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Amarone wine is different from regular dry wine due to the postharvest withering of Corvina, Corvinone and Rondinella grapes. Grapes were withered in a commercial facility with variability in terms of temperature and relative humidity (R.H.). Sugar content reached 230–240 g L −1 and 280 g L −1 at 20% and 30% mass loss, respectively. Most of VOCs (volatile organic compounds) decreased during withering but few VOCs increased during withering and we considered as markers; in Corvinone they were methylhexanoate, dimethylsuccinate, nerol, nonanoic acid, and benzyl alcohol; in Corvina, benzyl alcohol, isoamyl alcohol, 1-hexanol, p-cymen-8-ol, 2,3 pinanediol, 3-oxo-ionol and 3-methyl-1-pentanol, coumaran and damascenone; in Rondinella, hexanol, nonanoic acid, methyl vanillate, damascenone, 3-oxo-ionol, eugenol, p-cymen-8-ol, 2,3 pinanediol, coumaran and raspberry keton. Olfactive descriptors of the wines and the potential aroma of the combination of Corvina wine with the wines of the other two varieties at different percentages of mass loss are reported. [ABSTRACT FROM AUTHOR]

Trichoderma gamsii 6085 is known for its features as antagonist, mycoparasite and competitor on natural substrates of mycotoxigenic Fusarium graminearum and Fusarium culmorum. This beneficial isolate is also able to significantly reduce deoxynivalenol (DON) production by these pathogens. In the present study, the ability of T. gamsii 6085 to grow in the presence of DON was investigated. On the basis of HPLC results showing no differences in the amount of DON in the growth medium after 72 h of incubation, the possible role of some PDR-ABC transporters in DON resistance by T. gamsii 6085 was investigated. T. gamsii 6085 was then tested as biocontrol agent under field conditions for two growing seasons (2011 and 2012), in order to evaluate its ability to reduce FHB symptoms on spikes and prevent mycotoxin production in kernels. The isolate was inoculated into soil before sowing or on spikes during anthesis. Spike application of T. gamsii 6085 reduced FHB severity by 57%. Colonization of spikes and spikelet components by the antagonist was assessed and discussed in connection with disease control. Results concerning spike colonization and the effects on the disease are encouraging. These preliminary results suggest that T. gamsii 6085 deserves further attention as a potential biocontrol agent of FHB. [ABSTRACT FROM AUTHOR]

Celiac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and malabsorption of nutrients. The major instigating factor for the immune response in celiac disease is the activation of gluten-specific CD4+ T cells expressing T cell receptors that recognize gluten peptides presented in the context of HLA-DQ2 and DQ8. Here we provide an in-depth characterization of 28 gluten-specific T cell clones. We assess their transcriptional and epigenetic response to T cell receptor stimulation and link this to genetic factors associated with celiac disease. Gluten-specific T cells have a distinct transcriptional profile that mostly resembles that of Th1 cells but also express cytokines characteristic of other types of T-helper cells. This transcriptional response appears not to be regulated by changes in chromatin state, but rather by early upregulation of transcription factors and non-coding RNAs that likely orchestrate the subsequent activation of genes that play a role in immune pathways. Finally, integration of chromatin and transcription factor binding profiles suggest that genes activated by T cell receptor stimulation of gluten‑specific T cells may be impacted by genetic variation at several genetic loci associated with celiac disease.

