for UTX was uncovered in promoting general chromatin remodeling in concert with the BRG1-containing SWI/SNF remodeling complex. Constitutional inactivation of UTX causes a specific hereditary disorder called the Kabuki syndrome, whereas somatic loss of UTX has been reported in a variety of human cancers. Here, we compile the breakthrough discoveries made from the first disclosure of UTX as a histone demethylase till the identification of disease-related UTX mutations and specific UTX inhibitors.

Studies into posttranslational modifications of histones, notably acetylation, have yielded important insights into the dynamic nature of chromatin structure and its fundamental role in gene expression. The roles of other covalent histone modifications remain poorly understood. To gain further insight into histone methylation, we investigated its occurrence and pattern of site utilization in Tetrahymena , yeast, and human HeLa cells. In Tetrahymena , transcriptionally active macronuclei, but not transcriptionally inert micronuclei, contain a robust histone methyltransferase activity that is highly selective for H3. Microsequence analyses of H3 from Tetrahymena, yeast, and HeLa cells indicate that lysine 4 is a highly conserved site of methylation, which to date, is the major site detected in Tetrahymena and yeast. These data document a nonrandom pattern of H3 methylation that does not overlap with known acetylation sites in this histone. In as much as H3 methylation at lysine 4 appears to be specific to macronuclei in Tetrahymena , we suggest that this modification pattern plays a facilitatory role in the transcription process in a manner that remains to be determined. Consistent with this possibility, H3 methylation in yeast occurs preferentially in a subpopulation of H3 that is preferentially acetylated.

Hematopoietic stem and progenitor cells maintain blood homeostasis by giving rise to all mature blood cells and lymphocytes. A recent study identified the trithorax group gene ash1l as a critical regulator of quiescence and self-renewal potential in adult hematopoietic stem cells [1]. Ash1l encodes a large protein with histone methyltransferase activity that cooperates with the leukemia-associated gene Mll1 to regulate hematopoiesis in mice. Epigenetic modifiers have essential functions in normal hematopoiesis and are frequently dysregulated in hematological malignancies. Trithorax group (TrxG) genes were discovered in Drosophila melanogaster for their role in inducing normal body patterning by positively regulating Homeobox (Hox) gene expression [2]. Hox genes also play critical roles in mammalian development and in specific adult tissues, including the hematopoietic system. Although multiple members of the TrxG gene network interact genetically in flies [3], much less is known about their crosstalk and cooperativity in mammals. Mixed Lineage Leukemia 1 (MLL1) is the mammalian homolog of fly trithorax. MLL1 was originally discovered as a recurrent translocation partner in acute leukemias (reviewed in [4]). MLL1 maintains expression of HOX genes during development and regulates the function of normal hematopoietic stem cells. MLL1-driven leukemias are characterized by upregulated expression of HOXA cluster genes. Endogenous MLL1 contains a Su(var)3-9, Enhancer-of-zeste and Trithorax (SET) domain with H3K4 histone methyltransferase (HMT) activity that is lost in MLL1 fusion proteins [4]. MLL1 functions as part of a multiprotein complex that includes RBP4, WDR5, ASH2L, and the cofactor menin. In contrast, MLL1 fusion proteins consist of the N-terminal domain of MLL1 fused to an oncogenic partner. This complex also associates with the cofactor menin, but lacks intrinsic H3K4 HMT activity, which can be provided by endogenous MLL1 encoded by the non-rearranged allele [4]. In addition, the H3K79 histone methyltransferase DOT1L is recruited to the oncogenic complex at least in part through interactions with the MLL1 fusion partners AF10, AF4, AF9 or ENL [4]. Multiple research groups are focusing on targeting members of this complex in leukemia. Careful consideration must be given to disrupting oncogenic transformation while maintaining normal hematopoiesis, for example by targeting regulatory features of the complex that are unique or dominant in malignant cells. Besides MLL1, other TrxG members are poorly characterized in mammals. The TrxG gene ash1 (absent, small, or homeotic discs 1) was discovered in flies and shown to interact genetically with other TrxG genes [3]. Its mammalian homolog Ash1-like (Ash1l) encodes a large protein that contains a SET domain with in vitro H3K36 histone methyltransferase activity [5]. We recently discovered that Ash1l is a critical regulator of adult hematopoietic stem cells (HSCs) [1]. Ash1l-deficient mice generate normal numbers of fetal and neonatal long-term HSCs, but these cells become rapidly depleted in young adult mice. Phenotypically defined Ash1l-deficient HSCs display decreased quiescence and fail to give rise to long-term tri-lineage hematopoietic output after transplantation to irradiated recipients, indicating profoundly defective function. Moreover, Ash1l-deficient HSCs compete poorly for niche space, as evidenced by potent engraftment of wild-type HSCs in Ash1l-deficient recipients even in the absence of irradiation. Despite these defects, Ash1l-deficient mice maintain steady-state hematopoiesis and display enhanced self-renewal of progenitors downstream of HSCs. As observed for Mll1 in normal and malignant hematopoiesis, Ash1l regulates expression of Hoxa cluster genes [1]. Individual deficiency of Ash1l or Mll1 results in decreased but not abolished Hoxa expression [1, 6]. Functionally, inactivation of both Mll1 and Ash1l but not either gene alone leads to rapid hematopoietic failure [1]. Altogether, this is the first in vivo demonstration of cooperativity between mammalian TrxG proteins. More work is needed to define the critical pathways operating downstream of Ash1l and Mll1 in HSCs and the molecular mechanisms of their cooperative effects. As the field moves forward in targeting TrxG proteins in leukemia, careful consideration should be given to understanding how these proteins function during steady-state hematopoiesis in order to exploit critical structural and functional differences. For example, recent work showed that small molecular inhibitors of the menin/MLL1 interaction have substantial in vivo therapeutic activity in mouse models of MLL1-driven leukemia, but minimal negative impact on normal hematopoiesis [7]. Menin is an MLL1 cofactor that is essential for its recruitment to a subset of target genes [6] and is part of the MLL1 complex in normal hematopoiesis and leukemia. Combined deficiency of Men1 and Ash1l in mice results in bone marrow hypocellularity and HSC loss [1]. Thus, the modest impact of Men1 loss alone or menin/MLL1 disruption in normal hematopoiesis may result from compensatory activity of the TrxG gene Ash1l. It is also possible that individual molecular pathways differentially regulate recruitment of MLL1 and MLL1 fusion proteins to target genes, opening a therapeutic window. Systematic studies of TrxG gene networks in normal and malignant hematopoiesis could point to the Achilles' heel of MLL1-driven leukemia.

