The role of extracellular vesicles in cancer.

Extracellular vesicles (EVs), which include small EVs such as exosomes, play a critical role in intercellular communication and are produced by both cancer and non-cancer cells. Several studies have shown that cancer cells exploit various strategies to regulate the biogenesis, composition, and functions of EVs primarily to promote cancer progression. Given that exosomes originate from major sorting hubs at the limiting membrane of endosomes, they are central to a signaling network that connects external stimuli with intrinsic tumor cell features. Exosomes contain diverse repertoires of molecular cargos, such as proteins, lipids, and nucleic acids, which determine their heterogeneity and functional properties in cancer progression. Therefore, targeting exosome biogenesis will enhance our understanding of tumorigenesis and also promote the discovery of novel approaches for cancer therapy. In this chapter we summarize the machinery of exosome biogenesis and the local, distant, and systemic effects of exosomes released by cancer cells. Furthermore, we explore how these exosomes regulate the anti-tumor immune response and epigenetic mechanisms to sustain cancer progression and their implications in cancer prevention and treatment.

Epigenetic modifiers as inducer of bioactive secondary metabolites in fungi.

Scientists are making efforts to search for new metabolites as they are essential lead molecules for the drug discovery, much required due to the evolution of multi drug resistance and new diseases. Moreover, higher production of known drugs is required because of the ever growing population. Microorganisms offer a vast collection of chemically distinct compounds that exhibit various biological functions. They play a crucial role in safeguarding crops, agriculture, and combating several infectious ailments and cancer. Research on fungi have grabbed a lot of attention after the discovery of penicillin, most of the compounds produced by fungi under normal cultivation conditions are discovered and now rarely new compounds are discovered. Treatment of fungi with the epigenetic modifiers has been becoming very popular since the last few years to boost the discovery of new molecules and enhance the production of already known molecules. Epigenetic literally means above genetics that actually does not alter the genome but alter its expression by altering the state of chromatin from heterochromatin to euchromatin. Chromatin in heterochromatin state usually doesn't express because it is closely packed by histones in this state. Epigenetic modifiers loosen the packing of chromatin by inhibiting DNA methylation and histone deacetylation and thus permit the expression of genes that usually remain dormant. This study delves into the possibility of utilizing epigenetic modifying agents to generate pharmacologically significant secondary metabolites from fungi.

The Role of Epigenetic Modifier Mutations in Peripheral T-Cell Lymphomas.

Peripheral T-cell lymphomas (PTCLs) are a group of diseases with a low incidence, high degree of heterogeneity, and a dismal prognosis in most cases. Because of the low incidence of these diseases, there have been few therapeutic novelties developed over time. Nevertheless, this fact is changing presently as epigenetic modifiers have been shown to be recurrently mutated in some types of PTCLs, especially in the cases of PTCLs not otherwise specified (PTCL-NOS), T follicular helper (TFH), and angioimmunoblastic T-cell lymphoma (AITL). These have brought about more insight into PTCL biology, especially in the case of PTCLs arising from TFH lymphocytes. From a biological perspective, it has been observed that ten-eleven translocators (TET2) mutated T lymphocytes tend to polarize to TFH, while Tregs lose their inhibitory properties. IDH2 R172 was shown to have inhibitory effects on TET2, mimicking the effects of TET2 mutations, as well as having effects on histone methylation. DNA methyltransferase 3A (DNMT3A) loss-of-function, although it was shown to have opposite effects to TET2 from an inflammatory perspective, was also shown to increase the number of T lymphocyte progenitors. Aside from bringing about more knowledge of PTCL biology, these mutations were shown to increase the sensitivity of PTCLs to certain epigenetic therapies, like hypomethylating agents (HMAs) and histone deacetylase inhibitors (HDACis). Thus, to answer the question from the title of this review: We found the Achilles heel, but only for one of the Achilles.

Histone modifiers at the crossroads of oncolytic and oncogenic viruses.

Cancer is a disease caused by loss of regulatory processes that control the cell cycle, resulting in increased proliferation. The loss of control can deregulate both tumor suppressors and oncogenes. Apart from cell intrinsic gene mutations and environmental factors, infection by cancer-causing viruses also induces changes that lead to malignant transformation. This can be caused by both expression of oncogenic viral proteins and also by changes in cellular genes and proteins that affect the epigenome. Thus, these epigenetic modifiers are good therapeutic targets, and several epigenetic inhibitors are approved for the treatment of different cancers. In addition to small molecule drugs, biological therapies, such as antibodies and viral therapies, are also increasingly being used to treat cancer. An HSV-1-derived oncolytic virus is currently approved by the US FDA and the European Medicines Agency. Similarly, an adenovirus-based therapeutic is approved for use in China for some cancer types. Because viruses can affect cellular epigenetics, the interaction of epigenome-targeting drugs with oncogenic and oncolytic viruses is a highly significant area of investigation. Here, we will review the current knowledge about the impact of using epigenetic drugs in tumors positive for oncogenic viruses or as therapeutic combinations with oncolytic viruses.

