Level Of Epigenetic Regulation Research Articles

Quercetin alleviates diastolic dysfunction and suppresses adverse pro-hypertrophic signaling in diabetic rats.

Quercetin (Que) is a potent anti-inflammatory and antioxidant flavonoid with cardioprotective potential. However, very little is known about the signaling pathways and gene regulatory proteins Que may interfere with, especially in diabetic cardiomyopathy. Therefore, we aimed to study the potential cardioprotective effects of Que on the cardiac phenotype of type 2 diabetes mellitus (T2DM) accompanied by obesity. For this experiment, we used Zucker Diabetic Fatty rats (fa/fa) and their age-matched lean controls (fa/+) that were treated with either vehicle or 20 mg/kg/day of Que for 6 weeks. Animals underwent echocardiographic (echo) examination before the first administration of Que and after 6 weeks. After the initial echo examination, the diabetic rats showed increased E/A ratio, a marker of left ventricular (LV) diastolic dysfunction, in comparison to the control group which was selectively reversed by Que. Following the echo analysis, Que reduced LV wall thickness and exhibited an opposite effect on LV luminal area. In support of these results, the total collagen content measured by hydroxyproline assay was decreased in the LVs of diabetic rats treated with Que. The follow-up immunoblot analysis of proteins conveying cardiac remodeling pathways revealed that Que was able to interfere with cardiac pro-hypertrophic signaling. In fact, Que reduced relative protein expression of pro-hypertrophic transcriptional factor MEF2 and its counter-regulator HDAC4 along with pSer246-HDAC4. Furthermore, Que showed potency to decrease GATA4 transcription factor, NFAT3 and calcineurin, as well as upstream extracellular signal-regulated kinase Erk5 which orchestrates several pro-hypertrophic pathways. In summary, we showed for the first time that Que ameliorated pro-hypertrophic signaling on the level of epigenetic regulation and targeted specific upstream pathways which provoked inhibition of pro-hypertrophic signals in ZDF rats. Moreover, Que mitigated T2DM and obesity-induced diastolic dysfunction, therefore, might represent an interesting target for future research on novel cardioprotective agents.

Genetic and epigenetic regulation of growth, reproduction, disease resistance and stress responses in aquaculture.

Major progress has been made with genomic and genetic studies in aquaculture in the last decade. However, research on epigenetic regulation of aquaculture traits is still at an early stage. It is apparent that most, if not all, aquaculture traits are regulated at both genetic and epigenetic levels. This paper reviews recent progress in understanding of genetic and epigenetic regulation of important aquaculture traits such as growth, reproduction, disease resistance, and stress responses. Although it is challenging to make generalized statements, DNA methylation is mostly correlated with down-regulation of gene expression, especially when at promoters and enhancers. As such, methylation of growth factors and their receptors is negatively correlated with growth; hypomethylation of genes important for stress tolerance is correlated with increased stress tolerance; hypomethylation of genes important for male or female sex differentiation leads to sex differentiation into males or females, respectively. It is apparent that environmental regulation of aquaculture traits is mediated at the level of epigenetic regulation, and such environment-induced epigenetic changes appeared to be intergenerationally inherited, but evidences for transgenerational inheritance are still limited.

Involvement of histone deacetylase CsHDA2 in regulating (E)-nerolidol formation in tea (Camellia sinensis) exposed to tea green leafhopper infestation

Herbivore-induced plant volatiles (HIPVs) help the tea plant (Camellia sinensis) adapt to environmental stress, and they are also quality-related components of tea. However, the upstream mechanism regulating the herbivore-induced expression of volatile biosynthesis genes is unclear, especially at the level of epigenetic regulation. In this study, similar to the effects of a tea green leafhopper infestation, treatments with exogenous jasmonic acid (JA) and histone deacetylase inhibitors significantly increased the (E)-nerolidol content in tea and induced the expression of the associated biosynthesis gene CsNES. Furthermore, a key transcription factor related to JA signaling, myelocytomatosis 2 (CsMYC2), interacted with histone deacetylase 2 (CsHDA2) in vitro and in vivo. A tea green leafhopper infestation inhibited CsHDA2 expression and decreased CsHDA2 abundance. Moreover, the tea green leafhopper infestation increased H3 and H4 acetylation levels in the promoter region of CsNES, which in turn upregulated the expression of CsNES and increased the (E)-nerolidol content. In this study, we revealed the effects of histone acetylations on the accumulation of HIPVs, while also confirming that CsHDA2-CsMYC2 is an important transcriptional regulatory module for the accumulation of (E)-nerolidol induced by tea green leafhoppers. The results of this study may be useful for characterizing plant aromatic compounds and the main upstream stress-responsive signaling molecules. Furthermore, the study findings will assist researchers clarify the epigenetic regulation influencing plant secondary metabolism in response to external stress.