A subgroup of leukemogenic mixed-lineage leukemia (MLL) fusion proteins (MFPs) including MLL-AF9 activates the Mecom locus and exhibits extremely poor clinical prognosis. Mecom encodes EVI1 and MDS1-EVI1 (ME) proteins via alternative transcription start sites; these differ by the presence of a PRDI-BF1-RIZ1 (PR) domain with histone methyltransferase activity in the ME isoform. Using an ME-deficient mouse, we show that ME is required for MLL-AF9-induced transformation both in vitro and in vivo. And, although Nup98-HOXA9, MEIS1-HOXA9, and E2A-Hlf could transform ME-deficient cells, both MLL-AF9 and MLL-ENL were ineffective, indicating that the ME requirement is specific to MLL fusion leukemia. Further, we show that the PR domain is essential for MFP-induced transformation. These studies clearly indicate an essential role of PR-domain protein ME in MFP leukemia, suggesting that ME may be a novel target for therapeutic intervention for this group of leukemias.

Cancers of glial cells, known as gliomas, make up ~30% of all tumors of the brain and spinal cord. Pediatric gliomas, such as glioblastoma mutiforme (GBM) and diffuse intrinsic pontine gliomas (DIPG), are devastating brain tumors without effective therapies that most frequently occur in children. Recent advances in tumor sequencing have revealed an abundance of a particular somatic mutation in ~60% of pediatric GBMs and DIPGs in a protein involved in packaging DNA: histone H3. Now, Lewis et al. reveal that the H3-specific substitution of the amino acid lysine (K) for methionine (M) at position 27 (H3K27) results in a “poisonous” gain-of-function that causes global inhibition of H3K27 methylation in these tumors. In this newly discovered pathway, minor amounts of mutated histones become the cause of enzymatic impairment followed by global defects in histone methylation that may underlie disease pathology. Histones, which comprise the bulk of nuclear proteins, are required for packaging of the DNA into chromatin and for regulation of gene function. Posttranslational H3K27 methylation could be justly considered as a holy grail of gene regulation; it controls cell differentiation, cell division, and X-chromosome inactivation. The specific K to M substitution identified in brain tumors by the authors affects only a small fraction of total H3K27 residues. However, this relatively minor imperfection, as compared with the overall bulk of histone, turns out to be a powerful poison for global H3K27 methylation. Using an in vitro cell culture system, Lewis et al. showed that the presence of small amounts (~1%) of the mutant H3 extracted from human cancer cells was sufficient to nearly ablate endogenous H3K27me2/3 levels. This global effect on H3K27me was caused by the ability of the mutant histone to reduce the catalytic activity of the enzyme Ezh2, which is solely responsible for H3K27me2/3. Although this reduction in PRC2-dependent histone methyltransferase activity was restricted to the H3K27M mutation (none of the other K27X mutations tested caused a decrease in endogenous H3K27 methylation), Lewis et al. revealed a surprising conservation; the histone methyltransferase K to M mutation caused a similar inhibition at two additional, functionally important H3 lysine methylation sites. Although the H3K9M or H3K36M mutations have not yet been linked to human diseases, the conservation of the toxic gain-of-function mechanism exposes a chilling vulnerability of histone-dependent gene expression regulation. The detailed stoichiometry for the described events remains elusive because it is not clear how a H3 minority gains access to most of the cellular Ezh2. It cannot be entirely excluded that the “intoxicated” Ezh2 acquires novel properties that can amplify the inactivation reaction. With many questions to be answered, this newly discovered epigenetic paradigm reveals a potent regulatory circuitry in which histone gene mutation or histone RNA editing may provide powerful mechanisms for the regulation of gene expression in health and disease. P. W. Lewis et al. , Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science , published online 28 March 2013 (10.1126/science.1232245). [[Abstract]][1] [1]: http://www.sciencemag.org/content/early/2013/03/27/science.1232245

Colorectal cancer is among the leading causes of cancer death in the USA. The polycomb repressive complex 2 (PRC2), including core components SUZ12 and EZH2, represents a key epigenetic regulator of digestive epithelial cell physiology and was previously shown to promote deleterious effects in a number of human cancers, including colon. Using colon cancer stem cells (CCSC) isolated from human primary colorectal tumors, we demonstrate that SUZ12 knockdown and treatment with DZNep, one of the most potent EZH2 inhibitors, increase apoptosis levels, marked by decreased Akt phosphorylation, in CCSCs, while embryonic stem (ES) cell survival is not affected. Moreover, DZNep treatments lead to increased PTEN expression in these highly tumorigenic cells. Taken together, our findings suggest that pharmacological inhibition of PRC2 histone methyltransferase activity may constitute a new, epigenetic therapeutic strategy to target highly tumorigenic and metastatic colon cancer stem cells.

The role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.

Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes that regulate cancer cell growth and survival. It is overexpressed in hepatocellular carcinoma (HCC) with a clinical significance that remains obscure. Sorafenib, a multikinase inhibitor, has been used as a first-line therapeutic drug and shown clinical efficiency for advanced-stage HCC patients. In the present study, we found that sorafenib lowered the protein level of EZH2 through accelerating proteasome-mediated EZH2 degradation in hepatoma cells. Overexpression of EZH2 reversed sorafenib-induced cell growth arrest, cell cycle arrest, and cell apoptosis dependent on histone methyltransferase activity in hepatoma cells. More importantly, shRNA-mediated EZH2 knockdown or EZH2 inhibition with 3-deazaneplanocin A treatment promoted sorafenib-induced hepatoma cell growth arrest and apoptosis. Sorafenib altered the hepatoma epigenome by reducing EZH2 and H3K27 trimethylation. These results revealed a novel therapeutic mechanism underlying sorafenib treatment in suppressing hepatoma growth and survival by accelerating EZH2 degradation. Genetic deletion or pharmacological ablation of EZH2 made hepatoma cells more sensitive to sorafenib, which helps provide a strong framework for exploring innovative combined therapies for advanced-stage HCC patients.

Epigenetic regulators represent a new class of therapeutic targets for cancer [1]. Substantial studies suggest that the enhancer of zeste homolog 2 (EZH2) is one of such promising targets [2-4]. The current model of EZH2 oncogenic activity primarily focuses on its function as a subunit of Polycomb repressive complex 2 (PRC2), which silences gene expression via EZH2 histone methyltransferase activity [5,6]. Using a genome-wide approach we found that the oncogenic function of EZH2 in castration resistant prostate cancer (CRPC) is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a co-activator for critical transcription factors including the androgen receptor (AR). This functional switch is dependent on phosphorylation of EZH2, and requires an intact methyltransferase domain. Given that the loss-of-function mutations of EZH2 were observed in myelodysplastic syndrome and acute leukemia [7,8], our discovery of the non-PRC2 function of EZH2 in CRPC raises the potential to develop inhibitors that specifically target the EZH2 activation function while sparing its PRC2 repressive function to avoid the potential hematologic side effects. In addition, our finding that EZH2 cooperates with AR-associated complexes and requires phosphorylation to support CRPC growth suggests novel combination therapies for the treatment of metastatic, hormone-refractory prostate cancer.