Combination of the modulators of epigenetic machinery and specific cell signaling pathways as a promising approach for cell reprogramming.

During embryogenesis and further development, mammalian epigenome undergoes global remodeling, which leads to the emergence of multiple fate-restricted cell lines as well as to their further differentiation into different specialized cell types. There are multiple lines of evidence suggesting that all these processes are mainly controlled by epigenetic mechanisms such as DNA methylation, histone covalent modifications, and the regulation of ATP-dependent remolding of chromatin structure. Based on the histone code hypothesis, distinct chromatin covalent modifications can lead to functionally distinct chromatin structures and thus distinctive gene expression that determine the fate of the cells. A large amount of recently accumulated data showed that small molecule biologically active compounds that involved in the regulation of chromatin structure and function in discriminative signaling environments can promote changes in cells fate. These data suggest that agents that involved in the regulation of chromatin modifying enzymes combined with factors that modulate specific cell signaling pathways could be effective tools for cell reprogramming. The goal of this review is to gather the most relevant and most recent literature that supports this proposition.

Health-promoting role of dietary bioactive compounds through epigenetic modulations: a novel prophylactic and therapeutic approach.

The epigenome is an overall epigenetic state of an organism, which is as important as that of the genome for normal development and functioning of an individual. Epigenetics involves heritable but reversible changes in gene expression through alterations in DNA methylation, histone modifications and regulation of non-coding RNAs in cells, without any change in the DNA sequence. Epigenetic changes are owned by various environmental factors including pollution, microbiota and diet, which have profound effects on epigenetic modifiers. The bioactive compounds present in the diet mainly include curcumin, resveratrol, catechins, quercetin, genistein, sulforaphane, epigallocatechin-3-gallate, alkaloids, vitamins, and peptides. Bioactive compounds released during fermentation by the action of microbes also have a significant effect on the host epigenome. Besides, recent studies have explored the new insights in vitamin's functions through epigenetic regulation. These bioactive compounds exert synergistic, preventive and therapeutic effects when combined as well as when used with chemotherapeutic agents. Therefore, these compounds have potential of therapeutic agents that could be used as "Epidrug" to treat many inflammatory diseases and various cancers where chemotherapy results have many side effects. In this review, the effect of diet derived bioactive compounds through epigenetic modulations on in vitro and in vivo models is discussed.

The JmjC-domain protein NO66/RIOX-1 affects the balance between proliferation and maturation in acute myeloid leukemia.

As epigenetic regulators are frequently dysregulated in acute myeloid leukemia (AML) we determined expression levels of the JmjC-protein NO66 in AML cell lines and sub fractions of healthy human hematopoietic cells. NO66 is absent in the AML cell lines KG1/KG1a which consist of cells with the immature CD34+/CD38- phenotype and is regarded as a "stem cell-like" model system. Similarly, NO66 is not detectable in CD34+/CD38- cells purified from healthy donors but is clearly expressed in the more committed CD34+/CD38+ cell population. Loss of NO66 expression in KG1/KG1a cells is due to hyper-methylation of its promoter and is released by DNA-methyltransferase inhibitors. In KG1a cells stably expressing exogenous wild type (KG1a66wt) or enzymatically inactive mutant (KG1a66mut) NO66, respectively, the wild type protein inhibited proliferation and rDNA transcription. Gene expression profiling revealed that the expression of NO66 induces a transcriptional program enriched for genes with roles in proliferation and maturation (e.g.EPDR1, FCER1A, CD247, MYCN, SNORD13). Genes important for the maintenance of stem cell properties are downregulated (e.g. SIRPA, Lin28B, JAML). Our results indicate that NO66 induces lineage commitment towards myeloid progenitor cell fate and suggest that NO66 contributes to loss of stem cell properties.

Trichostatin A-Mediated Epigenetic Modulation Predominantly Triggers Transcriptomic Alterations in the Ex Vivo Expanded Equine Chondrocytes.