New knowledge about the biosynthesis of lovastatin and its production by fermentation of Aspergillus terreus.

Lovastatin, and its semisynthetic derivative simvastatine, has great medical and economic importance, besides great potential for other uses. In the last years, a deeper and more complex view of secondary metabolism regulation has emerged, with the incorporation of cluster-specific and global transcription factors, and their relation to signaling cascades, as well as the new level of epigenetic regulation. Recently, a new mechanism, which regulates lovastatin biosynthesis, at transcriptional level, has been discovered: reactive oxygen species (ROS) regulation; also new unexpected environmental stimuli have been identified, which induce the synthesis of lovastatin, like quorum sensing-type molecules and support stimuli. The present review describes this new panorama and uses this information, together with the knowledge on lovastatin biosynthesis and genomics, as the foundation to analyze literature on optimization of fermentation parameters and medium composition, and also to fully understand new strategies for strain genetic improvement. This new knowledge has been applied to the development of more effective culture media, with the addition of molecules like butyrolactone I, oxylipins, and spermidine, or with addition of ROS-generating molecules to increase internal ROS levels in the cell. It has also been applied to the development of new strategies to generate overproducing strains of Aspergillus terreus, including engineering of the cluster-specific transcription factor (lovE), global transcription factors like the ones implicated in ROS regulation (or even mitochondrial alternative respiration aox gen), or the global regulator LaeA. Moreover, there is potential to apply some of these findings to the development of novel unconventional production systems. KEY POINTS: • New findings in regulation of lovastatin biosynthesis, like ROS regulation. • Induction by unexpected stimuli: autoinducer molecules and support stimuli. • Recent reports on culture medium and process optimization from this stand point. • Applications to molecular genetic strain improvement methods and production systems.

Umwelt und Epigenetik

Environmental factors are among the strongest risk factors for psychiatric disorders. Differences in exposure to such environments have been associated with lasting biological changes. In recent years epigenetic mechanisms have been brought to the forefront as central in mediating alasting embedding of environmental risk factors. This article first summarizes the different levels of epigenetic regulation and then focuses on mechanisms transducing environmental signals into lasting epigenetic changes. This is followed by examples of how environmentally induced epigenetic changes relate to risk and resilience to psychiatric disorders and their treatment.

BET inhibition in advanced cutaneous T cell lymphoma is synergistically potentiated by BCL2 inhibition or HDAC inhibition.

While several systemic therapies are approved for cutaneous T cell lymphoma (CTCL), a non-Hodgkin lymphoma of skin-homing T cells that may involve lymph nodes and peripheral blood in advanced stages, relapses are common. Mutational analysis of CTCL cells has revealed frequent amplification of the MYC oncogene, and bromodomain and extraterminal (BET) protein inhibitors have been shown to repress MYC expression in various malignancies. Towards a potential novel therapy, we thus sought to examine the effect of BET inhibition on CTCL cells in vitro. Each of the four tested BET inhibitors (JQ1, ABBV-075, I-BET762, CPI-0610) consistently induced dose-dependent decreases in viability of isolated patient-derived CTCL cells and established CTCL cell lines (MyLa, Sez4, HH, Hut78). This effect was synergistically potentiated by combination of BET inhibition with BCL2 inhibition (e.g. venetoclax) or histone deacetylase (HDAC) inhibition (e.g. vorinostat or romidepsin). There was also a marked increase in caspase 3/7 activation when JQ1 was combined with either vorinostat or romidepsin, confirming that the observed synergies are due in major part to induction of apoptosis. Furthermore, MYC and BCL2 expression were each synergistically repressed when CTCL cells were treated with JQ1 plus HDAC inhibitors, suggesting cooperative activities at the level of epigenetic regulation. Taken together, these data indicate that targeting BET proteins in CTCL represents a promising novel therapeutic strategy that may be substantially potentiated by combination with BCL2 or HDAC inhibition.