Proper regulation of gene expression is essential for cellular viability, and the integrity of chromatin is a fundamental factor in maintaining proper gene regulation. In cancer cells, this precise regulation is disrupted and aberrant gene expression ensues - a hallmark of the disease. In many forms of hematological malignancy, aberrant expression, activation or deletion of a critical enzymatic regulator of chromatin modification underlies the disease. One such regulator is MMSET (multiple myeloma SET domain) protein, a histone methyltransferase, that is rearranged and joined with the immunoglobulin locus associated with t(4:14) due to aberrant class switch DNA recombination in approximately 20% of multiple myeloma cases and is associated with particularly poor prognosis. Our lab has previously shown that an increase in MMSET expression results in global histone modification changes and up-regulation of hundreds of genes. We believe that these changes are due to radical reprogramming of chromatin, including repositioning of nucleosomes in MMSET-overexpressed cells. Mechanistic insight into epigenetic changes during MMSET-mediated gene expression will provide deeper understanding of disease progression in multiple myeloma, and potentially other similar malignancies. To begin understanding the changes in chromatin structure when MMSET is overexpressed, we generated a genome wide nucleosome map in cells expressing high and low levels of MMSET. Initial analysis of this mapping reveals changes in nucleosome positioning in promoters where gene expression and histone modifications had been identified to be significantly altered in cells expressing high and low levels of MMSET. Generally, overexpression of MMSET results in an increase in H3K36 dimethylation, a histone modification associated with actively transcribed genes, and a concomitant genome-wide loss of H3K27 trimethylation, a histone modification associated with repressed genes. Indeed, in cells expressing high levels of MMSET, genes that have been identified to have an increase in expression and an increase in H3K36 dimethylation have a distinct change in nucleosome positioning compared to the cells expressing low levels of MMSET. Further, differences in nucleosome positioning can be detected in promoter regions with increased H3K27 trimethylation and decreased gene expression. We are currently examining whether these changes in nucleosome positioning are due to the presence of MMSET and require MMSET histone methyltransferase activity. Through these and related studies, our aim is to determine how MMSET behaves in multiple myeloma through a likely radical reprogramming of chromatin. We believe that mechanistic insight into MMSET-mediated gene expression changes will provide significant avenues for potential therapeutic strategies, as MMSET overexpression has been implicated in myeloma and other solid tumors. Moreover, our work will provide insight into mechanisms that may be shared amongst various diseases that are initiated through epigenetic misregulation.

Epigenetics, or the reversible and heritable marks of gene regulation not including DNA sequence, encompasses chromatin modifications on both the DNA and histones and is as important as the DNA sequence itself. Chromatin-modifying factors are playing an increasingly important role in tumorigenesis, particularly among pediatric rhabdomyosarcomas (RMS), revealing potential novel therapeutic targets. We performed an overexpression screen of chromatin-modifying factors in a KRAS(G12D)-driven zebrafish model for RMS. Here, we describe the identification of a histone H3 lysine 9 histone methyltransferase, SUV39H1, as a suppressor of embryonal RMS formation in zebrafish. This suppression is specific to the histone methyltransferase activity of SUV39H1, as point mutations in the SET domain lacked the effect. SUV39H1-overexpressing and control tumors have a similar proliferation rate, muscle differentiation state, and tumor growth rate. Strikingly, SUV39H1-overexpressing fish initiate fewer tumors, which results in the observed suppressive phenotype. We demonstrate that the delayed tumor onset occurs between 5 and 7 days post fertilization. Gene expression profiling at these stages revealed that in the context of KRAS(G12D) overexpression, SUV39H1 may suppress cell cycle progression. Our studies provide evidence for the role of SUV39H1 as a tumor suppressor.

LATS2, a pivotal Ser/Thr kinase of the Hippo pathway, plays important roles in many biological processes. LATS2 also function in Hippo-independent pathway, including mitosis, DNA damage response and epithelial to mesenchymal transition. However, the physiological relevance and molecular basis of these LATS2 functions remain obscure. To understand novel functions of LATS2, we constructed a LATS2 knockout HeLa-S3 cell line using TAL-effector nuclease (TALEN). Integrated omics profiling of this cell line revealed that LATS2 knockout caused genome-wide downregulation of Polycomb repressive complex 2 (PRC2) and H3K27me3. Cell-cycle analysis revealed that downregulation of PRC2 was not due to cell cycle aberrations caused by LATS2 knockout. Not LATS1, a homolog of LATS2, but LATS2 bound PRC2 on chromatin and phosphorylated it. LATS2 positively regulates histone methyltransferase activity of PRC2 and their expression at both the mRNA and protein levels. Our findings reveal a novel signal upstream of PRC2, and provide insight into the crucial role of LATS2 in coordinating the epigenome through regulation of PRC2.

Human colon cancer-initiating cells (CCICs) are responsible for the unrestrained cell proliferation and chemoresistance of colorectal tumors. Notch signaling is critical for the maintenance and self-renewal of cancer-initiating cells in colon cancer. We have shown that the serine-threonine kinase receptor-associated protein (STRAP), a ubiquitous WD40 protein, is involved in promoting tumorigenesis through activated MAPK/ERK pathway and pRb phosphorylation. In addition, our previous studies uncovered that STRAP is up-regulated in 60% colon carcinomas and behaves as an oncogene in a TGF-β-dependent or -independent manner. Based on our new finding that STRAP stabilizes intracellular fragment of Notch3 (ICN3) via post-translational modification, we sought to explore the cross-talk between STRAP and Notch pathway in regulating stemness of CCICs. Here, we have generated STRAP knock down (KD) colorectal cancer cell lines (RKO, DLD1, HCT116, HT29, WiDr, SW480, SW620, and LOVO) to show how STRAP abrogates apoptosis by up-regulating CD44+/CD133+ subpopulations of CCICs. Heterozygous Strap KO inhibits tumorigenesis accompanied by the reduction of stemness phenotype (CD44+, Lgr5+) in a colitis-associated mouse model. Western blot assays and immunofluorescene staining demonstrated that the expression of stemness biomarkers (Nanog, Oct4 and Sox2) is decreased in STRAP KD cells. Furthermore, the transcriptional activation of Notch pathway related genes is downregulated in the STRAP KD cells. EpiTect Chip qPCR Arrays profiled that 17 out of 84 genes key to Notch signal transduction had higher activating (H3K4me3) and lower repressive (H3K27me3) histone modifications in WT cells compared to STRAP KD cells. Among these genes, we identified two transcription factors, Hes1 and Hes5, that are transcriptionally regulated by STRAP as demonstrated by luciferase assays and qPCR. We further demonstrated that Hes1 and Hes5 were transcriptionally activated by STRAP through Polycomb Repressive Complex 2 (PRC2)-dependent mechanism. Immunoprecipitation experiments showed that STRAP interacts with the key components, EZH2 and SUZ12 of PRC2 complex in vivo, which antagonizes the histone methyltransferase activity in H3K27 on the promoters of Hes1 and Hes5, resulting in activating these two genes. Of interest, ectopic expression of either Hes1 or Hes5 can rescue the stemness phenotype in STRAP KD cells. Together, our findings provide a novel mechanistic insight into the regulation of CCICs via STRAP-PRC2-Hes1/5 regulatory axis, which represents a potential target for CCICs, thus overcoming cancer chemoresistance and relapse. Citation Format: Lin Jin, Trung Tam Vu, Pran K. Datta. STRAP mediates the stemness of human colorectal cancer cells by epigenetic regulation of Notch pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1709.