Epigenetic mechanisms of gene regulation are important for the proper differentiation of cells used for therapeutic and regenerative purposes. The primary goal of the present study was to investigate the impacts of 5-aza-2' deoxycytidine (5-AZA-dc)- and/or trichostatin A (TSA)-mediated approaches applied to epigenomically modulate the ex vivo expanded equine chondrocytes maintained in monolayer culture on the status of chondrogenic cytodifferentiation at the transcriptome level. The results of next-generation sequencing of 3' mRNA-seq libraries on stimulated and unstimulated chondrocytes of the third passage showed no significant influence of 5-AZA-dc treatment. Chondrocytes stimulated with TSA or with a combination of 5-AZA-dc+TSA revealed significant expressional decline, mainly for genes encoding histone and DNA methyltransferases, but also for other genes, many of which are enriched in canonical pathways that are important for chondrocyte biology. The TSA- or 5-AZA-dc+TSA-induced upregulation of expanded chondrocytes included genes that are involved in histone hyperacetylation and also genes relevant to rheumatoid arthritis and inflammation. Chondrocyte stimulation experiments including a TSA modifier also led to the unexpected expression incrementation of genes encoding HDAC3, SIRT2, and SIRT5 histone deacetylases and the MBD1 CpG-binding domain protein, pointing to another function of the TSA agent besides its epigenetic-like properties. Based on the transcriptomic data, TSA stimulation seems to be undesirable for chondrogenic differentiation of passaged cartilaginous cells in a monolayer culture. Nonetheless, obtained transcriptomic results of TSA-dependent epigenomic modification of the ex vivo expanded equine chondrocytes provide a new source of data important for the potential application of epigenetically altered cells for transplantation purposes in tissue engineering of the equine skeletal system.

Histone Deacetylases Cooperate with NF-κB to Support the Immediate Migratory Response after Zebrafish Pronephros Injury.

Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability to recover spontaneously from AKI; however, little progress has been made in the development of supportive treatments. Increasing evidence suggest that histone deacetylases (HDAC) and NF-κB promote the pathogenesis of AKI, and inhibition of Hdac activity has a protective effect in murine models of AKI. However, the role of HDAC at the early stages of recovery is unknown. We used the zebrafish pronephros model to study the role of epigenetic modifiers in the immediate repair response after injury to the tubular epithelium. Using specific inhibitors, we found that the histone deacetylase Hdac2, Hdac6, and Hdac8 activities are required for the repair via collective cell migration. We found that hdac6, hdac8, and nfkbiaa expression levels were upregulated in the repairing epithelial cells shortly after injury. Depletion of hdac6, hdac8, or nfkbiaa with morpholino oligonucleotides impaired the repair process, whereas the combined depletion of all three genes synergistically suppressed the recovery process. Furthermore, time-lapse video microscopy revealed that the lamellipodia and filopodia formation in the flanking cells was strongly reduced in hdac6-depleted embryos. Our findings suggest that Hdac activity and NF-κB are synergistically required for the immediate repair response in the zebrafish pronephros model of AKI, and the timing of HDAC inhibition might be important in developing supportive protocols in the human disease.

Dynamics in the expression of epigenetic modifiers and histone modifications in perinatal rat germ cells during de novo DNA methylation†.

Epigenetic reprogramming during perinatal germ cell development is essential for genomic imprinting and cell differentiation; however, the actors of this key event and their dynamics are poorly understood in rats. Our study aimed to characterize the expression patterns of epigenetic modifiers and the changes in histone modifications in rat gonocytes at the time of de novo DNA methylation. Using transgenic rats expressing Green Fluorescent Protein (GFP) specifically in germ cells, we purified male gonocytes by fluorescent activated cell sorting at various stages of perinatal development and established the transcriptomic profile of 165 epigenetic regulators. Using immunofluorescence on gonad sections, we tracked six histone modifications in rat male and female perinatal germ cells over time, including methylation of histone H3 on lysines 27, 9, and 4; ubiquitination of histone H2A on lysine119; and acetylation of histone H2B on lysine 20. The results revealed the dynamics in the expression of ten-eleven translocation enzymes and DNA methyltransferases in male gonocytes at the time of de novo DNA methylation. Moreover, our transcriptomic data indicate a decrease in histone ubiquitination and methylation coinciding with the beginning of de novo DNA methylation. Decreases in H2AK119Ub and H3K27me3 were further confirmed by immunofluorescence in the male germ cells but were not consistent for all H3 methylation sites examined. Together, our data highlighted transient chromatin remodeling involving histone modifications during de novo DNA methylation. Further studies addressing how these dynamic changes in histone posttranslational modifications could guide de novo DNA methylation will help explain the complex establishment of the male germ cell epigenome.