Global investigation of histone marks in acute myeloid leukemia (AML) remains limited. Analyses of 38 AML samples through integrated transcriptional and chromatin mark analysis exposes 2 major subtypes. One subtype is dominated by patients with NPM1 mutations or MLL-fusion genes, shows activation of the regulatory pathways involving HOX-family genes as targets, and displays high self-renewal capacity and stemness. The second subtype is enriched for RUNX1 or spliceosome mutations, suggesting potential interplay between the 2 aberrations, and mainly depends on IRF family regulators. Cellular consequences in prognosis predict a relatively worse outcome for the first subtype. Our integrated profiling establishes a rich resource to probe AML subtypes on the basis of expression and chromatin data., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Methyl jasmonate (MeJA) was applied in a vineyard on leaves and grape clusters of cv Sangiovese to test its ability to stimulate the production of aromas and identify the main genes involved in the biosynthetic pathways switched on by the elicitor. MeJA application led to a delay in grape technological maturity and a significant increase in the concentration of several berry aroma classes (about twice the total aroma: from around 3 to 6μg/g of berry). Of these, monoterpenes showed the most significant increase. An analysis of the expression of terpenoid biosynthesis genes confirmed that the MeJA application activated the related biosynthetic pathway. The expression of all the TPS genes analyzedwas higher in samples treated with MeJA. Also the wines produced by microvinification of Sangiovese treated and untreated grapes showed a rise in the aroma concentration as in berries, with an important impact on longevity and sensorial characters of wines., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Deregulation of epigenetic mechanisms, including microRNA, contributes to leukemogenesis and drug resistance by interfering with cancer-specific molecular pathways. Here, we show that the balance between miR-194-5p and its newly discovered target BCL2-associated transcription factor 1 (BCLAF1) regulates differentiation and survival of normal hematopoietic progenitors. In acute myeloid leukemias this balance is perturbed, locking cells into an immature, potentially 'immortal' state. Enhanced expression of miR-194-5p by treatment with the histone deacetylase inhibitor SAHA or by exogenous miR-194-5p expression re-sensitizes cells to differentiation and apoptosis by inducing BCLAF1 to shuttle between nucleus and cytosol. miR-194-5p/BCLAF1 balance was found commonly deregulated in 60 primary acute myeloid leukemia patients and was largely restored by ex vivo SAHA treatment. Our findings link treatment responsiveness to re-instatement of miR-194-5p/BCLAF1 balance.

We have developed a machine learning approach to predict stimulation-dependent enhancer-promoter interactions using evidence from changes in genomic protein occupancy over time. The occupancy of estrogen receptor alpha (ERα), RNA polymerase (Pol II) and histone marks H2AZ and H3K4me3 were measured over time using ChIP-Seq experiments in MCF7 cells stimulated with estrogen. A Bayesian classifier was developed which uses the correlation of temporal binding patterns at enhancers and promoters and genomic proximity as features to predict interactions. This method was trained using experimentally determined interactions from the same system and was shown to achieve much higher precision than predictions based on the genomic proximity of nearest ERα binding. We use the method to identify a genome-wide confident set of ERα target genes and their regulatory enhancers genome-wide. Validation with publicly available GRO-Seq data demonstrates that our predicted targets are much more likely to show early nascent transcription than predictions based on genomic ERα binding proximity alone., Competing Interests: The authors declare there are no competing interests. Korbinain Grote is employed by Genomatix Software GmbH.

Interactions between DNA and proteins are mainly studied through chemical procedures involving bi-functional reagents, mostly formaldehyde. Chromatin immunoprecipitation is used to identify the binding between transcription factors (TFs) and chromatin, and to evaluate the occurrence and impact of histone/DNA modifications. The current bottleneck in probing DNA-protein interactions using these approaches is caused by the fact that chemical crosslinkers do not discriminate direct and indirect bindings or short-lived chromatin occupancy. Here, we describe a novel application of UV laser-induced (L-) crosslinking and demonstrate that a combination of chemical and L-crosslinking is able to distinguish between direct and indirect DNA-protein interactions in a small number of living cells. The spatial and temporal dynamics of TF bindings to chromatin and their role in gene expression regulation may thus be assessed. The combination of chemical and L-crosslinking offers an exciting and unprecedented tool for biomedical applications.