Epigenetic mechanisms, processes that alter gene expression without changes in DNA sequence, precisely regulate gene expression by dynamic remodeling of chromatin. Eukaryotic DNA is wrapped around a histone octamer (H3/H4 heterotetramer and two H2A/H2B dimers) to form the nucleosome, the fundamental building block of chromatin. Histone proteins are subject to covalent modifications that modulate the local structure of chromatin and influence gene expression by regulating the accessibility of gene loci to transcriptional machinery. The dynamic remodeling of chromatin through histone modifications by nuclear proteins including histone acetyltransferases, deacetylases, and methyltransferases, as well as by demethylases, modulate spatiotemporal gene expression during cartilage development, homeostasis and disease. Disruptor of telomeric silencing 1-like (DOT1L) is the main histone-modifying enzyme that catalyzes the methylation on lysine-79 of histone 3 (H3K79). The human protein DOT1L contains 1537 aminoacids, with its N-terminal part responsible for the methyltransferase activity. The remaining C-terminal part is involved in physical interactions with many transcription relevant proteins. The general function of DOT1L is to methylate H3K79 as a member of a large protein complex, which can influence the transcriptional state. DOT1L9s histone methyltransferase activity has been linked to active transcription, and plays a role in many biological processes including the DNA damage response, the cell cycle, embryonic development and cell reprogramming. In cancer biology, DOT1L is involved in MLL-rearranged leukemia and in other tumors. We reported a strong genetic association between polymorphisms in the human DOT1L gene and hip cartilage thickness as well as osteoarthritis (OA). Variations in the DOT1L gene are also associated with human height. Furthermore, we earlier demonstrated that silencing of Dot1L inhibited chondrogenic differentiation of murine progenitor cells. Interestingly, DOT1L-associated H3K79 methylation has been linked to the Wingless-type (Wnt) cascade. Gain and loss of function studies for Wnt signaling mediator beta-catenin or Wnt antagonists such as Frizzled related protein have demonstrated that tight regulation of this pathway is key to joint health. This could specifically identify DOT1L as an attractive target for joint diseases, more particularly for OA. DOT1L is an enzyme and, thus, can be pharmacologically modulated. We therefore hypothesized that DOT1L plays a major role in cartilage homeostasis by tightly regulating gene expression patterns of key signaling pathways, such as the Wnt cascade. New insights into the transcriptional mechanisms may contribute to the development of new epigenetics-based strategies for the treatment of joint diseases, adding a new way to modulate fundamental signaling pathways in joint disease and repair. Disclosure of Interest S. Monteagudo Grant/research support from: Marie Curie Fellowship

Histone methylation at specific lysine residues is a crucial regulatory process in transcriptional regulation. Using chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis, we found that the H3K9-me2 target gene JAK2 was an important factor during differentiation of the HL-60 promyelocytic leukemia cell line by all-trans-retinoic acid (ATRA) treatment. Here, we report that the H3K9 methyltransferase G9a negatively regulated JAK2 transcription in histone methyltransferase activity and in a YY1-dependent manner during ATRA-mediated leukemia cell differentiation. We found that G9a knockdown repressed ATRA-mediated HL-60 cell differentiation. We demonstrated that G9a interacts with YY1 and is recruited to the JAK2 promoter along with corepressors, including histone deacetylase, that induced H3K9-me2. Repression of JAK2 transcription by G9a decreased H3Y41 phosphorylation and promoted inhibition of the recently identified JAK2-H3Y41P-HP1α pathway-mediated leukemogenesis.

Colorectal cancer is ranked among the top leading causes of cancer death in industrialized populations. Polycomb group proteins, including Suz12 and Ezh2, are epigenetic regulatory proteins that act as transcriptional repressors of many differentiation-associated genes and are overexpressed in a large subset of colorectal cancers. Retinoic acid (RA) acts as a negative regulator of PcG actions in stem cells, but has shown limited therapeutic potential in some solid tumors, including colorectal cancer, in part because of retinoic acid receptor β silencing. Through treatment with RA, Suz12 shRNA knockdown, or Ezh2 pharmacological inhibition with 3-deazaneplanocin A (DZNep), we increased TRAIL-mediated apoptosis in human colorectal cancer cell lines. This increased apoptosis in human colon cancer cells after RA or DZNep treatment was associated with a ~2.5-fold increase in TNFRSF10B (DR5) transcript levels and a 42% reduction in the H3K27me3 epigenetic mark at the TNFRSF10B promoter after DZNep addition. Taken together, our findings indicate that pharmacological inhibition of Polycomb repressive complex 2 histone methyltransferase activity may constitute a new epigenetic therapeutic strategy to overcome RA non-responsiveness in a subset of colorectal tumors by increasing TRAIL-mediated apoptosis sensitivity.