Current Understandings of Myeloid Differentiation Inducers in Leukemia Therapy.

Differentiation therapy using all-trans retinoic acid for acute promyelocytic leukemia (APL) is well established. Several attempts have been made to treat non-APL, AML patients by employing differentiation inducers, such as hypomethylating agents (HMAs), and low-dose cytarabine (Ara-C) (LDAC), with encouraging results. Other than HMAs and LDAC, various inducers of myeloid cell differentiation have been identified. This review describes and categorizes these inducers, which include glycosylation modifiers, epigenetic modifiers, vitamin derivatives, cytokines, and chemotherapeutic agents. Some of these inducers are currently being used in clinical trials. I highlight the potential applications of glycosylation modifiers and epigenetic modifiers, which are attracting increasing attention in their use as differentiation therapy against AML. Among the agents described in this review, epigenomic modifiers seem particularly promising, and particular attention should also be paid to glycosylation modifiers. These drugs may signal a new era for AML differentiation therapy.

Genetic and epigenetic stability of stem cells: Epigenetic modifiers modulate the fate of mesenchymal stem cells.

Stem cell research has progressed widely and has been receiving a considerable attention for its advantages and drawbacks. Despite their extensive therapeutic potential in regenerative medicine, they are debatable for their genetic and epigenetic stability. In fact lineage specific differentiation is mediated via epigenetic changes in DNA methylation, acetylation, histone modifications etc. Thus epigenetics plays an important role in stem cell biology. For therapeutic interventions stem cells need to be genetically and epigenetically stable for their maximum paracrine secretions for bringing about expected tissue repair and regeneration. In this review we have focused on the current status of genetic and epigenetic stability in stem cells and their importance in regenerative medicine. We have also touched upon the possibility of considering tissue resident mesenchymal stem cells as epigenetic modifiers. This is likely to open a new era in stem cell therapeutic intervention by reversing disease inducing epigenetic changes.

Gene Expression Analysis of Pediatric Acute Myeloid Leukemia Identified a Hyperactive ASXL1/BAP1 Axis Linked with Poor Prognosis and over Expressed Epigenetic Modifiers.

Genetic aberrations in the epigenome are rare in pediatric AML, hence expression data in epigenetic regulation and its downstream effect is lacking in childhood AML. Our pilot study screened epigenetic modifiers and its related oncogenic signal transduction pathways concerning clinical outcomes in a small cohort of pediatric AML in KSA. RNA from diagnostic BM biopsies (n = 35) was subjected to expression analysis employing the nCounter Pan-Cancer pathway panel. The patients were dichotomized into low ASXL1 (17/35; 49%) and high ASXL1 (18/35; 51%) groups based on ROC curve analysis. Age, gender, hematological data or molecular risk factors (FLT3 mutation/molecular fusion) exposed no significant differences across these two distinct ASXL1 expression groups (P > 0.05). High ASXL1 expression showed linkage with high expression of other epigenetic modifiers (TET2/EZH2/IDH1&2). Our data showed that high ASXL1 mRNA is interrelated with increased BRCA1 associated protein-1 (BAP1) and its target gene E2F Transcription Factor 1 (E2F1) expression. High ASXL1 expression was associated with high mortality {10/18 (56%) vs. 1/17; (6%) P < 0 .002}. Low ASXL1 expressers showed better OS {740 days vs. 579 days; log-rank P= < 0.023; HR 7.54 (0.98-54.1)}. The association between high ASXL1 expression and epigenetic modifiers is interesting but unexplained and require further investigation. High ASXL1 expression is associated with BAP1 and its target genes. Patients with high ASXL1 expression showed poor OS without any association with a conventional molecular prognostic marker.

Regulatory T Cells-Related Genes Are under DNA Methylation Influence.

Regulatory T cells (Tregs) exert a highly suppressive function in the immune system. Disturbances in their function predispose an individual to autoimmune dysregulation, with a predominance of the pro-inflammatory environment. Besides Foxp3, which is a master regulator of these cells, other genes (e.g., Il2ra, Ctla4, Tnfrsf18, Ikzf2, and Ikzf4) are also involved in Tregs development and function. Multidimensional Tregs suppression is determined by factors that are believed to be crucial in the action of Tregs-related genes. Among them, epigenetic changes, such as DNA methylation, tend to be widely studied over the past few years. DNA methylation acts as a repressive mark, leading to diminished gene expression. Given the role of increased CpG methylation upon Tregs imprinting and functional stability, alterations in the methylation pattern can cause an imbalance in the immune response. Due to the fact that epigenetic changes can be reversible, so-called epigenetic modifiers are broadly used in order to improve Tregs performance. In this review, we place emphasis on the role of DNA methylation of the genes that are key regulators of Tregs function. We also discuss disease settings that have an impact on the methylation status of Tregs and systematize the usefulness of epigenetic drugs as factors able to influence Tregs functions.