Epigenetic Reprogramming Strategies to Reverse Global Loss of 5-Hydroxymethylcytosine, a Prognostic Factor for Poor Survival in High-grade Serous Ovarian Cancer.

Purpose: A major challenge in platinum-based cancer therapy is the clinical management of chemoresistant tumors, which have a largely unknown pathogenesis at the level of epigenetic regulation.Experimental Design: We evaluated the potential of using global loss of 5-hydroxymethylcytosine (5-hmC) levels as a novel diagnostic and prognostic epigenetic marker to better assess platinum-based chemotherapy response and clinical outcome in high-grade serous tumors (HGSOC), the most common and deadliest subtype of ovarian cancer. Furthermore, we identified a targetable pathway to reverse these epigenetic changes, both genetically and pharmacologically.Results: This study shows that decreased 5-hmC levels are an epigenetic hallmark for malignancy and tumor progression in HGSOC. In addition, global 5-hmC loss is associated with a decreased response to platinum-based chemotherapy, shorter time to relapse, and poor overall survival in patients newly diagnosed with HGSOC. Interestingly, the rescue of 5-hmC loss restores sensitivity to platinum chemotherapy in vitro and in vivo, decreases the percentage of tumor cells with cancer stem cell markers, and increases overall survival in an aggressive animal model of platinum-resistant disease.Conclusions: Consequently, a global analysis of patient 5-hmC levels should be included in future clinical trials, which use pretreatment with epigenetic adjuvants to elevate 5-hmC levels and improve the efficacy of current chemotherapies. Identifying prognostic epigenetic markers and altering chemotherapeutic regimens to incorporate DNMTi pretreatment in tumors with low 5-hmC levels could have important clinical implications for newly diagnosed HGSOC disease. Clin Cancer Res; 24(6); 1389-401. ©2017 AACR.

Stochastics of Cellular Differentiation Explained by Epigenetics: The Case of T-Cell Differentiation and Functional Plasticity.

Epigenetic marks including histone modifications and DNA methylation are associated with the regulation of gene expression and activity. In addition, an increasing number of non-coding RNAs with regulatory activity on gene expression have been identified. Alongside, technological advancements allow for the analysis of these mechanisms with high resolution up to the single-cell level. For instance, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) simultaneously probes for chromatin accessibility and nucleosome positioning. Thus, it provides information on two levels of epigenetic regulation. Development and differentiation of T cells into functional subset cells including memory T cells are dynamic processes driven by environmental signals. Here, we briefly review the current knowledge of how epigenetic regulation contributes to subset specification, differentiation and memory development in T cells. Specifically, we focus on epigenetic mechanisms differentially active in the two distinct T cell populations expressing αβ or γδ T cell receptors. We also discuss examples of epigenetic alterations of T cells in autoimmune diseases. DNA methylation and histone acetylation are subject to modification by several classes of 'epigenetic modifiers', some of which are in clinical use or in preclinical development. Therefore, we address the impact of some epigenetic modifiers on T-cell activation and differentiation, and discuss possible synergies with T cell-based immunotherapeutic strategies.

Genome technologies in pulmonology: a role of microRNA in asthma and COPD development

MicroRNAs (miRNAs) are small noncoding RNA molecules that affect gene expression and thus take part in the epigenetic regulation of almost all physiological and pathological processes. About 1,800 human miRNAs have been discovered to date; however, biological functions and protein targets for the majority remain to be unknown. Within the respiratory system, miRNAs contribute to the lung growth and lifelong maintenance of pulmonary homeostasis. Recently, the leading role of miRNAs in pathogenesis of various pulmonary diseases has been found, including asthma, chronic obstructive pulmonary disease (COPD) and lung cancer. Due to a significant progress in studying interactions between genes and their products and environmental factors, a great role of epigenetic variability, which is gene expression change not related to DNA damage, but could be inherited consistently, became apparent. There are three levels of epigenetic regulation corresponding to three main mechanisms: genomic (DNA methylation), proteomic (histone modification) and transcriptomic (regulation through RNA, primarily miRNA). Extending our knowledge on a role of miRNAs for the respiratory system could open new therapeutic targets and diagnostic markers for respiratory diseases, particularly asthma and COPD.