Protein lysine methylation controls gene expression and repair of deoxyribonucleic acid in the nucleus but also occurs in the cytoplasm, where the role of this posttranslational modification is less understood. Members of the Smyd protein family of lysine methyltransferases are particularly abundant in the cytoplasm, with Smyd1 and Smyd2 being most highly expressed in the heart and in skeletal muscles. Smyd1 is a crucial myogenic regulator with histone methyltransferase activity but also associates with myosin, which promotes sarcomere assembly. Smyd2 methylates histones and non-histone proteins, such as the tumor suppressors, p53 and retinoblastoma protein, RB. Smyd2 has an intriguing function in the cytoplasm of skeletal myocytes, where it methylates the chaperone Hsp90, thus promoting the interaction of a Smyd2–methyl-Hsp90 complex with the N2A-domain of titin. This complex protects the sarcomeric I-band region and myocyte organization. We briefly summarize some novel functions of Smyd family members, with a focus on Smyd2, and highlight their role in striated muscles and cytoplasmic actions. We then provide experimental evidence that Smyd2 is also important for cardiac function. In the cytoplasm of cardiomyocytes, Smyd2 was found to associate with the sarcomeric I-band region at the titin N2A-domain. Binding to N2A occurred in vitro and in yeast via N-terminal and extreme C-terminal regions of Smyd2. Smyd2-knockdown in zebrafish using an antisense oligonucleotide morpholino approach strongly impaired cardiac performance. We conclude that Smyd2 and presumably several other Smyd family members are lysine methyltransferases which have, next to their nuclear activity, specific regulatory functions in the cytoplasm of heart and skeletal muscle cells. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

EZH2 plays an important role in stem cell renewal and maintenance by inducing gene silencing via its histone methyltransferase activity. Previously, we showed that EZH2 downregulation enhances neuron differentiation of human mesenchymal stem cells (hMSCs); however, the underlying mechanisms of EZH2-regulated neuron differentiation are still unclear. Here, we identify Smurf2 as the E3 ubiquitin ligase responsible for the polyubiquitination and proteasome-mediated degradation of EZH2, which is required for neuron differentiation. A ChIP-on-chip screen combined with gene microarray analysis revealed that PPARγ was the only gene involved in neuron differentiation with significant changes in both its modification and expression status during differentiation. Moreover, knocking down PPARγ prevented cells from undergoing efficient neuron differentiation. In animal model, rats implanted with intracerebral EZH2-knocked-down hMSCs or hMSCs plus treatment with PPARγ agonist (rosiglitazone) showed better improvement than those without EZH2 knockdown or rosiglitazone treatment after a stroke. Together, our results support Smurf2 as a regulator of EZH2 turnover to facilitate PPARγ expression, which is specifically required for neuron differentiation, providing a molecular mechanism for clinical applications in the neurodegenerative diseases.

Runx2, best known for its role in regulating osteoblast-specific gene expression, also plays an increasingly recognized role in prostate and breast cancer metastasis. Using the C4-2B/Rx2(dox) prostate cancer cell line that conditionally expressed Runx2 in response to doxycycline treatment, we identified and characterized G9a, a histone methyltransferase, as a novel regulator for Runx2 activity. G9a function was locus-dependent. Whereas depletion of G9a reduced expression of many Runx2 target genes, including MMP9, CSF2, SDF1, and CST7, expression of others, such as MMP13 and PIP, was enhanced. Physical association between G9a and Runx2 was indicated by co-immunoprecipitation, GST-pulldown, immunofluorescence, and fluorescence recovery after photobleaching (FRAP) assays. Since G9a makes repressive histone methylation marks and is primarily known as a corepressor, we further investigated the mechanism by which G9a functioned as a positive regulator for Runx2 target genes. Transient reporter assays indicated that the histone methyltransferase activity of G9a was not required for transcriptional activation by Runx2. Chromatin immunoprecipitation assays for Runx2 and G9a showed that G9a was recruited to endogenous Runx2 binding sites. We conclude that a subset of cancer-related Runx2 target genes require recruitment of G9a for their expression, but do not depend on its histone methyltransferase activity.

The mechanisms that give rise to androgen-independent prostate cancer (AIPC)are incompletely understood, resulting in a lack of treatment options for this fatal form of the disease. It is believed that most cases of AIPC are the result of constitutive activation of the androgen receptor (AR) pathway, leading us to hypothesize that chronic, prostatic infection could promote cancer progression as a result of pathogen virulence factors activating cell signaling pathways known to induce AR signaling. We found that humanherpesvirus-8 (HHV-8) infection of androgen-sensitive LNCaP cells promotes AI cell proliferation. Contrary to our initial hypothesis, we found that, despite enhanced AI cell proliferation, HHV-8-infected LNCaP (LNCaPv219) cells have decreased expression of both AR and AR-regulated genes such as prostate-specific antigen (PSA), implying that the phenotype induced by HHV-8 occurs concomitantly with decreased AR signaling. It is known that HHV-8 maintains latency by epigenetically suppressing lytic gene expression though histone methyltransferase activity by the Polycomb group protein EZH2, which coincidentally has been shown to be up-regulated in advanced, metastatic prostate cancer tissues. We found that increased EZH2 expression in LNCaPv219 cells results in epigenetic silencing of two tumor-suppressing proteins, microseminoprotein-(MSMB) and DAB2-interacting protein (DAB2IP). Additionally, we found thatLNCaPv219 cells have adopted a pro-mesenchymal phenotype, evidenced by increased expression of mesenchymal markers (N-cadherin and vimentin) and decreased expression of the epithelial marker E-cadherin. Collectively, these results suggest that HHV-8infection promotes an AI phenotype that mimics the EZH2-mediated transcriptional profile of poorly differentiated prostate tumors.

EZH2 (enhancer of zeste homolog 2) is the catalytic subunit of PRC2 (polycomb repressive complex 2), which mediates histone methyltransferase activity and functions as transcriptional repressor involved in gene silencing. EZH2 is involved in malignant transformation and biological aggressiveness of several human malignancies. We previously demonstrated that non-small cell lung cancers (NSCLCs) also overexpress EZH2 and that high expression of EZH2 correlates with poor prognosis. Growing evidence indicates that EZH2 may be an appropriate therapeutic target in malignancies, including NSCLCs. Recently, an S-adenosyl-l-homocysteine hydrolase inhibitor, 3-Deazaneplanocin A (DZNep), has been shown to deplete and inhibit EZH2. The aim of this study was to determine the effect of DZNep in NSCLC cells. Knockdown of EZH2 by small-interfering RNA (siRNA) resulted in decreased growth of four NSCLC cell lines. MTT assays demonstrated that DZNep treatment resulted in dose-dependent inhibition of proliferation in the NSCLC cell lines with a half maximal inhibitory concentration (IC50) ranging from 0.08 to 0.24 μM. Immortalized but non-cancerous bronchial epithelial and fibroblast cell lines were less sensitive to DZNep than the NSCLC cell lines. Soft agarose assays demonstrated that anchorage-independent growth was also reduced in all three NSCLC cell lines that were evaluated using this assay. Flow cytometry analysis demonstrated that DZNep induced apoptosis and G1 cell cycle arrest in NSCLC cells, which was partially associated with cyclin A decrease and p27(Kip1) accumulation. DZNep depleted cellular levels of EZH2 and inhibited the associated histone H3 lysine 27 trimethylation. These results indicated that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treatment of NSCLCs.