Inactivation of CREBBP expands the germinal center B cell compartment, down-regulates MHCII expression and promotes DLBCL growth.

The genes encoding the histone acetyl-transferases (HATs) CREB binding protein (CREBBP) and EP300 are recurrently mutated in the activated B cell-like and germinal center (GC) B cell-like subtypes of diffuse large B cell lymphoma (DLBCL). Here, we introduced a patient mutation into a human DLBCL cell line using CRISPR and deleted Crebbp and Ep300 in the GC B cell compartment of mice. CREBBP-mutant DLBCL clones exhibited reduced histone H3 acetylation, expressed significantly less MHCII, and grew faster than wild-type clones in s.c. and orthotopic xenograft models. Mice lacking Crebbp in GC B cells exhibited hyperproliferation of their GC compartment upon immunization, had reduced MHCII surface expression on GC cells, and developed accelerated MYC-driven lymphomas. Ep300 inactivation reproduced some, but not all, consequences of Crebbp inactivation. MHCII deficiency phenocopied the effects of CREBBP loss in spontaneous and serial transplantation models of MYC-driven lymphomagenesis, supporting the idea that the mutational inactivation of CREBBP promotes immune evasion. Indeed, the depletion of CD4+ T cells greatly facilitated the engraftment of lymphoma cells in serial transplantation models. In summary, we provide evidence that both HATs are bona fide tumor suppressors that control MHCII expression and promote tumor immune control; mutational inactivation of CREBBP, but not of EP300, has additional cell-intrinsic engraftment and growth-promoting effects.

Genome-Wide Identification of Epigenetic Regulators in Quercus suber L.

Modifications of DNA and histones, including methylation and acetylation, are critical for the epigenetic regulation of gene expression during plant development, particularly during environmental adaptation processes. However, information on the enzymes catalyzing all these modifications in trees, such as Quercus suber L., is still not available. In this study, eight DNA methyltransferases (DNA Mtases) and three DNA demethylases (DDMEs) were identified in Q. suber. Histone modifiers involved in methylation (35), demethylation (26), acetylation (8), and deacetylation (22) were also identified in Q. suber. In silico analysis showed that some Q. suber DNA Mtases, DDMEs and histone modifiers have the typical domains found in the plant model Arabidopsis, which might suggest a conserved functional role. Additional phylogenetic analyses of the DNA and histone modifier proteins were performed using several plant species homologs, enabling the classification of the Q. suber proteins. A link between the expression levels of each gene in different Q. suber tissues (buds, flowers, acorns, embryos, cork, and roots) with the functions already known for their closest homologs in other species was also established. Therefore, the data generated here will be important for future studies exploring the role of epigenetic regulators in this economically important species.

The success and the challenge of all-trans retinoic acid in the treatment of cancer.

All-trans retinoic acid (ATRA), an active metabolite of vitamin A, plays important roles in cell proliferation, cell differentiation, apoptosis, and embryonic development. The effects of ATRA are mediated by nuclear retinoid receptors as well as non-genomic signal pathway, such as MAPK and PKA. The great success of differentiation therapy with ATRA in acute promyelocytic leukemia (APL) not only improved the prognosis of APL but also spurred the studies of ATRA in the treatment of other tumors. Since the genetic and physiopathological simplicity of APL is not common in human malignancies, the combination of ATRA with other agents (chemotherapy, epigenetic modifiers, and arsenic trioxide, etc) had been extensively investigated in a variety of tumors. In this review, we will discuss in details about ATRA and its role in cancer treatment.

MicroRNA-495-3p functions as a tumor suppressor by regulating multiple epigenetic modifiers in gastric carcinogenesis.