Background: In this work, we evaluated the aromatic composition of berries at harvest and during the whole berry development of Aleatico, Brachetto, Malvasia di Candia aromatica and Moscato bianco grapevine varieties, which were cultivated in the same vineyard and under the same agricultural conditions., Results: Malvasia had a total concentration of aroma which was significantly higher than the other grapes that showed comparable amounts with each other. The class of monoterpenes was quantitatively predominant for all four grapes, in the free and in the most plentiful bound form. In Malvasia and Aleatico there was a high prevalence of geraniol derivatives throughout the entire berry development while in Brachetto and in Moscato there was a prevalence of linalool derivatives during the green phase and geraniol derivatives during ripening., Conclusion: The study of the monoterpene profile allowed us to highlight similarities and differences among the four aromatic varieties and to present a hypothesis about their biosynthetic dynamics. This information is useful for further studies on gene functional characterisation and the regulation of these important berry pathways. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Background: Environmental factors have been acknowledged to greatly influence grape and wine aromas. Among them, the effect of altitude on grape aroma compounds has scarcely been debated in literature available to date. In the present study, we investigated the influence of altitude on grape composition and aroma evolution during ripening of Vitis vinifera L. cultivar Glera grown in Conegliano-Valdobbiadene DOCG area (Italy)., Results: The site at highest altitude (380 m above sea level) was warmer than the lowest site (200 m above sea level) and, even with differences in temperature in the range 1.5-2 °C, the impact of the cultivation site on grape ripening and aroma accumulation and preservation was significant. The lowest site demonstrated slower grape ripening, and grapes at harvest accumulated lower amounts of all of the main classes of aroma compounds typical of the Glera variety. Wines produced from the highest site were preferred in tasting trials for their more patent floral notes and elegance., Conclusion: Altitude strongly influences grape ripening evolution and flavour accumulation in the Glera grape, and this result accounts for the different styles in the sparkling wines subsequently produced. Moreover, the present study shows that aroma compound biosynthesis, particularly that of benzenoides, starts before véraison in Glera. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Trichoderma spp. are opportunistic fungi some of which are commonly present in the rhizosphere. Several species, such as T. virens, are also efficient biocontrol agents against phytopathogenic fungi and exert beneficial effects on plants. These effects are the consequence of interactions between Trichoderma and plant roots, which trigger enhanced plant growth and induce plant resistance. We have previously shown that T. virens I10 expresses two endopolygalacturonase genes, tvpg1 and tvpg2, during the interaction with plant roots; tvpg1 is inducible while tvpg2 is constitutively transcribed. Using the same system, the tomato polygalacturonase-inhibitor gene Lepgip1 was induced at the same time as tvpg1. Here we show by gene disruption that TvPG2 performs a regulatory role on the inducible tvpg1 gene and in triggering the plant immune response. A tvpg2-knockout strain fails to transcribe the inducible tvpg1 gene in neither in vitro in inducing media containing pectin or plant cell walls, nor during the in vivo interaction with tomato roots. Likewise, the in vivo induction of Lepgip1 does not occur, and its defense against the pathogen Botrytis cinerea is significantly reduced. Our data prove the importance of a T. virens constitutively produced endopolygalacturonase in eliciting plant induced systemic resistance against pathogenic fungi.

Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, β-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo β-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes. VIDEO ABSTRACT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14 + monocytes, CD16 + neutrophils, and naive CD4 + T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Epigenome-wide association studies (EWAS) provide an alternative approach for studying human disease through consideration of non-genetic variants such as altered DNA methylation. To advance the complex interpretation of EWAS, we developed eFORGE (http://eforge.cs.ucl.ac.uk/), a new standalone and web-based tool for the analysis and interpretation of EWAS data. eFORGE determines the cell type-specific regulatory component of a set of EWAS-identified differentially methylated positions. This is achieved by detecting enrichment of overlap with DNase I hypersensitive sites across 454 samples (tissues, primary cell types, and cell lines) from the ENCODE, Roadmap Epigenomics, and BLUEPRINT projects. Application of eFORGE to 20 publicly available EWAS datasets identified disease-relevant cell types for several common diseases, a stem cell-like signature in cancer, and demonstrated the ability to detect cell-composition effects for EWAS performed on heterogeneous tissues. Our approach bridges the gap between large-scale epigenomics data and EWAS-derived target selection to yield insight into disease etiology., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic transcription factors LYL1 and LMO2. In contrast, ERG, FLI1, TAL1, and RUNX1 bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. We find that expression of wild-type transcription factors RUNX1 and ERG in AML is required to prevent this oncogene overexpression. Together our results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Grapevine (Vitis vinifera L.) is an economically important crop with a wide geographical distribution, reflecting its ability to grow successfully in a range of climates. However, many vineyards are located in regions with seasonal drought, and these are often predicted to be global climate change hotspots. Climate change affects the entire physiology of grapevine, with strong effects on yield, wine quality and typicity, making it difficult to produce berries of optimal enological quality and consistent stability over the forthcoming decades., Results: Here we investigated the reactions of two grapevine cultivars to water stress, the isohydric variety Montepulciano and the anisohydric variety Sangiovese, by examining physiological and molecular perturbations in the leaf and berry. A multidisciplinary approach was used to characterize the distinct stomatal behavior of the two cultivars and its impact on leaf and berry gene expression. Positive associations were found among the photosynthetic, physiological and transcriptional modifications, and candidate genes encoding master regulators of the water stress response were identified using an integrated approach based on the analysis of topological co-expression network properties. In particular, the genome-wide transcriptional study indicated that the isohydric behavior relies upon the following responses: i) faster transcriptome response after stress imposition; ii) faster abscisic acid-related gene modulation; iii) more rapid expression of heat shock protein (HSP) genes and iv) reversion of gene-expression profile at rewatering. Conversely, that reactive oxygen species (ROS)-scavenging enzymes, molecular chaperones and abiotic stress-related genes were induced earlier and more strongly in the anisohydric cultivar., Conclusions: Overall, the present work found original evidence of a molecular basis for the proposed classification between isohydric and anisohydric grapevine genotypes.

NuRD (nucleosome remodeling and histone deacetylase) is a versatile multi-protein complex with roles in transcription regulation and the DNA damage response. Here, we show that ZMYND8 bridges NuRD to a number of putative DNA-binding zinc finger proteins. The MYND domain of ZMYND8 directly interacts with PPPLΦ motifs in the NuRD subunit GATAD2A. Both GATAD2A and GATAD2B exclusively form homodimers and define mutually exclusive NuRD subcomplexes. ZMYND8 and NuRD share a large number of genome-wide binding sites, mostly active promoters and enhancers. Depletion of ZMYND8 does not affect NuRD occupancy genome-wide and only slightly affects expression of NuRD/ZMYND8 target genes. In contrast, the MYND domain in ZMYND8 facilitates the rapid, poly(ADP-ribose)-dependent recruitment of GATAD2A/NuRD to sites of DNA damage to promote repair by homologous recombination. Thus, these results show that a specific substoichiometric interaction with a NuRD subunit paralogue provides unique functionality to distinct NuRD subcomplexes., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor α activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes.

Photosynthetic performances and energy dissipation mechanisms were evaluated on the anisohydric cv. Sangiovese and on the isohydric cv. Montepulciano (Vitis vinifera L.) under conditions of multiple summer stresses. Potted vines of both cultivars were maintained at 90% and 40% of maximum water availability from fruit-set to veraison. One week before veraison, at predawn and midday, main gas-exchange and chlorophyll fluorescence parameters, chlorophyll content, xanthophyll pool and cycle and catalase activity were evaluated. Under water deficit and elevated irradiance and temperature, contrary to cv. Montepulciano and despite a significant leaf water potential decrease, Sangiovese's leaves kept their stomata more open and continued to assimilate CO2 while also showing higher water use efficiency. Under these environmental conditions, in comparison with the isohydric cv. Montepulciano, the protective mechanisms of energy dissipation exerted by the anisohydric cv. Sangiovese were: (i) higher stomatal conductance and thermoregulation linked to higher transpiration rate; (ii) greater ability at dissipating more efficiently the excess energy via the xanthophylls cycle activity (thermal dissipation) due to higher VAZ pool and greater increase of de-epoxidation activity., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Monocyte differentiation into macrophages represents a cornerstone process for host defense. Concomitantly, immunological imprinting of either tolerance or trained immunity determines the functional fate of macrophages and susceptibility to secondary infections. We characterized the transcriptomes and epigenomes in four primary cell types: monocytes and in vitro-differentiated naïve, tolerized, and trained macrophages. Inflammatory and metabolic pathways were modulated in macrophages, including decreased inflammasome activation, and we identified pathways functionally implicated in trained immunity. β-glucan training elicits an exclusive epigenetic signature, revealing a complex network of enhancers and promoters. Analysis of transcription factor motifs in deoxyribonuclease I hypersensitive sites at cell-type-specific epigenetic loci unveiled differentiation and treatment-specific repertoires. Altogether, we provide a resource to understand the epigenetic changes that underlie innate immunity in humans., (Copyright © 2014, American Association for the Advancement of Science.)