Polycomb group (PcG) and trithorax group (trxG) proteins are key regulators of homeotic genes and have central roles in cell proliferation, growth and development. In animals, PcG and trxG proteins form higher order protein complexes that contain SET domain proteins with histone methyltransferase activity, and are responsible for the different types of lysine methylation at the N-terminal tails of the core histone proteins. However, whether H3K4 methyltransferase complexes exist in Arabidopsis thaliana remains unknown. Here, we make use of the yeast two-hybrid system and the bimolecular fluorescence complementation assay to provide evidence for the self-association of the Arabidopsis thaliana SET-domain-containing protein SET DOMAIN GROUP 26 (SDG26), also known as ABSENT, SMALL, OR HOMEOTIC DISCS 1 HOMOLOG 1 (ASHH1). In addition, we show that the ASHH1 protein associates with SET-domain-containing sequences from two distinct histone lysine methyltransferases, the ARABIDOPSIS HOMOLOG OF TRITHORAX-1 (ATX1) and ASHH2 proteins. Furthermore, after screening a cDNA library we found that ASHH1 interacts with two proteins from the heat shock protein 40 kDa (Hsp40/DnaJ) superfamily, thus connecting the epigenetic network with a system sensing external cues. Our findings suggest that trxG complexes in Arabidopsis thaliana could involve different sets of histone lysine methyltransferases, and that these complexes may be engaged in multiple developmental processes in Arabidopsis.

JAK-STAT-activated gene expression is both rapid and transient and requires dynamic post-translational modification of the chromatin template. Previously, we showed that following IFN-γ treatment, trimethylation of histone H3 at lysine 79 (H3K79me3) is rapidly and highly induced in the 5′-end of the STAT1-dependent gene interferon regulatory factor 1 (IRF1), but the role of this histone modification was unexplored. Here we report that DOT1L, the non-SET domain containing methyltransferase that modifies Lys-79, is localized across IRF1 in the uninduced state and is not further recruited by IFN-γ induction. RNAi-mediated depletion of DOT1L prevents the induction of H3K79me3 and lowers the transcription of IRF1 2-fold, as expected. Surprisingly, STAT1 binding to its DNA recognition element near the IRF1 promoter is diminished 2-fold in the DOT1L-depleted cell line. In vivo and in vitro protein interaction assays reveal a DOT1L-STAT1 interaction. Domain mapping identifies the middle region of DOT1L (amino acids 580–1183) as the STAT1 interaction domain. Overexpression of the DOT1L STAT1 interaction domain represses IRF1 transcription (2-fold) and interferes with STAT1 DNA binding at IRF1 and endogenous DOT1L histone methyltransferase activity. Collectively, our findings reveal a novel STAT1-DOT1L interaction that is required for the regulation JAK-STAT-inducible gene expression.

Rhabdomyosarcoma (RMS) is a pediatric tumor of skeletal muscle that bears a strong resemblance to myoblasts, the undifferentiated but committed cells of skeletal muscle. The two most common subtypes of RMS, alveolar (ARMS) and embryonal (ERMS), exhibit different morphologies as well as generally affecting different patient populations, but both share the common characteristic of a failure to properly execute the myogenic transcriptional differentiation program.1 While the most common genetic lesion in ERMS is a loss of 11p15, ARMS are notable for frequently exhibiting a gene fusion between either PAX3 or PAX7 and the forkhead transcription factor FOXO1A, a fusion that results in abnormally strong expression of PAX targets.2

Polycomb repressive complex two (PRC2) has been implicated in embryonic stem (ES) cell pluripotency; however, the mechanistic roles of this complex are unclear. It was assumed that ES cells contain PRC2 with the same subunit composition as that identified in HeLa cells and Drosophila embryos. Here, we report that PRC2 in mouse ES cells contains at least three additional subunits: JARID2, MTF2, and a novel protein denoted esPRC2p48. JARID2, MTF2, and esPRC2p48 are highly expressed in mouse ES cells compared to differentiated cells. Importantly, knockdowns of JARID2, MTF2, or esPRC2p48 alter the level of PRC2-mediated H3K27 methylation and result in the expression of differentiation-associated genes in ES cells. Interestingly, expression of JARID2, MTF2, and esPRC2p48 together, but not individually, enhances Oct4/Sox2/Klf4-mediated reprograming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells, whereas knockdown or knockout of JARID2, MTF2, or esPRC2p48 significantly inhibits reprograming. JARID2, MTF2, and esPRC2p48 modulate H3K27 methylation and facilitate repression of lineage-associated gene expression when transduced into MEFs, and synergistically stimulate the histone methyltransferase activity of PRC2 in vitro. Therefore, these studies identify JARID2, MTF2, and esPRC2p48 as important regulatory subunits of PRC2 in ES cells and reveal critical functions of these subunits in modulating PRC2's activity and gene expression both in ES cells and during somatic cell reprograming.

Protection of the transcriptional "stemness" network is important to maintain a healthy hematopoietic stem cells (HSCs) compartment during the lifetime of the organism. Recent evidence shows that fundamental changes in the epigenetic status of HSCs might be one of the driving forces behind many age-related HSC changes and might pave the way for HSC malignant transformation and subsequent leukemia development, the incidence of which increases exponentially with age. Polycomb group (PcG) proteins are key epigenetic regulators of HSC cellular fate decisions and are often found to be misregulated in human hematopoietic malignancies. In this review, we speculate that PcG proteins balance HSC aging against the risk of developing cancer, since a disturbance in PcG genes and proteins affects several important cellular processes such as cell fate decisions, senescence, apoptosis, and DNA damage repair.

Absent, small, or homeotic disc1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addition, we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.

Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with transcription activation and is mainly catalyzed by the MLL/SET1 family histone methyltransferases. A common feature of the mammalian MLL/SET1 complexes is the presence of three core components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Unlike most other histone lysine methyltransferases, all four proteins are required for efficient H3 K4 methylation. Despite extensive efforts, mechanisms for how three core components regulate MLL/SET1 methyltransferase activity remain elusive. Here we show that a heterodimer of Ash2L and RbBP5 has intrinsic histone methyltransferase activity. This activity requires the highly conserved SPRY domain of Ash2L and a short peptide of RbBP5. We demonstrate that both Ash2L and the MLL1 SET domain are capable of binding to S-adenosyl-L- [methyl-(3)H] methionine in the MLL1 core complex. Mutations in the MLL1 SET domain that fail to support overall H3 K4 methylation also compromise SAM binding by Ash2L. Taken together, our results show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall catalysis of MLL1 mediated H3 K4 methylation. The results we describe here provide mechanistic insights for unique regulation of the MLL1 methyltransferase activity. It suggests that both Ash2L/RbBP5 and the MLL1 SET domain make direct contacts with the substrates and contribute to the formation of a joint catalytic center. Given the shared core configuration among all MLL/SET1 family HMTs, it will be interesting to test whether the mechanism we describe here can be generalized to other MLL/SET1 family members in the future.