Enhancer of zeste homolog-2 (EZH2) methyltransferase regulates transgelin/smooth muscle-22α expression in endothelial cells in response to interleukin-1β and transforming growth factor-β2

Smooth muscle-22α (SM22α), encoded by transgelin (TAGLN), is expressed in mesenchymal lineage cells, including myofibroblasts and smooth muscle cells. It is an F-actin binding protein that regulates the organization of actin cytoskeleton, cellular contractility and motility. SM22α is crucial for the maintenance of smooth muscle cell phenotype and its function. SM22α is also expressed in the processes of mesenchymal transition of epithelial (EMT) or endothelial cells (EndMT). The expression of TAGLN/SM22α is induced by transforming growth factor-β (TGFβ) signaling and enhanced by concomitant interleukin-1β (IL-1β) signaling. We investigated the epigenetic regulation of TAGLN expression by enhancer of zeste homolog-2 (EZH2), the methyltransferase of Polycomb, in the context of TGFβ and IL-1β signaling in endothelial cells. We demonstrate that the expression of EZH2 in endothelial cells was regulated by the inflammatory cytokine IL-1β. A decrease in both expression and activity of EZH2 led to an increase in TAGLN expression. Inhibition of EZH2 augmented TGFβ2-induced SM22α expression. The decrease of EZH2 levels in endothelial cells co-stimulated with IL-1β and TGFβ2 correlated with decreased H3K27me3 levels at the TAGLN proximal promoter. Moreover, the SM22α expression increased. Taken together, this suggests that EZH2 regulates the chromatin structure at the TAGLN promoter through tri-methylation of H3K27. EZH2 therefore acts as an epigenetic integrator of IL-1β and TGFβ2 signaling, providing an example of how cellular signaling can be resolved at the level of epigenetic regulation. Since IL-1β and TGFβ2 represent the pro-inflammatory and pro-fibrotic conditions during vascular fibroproliferative disease, we surmise that EZH2, as the molecule that integrates their signaling, could also be a promising target for development of future therapy.

Abstract 3084: Epigenetic deregulation in H3.3-K27M mutant pediatric high-grade gliomas

Abstract A recurrent mutation substituting lysine 27 with a methionine (K27M) on the amino-terminal tail of histone variant H3.3 was recently identified in about 25% of pediatric high-grade gliomas (pHGGs), the most common malignant brain tumor in childhood. K27M mutant tumors represent a clinically and biologically distinct subgroup of pHGGs, characterized by a unique genome-wide DNA methylation pattern as well as transcriptomic profile. Interestingly, tumors harboring the K27M mutation display a global reduction of the repressive histone modification H3K27me3, which is caused by a dominant-negative effect of K27M mutant H3.3 protein. The purpose of this study was to further elucidate the dominant-negative effect of K27M mutant H3.3 and investigate the connection between K27M-induced reduction of H3K27me3, DNA methylation and gene expression. Furthermore, we aimed to clarify if immunohistochemical (IHC) testing of H3K27me3 represents a reliable tool for clinical application to identify patients suffering from K27M mutant pHGGs, which might benefit from more intense treatment options. Co-immunoprecipitation (CoIP) experiments and histone methyltransferase (HMT) assays were conducted in three different cell lines to elucidate the dominant-negative function of K27M mutant H3.3. The potential impact on different levels of epigenetic gene regulation was analyzed by performing H3K27me3 chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq), whole genome bisulfite sequencing (WGBS) and gene expression profiling on fresh-frozen primary tumors with and without K27M mutations. As a result, we demonstrate that IHC reliably identifies K27M mutant pHGGs with high specificity and sensitivity, and confirm that H3K27me3 levels are globally reduced due to the dominant-negative effect of K27M mutant H3.3. As indicated by our CoIPs, this is a result of an increased interaction of K27M mutant H3.3 with several components of the Polycomb repressive complex 2 (PRC2), including enhancer of zeste homolog 2 (EZH2), which is responsible for di- and trimethylation of H3K27. EZH2 catalytic activity measured by HMT assay is significantly reduced in the presence of K27M mutant H3.3 protein. Genome-wide H3K27me3 ChIP-Seq data of fresh-frozen pHGG samples confirms globally reduced levels of H3K27me3, but also identifies a subset of loci with focal enrichment of this histone modification. By integrating with WGBS data, we show that K27M mutant pHGGs are characterized by reduced H3K27me3 occupancy and concomitant global DNA hypomethylation. Strikingly, 74% of genes found to be specifically upregulated in K27M mutant pHGGs show changes in one or both of these levels of epigenetic regulation. In conclusion, epigenetic deregulation in K27M mutant pHGGs reflected by reduced H3K27me3 occupancy and/or global DNA hypomethylation is a key modulator of gene activity in these highly aggressive brain tumors. Citation Format: Sebastian Bender, Yujie Tang, Anders M. Lindroth, Volker Hovestadt, Marc Zapatka, David T.W. Jones, Marcel Kool, Paul A. Northcott, Dominik Sturm, Peter Lichter, Christoph Plass, Yoon-Jae Cho. Epigenetic deregulation in H3.3-K27M mutant pediatric high-grade gliomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2014-3084