MicroRNAs (miRNAs) engage in complex interactions with the machinery that controls the transcriptome and concurrently target multiple mRNAs. Here, we demonstrate that microRNA-495-3p (miR-495-3p) functions as a potent tumor suppressor by governing ten oncogenic epigenetic modifiers (EMs) in gastric carcinogenesis. From the large cohort transcriptome datasets of gastric cancer (GC) patients available from The Cancer Genome Atlas (TCGA) and the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), we were able to recapitulate 15 EMs as significantly overexpressed in GC among the 51 EMs that were previously reported to be involved in cancer progression. Computational target prediction yielded miR-495-3p, which targets as many as ten of the 15 candidate oncogenic EMs. Ectopic expression of miRNA mimics in GC cells caused miR-495-3p to suppress ten EMs, and inhibited tumor cell growth and proliferation via caspase-dependent and caspase-independent cell death processing. In addition, in vitro metastasis assays showed that miR-495-3p plays a role in the metastatic behavior of GC cells by regulating SLUG, vimentin, and N-cadherin. Furthermore, treatment of GC cells with 5-aza-2'-deoxcytidine restored miR-495-3p expression; sequence analysis revealed hypermethylation of the miR-495-3p promoter region in GC cells. A negative regulatory loop is proposed, whereby DNMT1, among ten oncogenic EMs, regulates miR-495-3p expression via hypermethylation of the miR-495-3p promoter. Our findings suggest that the functional loss or suppression of miR-495-3p triggers overexpression of multiple oncogenic EMs, and thereby contributes to malignant transformation and growth of gastric epithelial cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epigenetic modifiers reduce inflammation and modulate macrophage phenotype during endotoxemia-induced acute lung injury.

Acute lung injury (ALI) during sepsis is characterized by bilateral alveolar infiltrates, lung edema and respiratory failure. Here, we examined the efficacy the DNA methyl transferase (DNMT) inhibitor 5-Aza 2-deoxycytidine (Aza), the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), as well as the combination therapy of Aza and TSA (Aza+TSA) provides in the protection of ALI. In LPS-induced mouse ALI, post-treatment with a single dose of Aza+TSA showed substantial attenuation of adverse lung histopathological changes and inflammation. Importantly, these protective effects were due to substantial macrophage phenotypic changes observed in LPS-stimulated macrophages treated with Aza+TSA as compared with untreated LPS-induced macrophages or LPS-stimulated macrophages treated with either drug alone. Further, we observed significantly lower levels of pro-inflammatory molecules and higher levels of anti-inflammatory molecules in LPS-induced macrophages treated with Aza+TSA than in LPS-induced macrophages treated with either drug alone. The protection was ascribed to dual effects by an inhibition of MAPK-HuR-TNF and activation of STAT3-Bcl2 pathways. Combinatorial treatment with Aza+TSA reduces inflammation and promotes an anti-inflammatory M2 macrophage phenotype in ALI, and has a therapeutic potential for patients with sepsis.

An Insight into the Secondary Metabolism of Muscodor yucatanensis: Small-Molecule Epigenetic Modifiers Induce Expression of Secondary Metabolism-Related Genes and Production of New Metabolites in the Endophyte.

Muscodor spp. are proficient producers of bioactive volatile organic compounds (VOCs) with many potential applications. However, all members of this genus produce varying amounts and types of VOCs which suggests the involvement of epigenetics as a possible explanation. The members of this genus are poorly explored for the production of soluble compounds (extrolites). In this study, the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes from an endophyte, Muscodor yucatanensis Ni30, were cloned and sequenced. The PKS genes belonged to reduced, partially reduced, non-reduced, and highly reduced subtypes. Strains over-expressing PKS genes were developed through the use of small-molecule epigenetic modifiers (suberoylanilide hydroxamic acid (SAHA) and 5-azacytidine). The putative epigenetic variants of this organism differed considerably from the wild type in morphological features and cultural characteristics as well as metabolites that were produced. Each variant produced a different set of VOCs distinct from the wild type, and several VOCs including methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)hexane-2,4-diol and 2-carboxymethyl-3-n-hexylmaleic appeared in the variant strains, the production of which could be attributed to the activity of otherwise silent PKS genes. The bioactive extrolite brefeldin A was isolated and characterized from the wild type. However, this metabolite was not detected in EV-1, but instead, two other products were isolated and characterized as ergosterol and xylaguaianol C. Hence, M. yucatanensis has the genetic potential to produce several previously undetectable VOCs and organic solvent soluble products. It is also the case that small-molecule epigenetic modifiers can be used to produce stable variant strains of fungi with the potential to produce new molecules. Finally, this work hints to the prospect that the epigenetics of an endophytic microorganism can be influenced by any number of environmental and chemical factors associated with its host plant which may help to explain the enormous chemical diversity of secondary metabolic products found in Muscodor spp.