The polycomb repressive complex 2 (PRC2) is the major methyltransferase for H3K27 methylation, a modification critical for maintaining repressed gene expression programs throughout development. It has been previously shown that PRC2 maintains histone methylation patterns during DNA replication in part through its ability to bind to H3K27me3. However, the mechanism by which PRC2 recognizes H3K27me3 is unclear. Here we show that the WD40 domain of EED, a PRC2 component, is a methyllysine histone-binding domain. The crystal structures of apo-EED and EED in complex respectively with five different trimethyllysine histone peptides reveal that EED binds these peptides via the top face of its β-propeller architecture. The ammonium group of the trimethyllysine is accommodated by an aromatic cage formed by three aromatic residues, while its aliphatic chain is flanked by a fourth aromatic residue. Our structural data provide an explanation for the preferential recognition of the Ala-Arg-Lys-Ser motif-containing trimethylated H3K27, H3K9, and H1K26 marks by EED over lower methylation states and other histone methyllysine marks. More importantly, we found that binding of different histone marks by EED differentially regulates the activity and specificity of PRC2. Whereas the H3K27me3 mark stimulates the histone methyltransferase activity of PRC2, the H1K26me3 mark inhibits PRC2 methyltransferase activity on the nucleosome. Moreover, H1K26me3 binding switches the specificity of PRC2 from methylating H3K27 to EED. In addition to determining the molecular basis of EED-methyllysine recognition, our work provides the biochemical characterization of how the activity of a histone methyltransferase is oppositely regulated by two histone marks.

The SmyD family represents a new class of chromatin regulators that is important in heart and skeletal muscle development. However, the critical questions regarding how they are regulated posttranslationally remain largely unknown. We previously suggested that the histone methyltransferase activity of SmyD1, a vital myogenic regulator, appears to be regulated by autoinhibition and that the possible hinge motion of the conserved C-terminal domain (CTD) might be central to the maintenance and release of the autoinhibition. However, the lack of direct evidence of the hinge motion has limited our further understanding of this autoinhibitory mechanism. Here, we report the crystal structure of full-length SmyD3 in complex with the methyltransferase inhibitor sinefungin at 1.7 A. SmyD3 has a two-lobed structure with the substrate binding cleft located at the bottom of a 15-A-deep crevice formed between the N- and C-terminal lobes. Comparison of SmyD3 and SmyD1 clearly suggests that the CTD can undergo a large hinge-bending motion that defines two distinct conformations: SmyD3 adopts a closed conformation with the CTD partially blocking the substrate binding cleft; in contrast, SmyD1 appears to represent an open form, where the CTD swings out by ∼ 12 A from the N-terminal lobe, forming an open cleft with the active site completely exposed. Overall, these findings provide novel structural insights into the mechanism that modulates the activity of the SmyD proteins and support the observation that a posttranslational activation, such as by molecular chaperon Hsp90, is required to potentiate the proteins.

Epigenetic abnormalities including abnormal histone methyltransferase activity contribute to breast cancer pathogenesis. An example is over expression of the polycomb repressive complex (PRC) 2 member enhancer of zeste homolog 2 (EZH2) which is linked to epigenetic silencing and poor prognosis. Recent evidence shows that S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors (AHI) such as 3-deazaneplanocin A (DZNep) modulate chromatin through indirect inhibition of histone methyltransferases including EZH2. We investigated the biological effects of AdoHcy hydrolase inhibition using DZNep and its structural analogues 3-deazaadenosine (DZA) and neplanocin A (Nep A) in breast cancer cells. EZH2 protein expression was decreased and dose dependent growth inhibition occurred with variable potencies in MCF7, MDA-MB-231 and SKBr3 breast cancer cells. Cellular proliferation was inhibited through G(2)/M cell cycle arrest and apoptosis. In addition breast cancer cells accumulated cytoplasmic lipid droplets in response to AdoHcy hydrolase inhibition consistent with a differentiating effect. Each analogue induced a similar pattern of biological activity against breast cancer cells but with differences in potency (DZA > DZNep > Nep A). Co-administration with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induced synergistic inhibition of breast cancer cell proliferation. Furthermore, the relatively AHI resistant human epidermal growth factor receptor 2 (HER2) positive cell line SKBr3 underwent synergistic growth inhibition in response to co-treatment with the HER2 directed therapeutic antibody trastuzumab. In conclusion, AHI induce growth inhibition, cell cycle arrest, apoptosis and differentiation in breast cancer cells and synergise with HDAC and HER2 inhibition. Targeting histone methyltransferase activity might be of therapeutic value in breast cancer.

In many animals and some fungi, mechanisms have been described that target unpaired chromosomes and chromosomal regions for silencing during meiotic prophase. These phenomena, collectively called “meiotic silencing,” target sex chromosomes in the heterogametic sex, for example, the X chromosome in male nematodes and the XY-body in male mice, and also target any other chromosomes that fail to synapse due to mutation or chromosomal rearrangement. Meiotic silencing phenomena are hypothesized to maintain genome integrity and perhaps function in setting up epigenetic control of embryogenesis. This review focuses on meiotic silencing in the nematode, Caenorhabditis elegans, including its mechanism and function(s), and its relationship to other gene silencing processes in the germ line. One hallmark of meiotic silencing in C. elegans is that unpaired/unsynapsed chromosomes and chromosomal regions become enriched for a repressive histone modification, dimethylation of histone H3 on lysine 9 (H3K9me2). Accumulation and proper targeting of H3K9me2 rely on activity of an siRNA pathway, suggesting that histone methyltransferase activity may be targeted/regulated by a small RNA-based transcriptional silencing mechanism.

LIM (Lin-11, Isl-1, Mec-3), RING (Really interesting new gene), PHD (Plant homology domain) and MYND (myeloid, Nervy, DEAF-1) domains are all zinc-binding domains that ligate two zinc ions. Unlike the better known classical zinc fingers, these domains do not bind DNA, but instead mediate interactions with other proteins. LIM-domain containing proteins have diverse functions as regulators of gene expression, cell adhesion and motility and signal transduction. RING finger proteins are generally associated with ubiquitination; the presence of such a domain is the defining feature of a class of E3 ubiquitin protein ligases. PHD proteins have been associated with SUMOylation but most recently have emerged as a chromatin recognition motif that reads the methylation state of histones. The function of the MYND domain is less clear, but MYND domains are also found in proteins that have ubiquitin ligase and/or histone methyltransferase activity. Here we review the structure-function relationships for these domains and discuss strategies to modulate their activity.