Abstract P3-04-01: Integrated Analysis of Epigenetic Signatures Associated with Inflammatory Breast Cancer

Abstract Inflammatory breast cancer (IBC) is the most aggressive manifestation of primary epithelial breast cancer and differs from non-IBC both from a clinical and a biological perspective. The high metastatic potential of IBC is highlighted by the low 5-year survival rate of 50%. The biological features that make IBC unique are its rapid local growth, its high angioinvasive capability with early metastasis and its aggressive behavior from inception. These differences could be the result of key changes in the epigenetic profile of IBC. To address this issue, we compared the levels of epigenetic regulation in IBC (N=19) and non-IBC (N=43) samples at the methylome (DNA methylation), miRome (miRNA) and transcriptome (mRNA) level using high-throughput molecular technologies. Differences in gene expression profiles allowed the classification of samples according to tumor subtype (IBC vs non-IBC). The analysis of 27,578 CpG loci demonstrated that the largest variation in methylation levels between samples was attributable to differences in metastatic status (P=0.026) and patients’ age (P=0.049). About 2% of all interrogated CpG loci was differentially methylated in IBC and non-IBC (FDR <0.1). Annotated gene functions for these CpG loci were enriched for biological processes related to inflammation and immune response. Using a whole genome approach, we observed significant, negative correlations between methylation and mRNA levels for 38% of genes. For 8% of genes, a significant, positive correlation was observed. Interrogated CpG loci for these genes were more frequently located in the gene body (>500 bp from TSS) (P=0.008). Our analysis of 322 miRNAs showed that the largest variation in miRNA levels between samples was attributable to differences in ER status (P=0.001) and histological grade (P=0.002). About 4% of all miRNAs was expressed at different levels in IBC and non-IBC. Using a whole genome approach, we identified 17,295 of highly correlated miRNA-mRNA pairs for these IBC-associated miRNAs. Based on this miRNA target gene information, we were able to detect a variety of biological processes that may indicate function of these miRNAs and to indicate potential associations of these miRNAs with prognosis in breast cancer. Furthermore, the comparison of mRNA levels of key miRNA processing genes revealed the upregulation of Ago2 (P=0.002) and the downregulation of Dicer (P=0.004) in IBC when compared to non-IBC, which may suggest higher RISC activities and, therefore, that miRNAs regulate target gene expression in IBC with higher efficiency. In conclusion, this study represents the first integrated analysis of epigenetic signatures in IBC. Using high-throughput technologies, we were able to identify a number of epigenetic markers specifically associated with IBC. A global initiative has been taken to set up an IBC tissue repository that will allow the confirmation of these kinds of studies on a larger IBC sample population. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-01.