Background Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemias (AML), and in mixed phenotype acute leukemias in infancy - a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type (wt) MLL possesses histone methyltransferase activity and functions at the level of chromatin organization by affecting the expression of specific target genes. While numerous MLL fusion proteins exert a diverse array of functions, they ultimately serve to induce transcription of specific genes. Hence, acute lymphoblastic leukemias (ALL) with MLL mutations (MLL mu) exhibit characteristic gene expression profiles including high-level expression of HOXA cluster genes. Here, we aimed to relate MLL mutational status and tumor suppressor gene (TSG) methylation/expression in acute leukemia cell lines. Results Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLL mu and MLL wt acute leukemia cell lines. On average, 1.8/24 TSG were methylated in MLL mu AML cells, while 6.2/24 TSG were methylated in MLL wt AML cells. Hypomethylation and expression of the TSG BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLL mu AML cell lines. MLL wt AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3, confirming epigenetic silencing of these genes in MLL wt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLL mu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLL mu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.

High expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34+ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that down-modulation of BMI1 in cord blood CD34+ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34+ cells, down-modulation of BMI1 impaired long-term expansion, whereby self-renewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and self-renewal of normal and leukemic stem and progenitor cells, and that expression of BMI1 protects cells against oxidative stress.

Histone deacetylases, such as silent information regulator 2 (Sir2) and Rpd3 are involved in chromatin silencing and implicated in lifespan determination in several organisms. The yeast Dot1 gene encoding a histone methyltransferase affects localization of silencing proteins including Sir2, and plays an essential role in the repair of damaged DNA. However, it is not known whether an alteration of a histone methyltransferase activity influences lifespan or stress resistance, which is often associated with extended lifespan. Here we investigated whether the Drosophilagrappa (gpp) gene, a Dot1 homolog influences lifespan and stress resistance using transgenic flies overexpressing gpp and those bearing a partial loss-of-function mutation. Overexpression of gpp throughout the adult stage did not extend the lifespan, but significantly enhanced resistances when they were kept on medium containing 1% H(2)O(2), or those with poor nutrients. As well, gpp-overexpressing flies were behaviourally more active than control flies. We investigated whether gpp overexpression induced anti-oxidant genes, Catalase, Sod, Sod2, GstD2, dhd, TrxT and Trx-2. However, none of these genes was induced. A partial loss-of-function mutations in gpp dramatically reduced the lifespan under oxidative and caloric stresses. Taken together, these results demonstrated that gpp is required for normal lifespan and stress resistance, and that its overexpression increases stress resistance in Drosophila, without obvious induction of representative anti-oxidant genes.

Since its discovery as a novel protein some 10 years ago, many cellular functions of menin have been identified. However, which ones of these relate specifically to menin’s role as a tumor suppressor and which ones not remains unclear. Menin is predominantly nuclear and acts as a scaffold protein to regulate gene transcription by coordinating chromatin remodeling. It is implicated in both histone deacetylase and histone methyltransferase activity and, via the latter, regulates the expression of cell cycle kinase inhibitor and homeobox domain genes. TGF-B family members are key cytostatic molecules and menin is a facilitator of the transcriptional activity of their signaling molecules, the Smads, thereby ensuring appropriate control of cell proliferation and differentiation.

Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification.

INTRODUCTIONHistone methyltransferases catalyze the addition of one or more methyl groups to a specific lysine or arginine residue within histones. Currently, there is a great deal of interest in histone methyltransferases, because mutations and misregulation of the genes encoding these proteins have been linked to various cancers and other diseases. Many genes encoding putative histone methyltransferases have been identified in eukaryotes, but the proteins they encode have not been functionally characterized. This protocol describes an in vitro assay for histone methyltransferase activity that uses bacterial cell extracts in which expression of a methyltransferase of interest is induced. In many cases, purification of the enzyme is unnecessary, making this experiment ideal for pilot studies. Bacterial cell extract containing the methyltransferase of interest is incubated with S-adenosyl-L-[methyl-3H]-methionine and various histone substrates, many of which are commercially available. Incorporation of the methyl-3H can be measured easily by scintillation counting. The labeled substrate is visualized by SDS-polyacrylamide gel electrophoresis (PAGE) followed by fluorography. This allows the substrate specificity and activity of a histone methyltransferase of interest to be readily characterized.

The mechanisms that regulate the differentiation program of multipotential stem cells remain poorly understood. In order to define the cues that delineate endothelial commitment from precursors, we screened for candidate regulatory genes in differentiating mouse embryoid bodies. We found that the PR/SET domain protein, PRDM6, is enriched in flk1(+) hematovascular precursor cells using a microarray-based approach. As determined by 5′ RACE, full length PRDM6 protein contains a PR domain and four Krüpple-like zinc fingers. In situ hybridization in mouse embryos demonstrates staining of the primitive streak, allantois, heart, outflow tract, para-aortic splanchnopleura (P-Sp)/aorto-gonadal-mesonephric (AGM) region and yolk sac, all sites known to be enriched in vascular precursor cells. PRDM6 is also detected in embryonic and adult-derived endothelial cell lines. PRDM6 is co-localized with histone H4 and methylates H4-K20 (but not H3) in vitro and in vivo, which is consistent with the known participation of PR domains in histone methyltransferase activity. Overexpression of PRDM6 in mouse embryonic endothelial cells induces apoptosis by activating caspase-3 and inducing G1 arrest. PRDM6 inhibits cell proliferation as determined by BrdU incorporation in endothelial cells, but not in rat aortic smooth muscle cells. Overexpression of PRDM6 also results in reduced tube formation in cultured endothelial cells grown in Matrigel. Taken together, our data indicate that PRDM6 is expressed by vascular precursors, has differential effects in endothelial cells and smooth muscle cells, and may play a role in vascular precursor differentiation and survival by modulating local chromatin-remodeling activity within hematovascular subpopulations during development.

p33ING2 belongs to the ING-gene family that is involved in tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. Most functions are dependent on the tumor suppressor p53. p33ING2 was also shown to bind to trimethylated lysine 4 of histone H3. Here, we show that p33ING2 contains a transferable silencing function, which is independent of p53. p33ING2-mediated gene silencing is resistant to the HDAC-inhibitor trichostatin A indicating that p33ING2 uses a non-HDAC class I or II pathway for gene repression in reporter assays. In line with that we show that p33ING2 is associated with histone methyltransferase (HMT) activity in vitro and in vivo, methylating specifically histone H3. Interestingly, the specificity is distinct from the MeCP2-recruited HMT. Mutation or methylation of lysine 9, a mark well known for repression, abrogates histone methylation by MeCP2 but not by the p33ING2 complex. Instead, the ING2-associated HMT shows an increased methylation activity if lysine 9 is methylated. In contrast, mutation or methylation of lysine 4, a methylation preferentially detected at active genes, led to a reduction of the ING2-associated HMT. Notably, also p33ING1 recruits HMT activity suggesting a more general biochemical interaction between members of p33ING family and HMT activity. Deletion analyses revealed that the ING2 C-terminus recruits HMT activity, which correlates with silencing function.