Plants produce a plethora of volatile organic compounds (VOCs) which are important in determining the quality and nutraceutical properties of horticultural food products, including the taste and aroma of wine. Given that some of the most prevalent grape aroma constituents are terpenoids, we investigated the possible variations in the relative expression of terpene synthase (TPS) genes that depend on the organ. We thus analysed mature leaves, young leaves, stems, young stems, roots, rachis, tendrils, peduncles, bud flowers, flowers and berries of cv Moscato bianco in terms of their VOC content and the expression of 23 TPS genes. In terms of the volatile characterization of the organs by SPME/GC-MS analysis, flower buds and open flowers appeared to be clearly distinct from all the other organs analysed in terms of their high VOC concentration. Qualitatively detected VOCs clearly separated all the vegetative organs from flowers and berries, then the roots and rachis from other vegetative organs and flowers from berries, which confirms the specialization in volatile production among different organs. Our real-time RT-PCR results revealed that the majority of TPS genes analysed exhibited detectable transcripts in all the organs investigated, while only some were found to be expressed specifically in one or just a few organs. In most cases, we found that the known products of the in vitro assay of VvTPS enzymes corresponded well to the terpenes found in the organs in which the encoding gene was expressed, as in the case of (E)-β-caryophyllene synthases, α-terpineol synthase and α-farnesene synthase. In addition, we found groups of homologous TPS genes, such as (E)-β-caryophyllene and β-ocimene synthases, expressed distinctively in the various tissues. This thus confirmed the subfunctionalization events and a specialization on the basis of the organs in which they are mostly expressed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MBD2 is a subunit of the NuRD complex that is postulated to mediate gene repression via recruitment of the complex to methylated DNA. In this study we adopted an MBD2 tagging-approach to study its genome wide binding characteristics. We show that in vivo MBD2 is mainly recruited to CpG island promoters that are highly methylated. Interestingly, MBD2 binds around 1 kb downstream of the transcription start site of a subset of ∼ 400 CpG island promoters that are characterized by the presence of active histone marks, RNA polymerase II (Pol2) and low to medium gene expression levels and H3K36me3 deposition. These tagged-MBD2 binding sites in MCF-7 show increased methylation in a cohort of primary breast cancers but not in normal breast samples, suggesting a putative role for MBD2 in breast cancer.

Gene transcription mediated by RNA polymerase II (pol-II) is a key step in gene expression. The dynamics of pol-II moving along the transcribed region influence the rate and timing of gene expression. In this work, we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data measured using ChIP-Seq. The model can be used to estimate transcription speed and to infer the temporal pol-II activity profile at the gene promoter. Model parameters are estimated using either maximum likelihood estimation or via Bayesian inference using Markov chain Monte Carlo sampling. The Bayesian approach provides confidence intervals for parameter estimates and allows the use of priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments. The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes. By clustering the inferred promoter activity time profiles, we are able to determine which genes respond quickly to stimuli and group genes that share activity profiles and may therefore be co-regulated. We apply our methodology to biological data obtained using ChIP-seq to measure pol-II occupancy genome-wide when MCF-7 human breast cancer cells are treated with estradiol (E2). The transcription speeds we obtain agree with those obtained previously for smaller numbers of genes with the advantage that our approach can be applied genome-wide. We validate the biological significance of the pol-II promoter activity clusters by investigating cluster-specific transcription factor binding patterns and determining canonical pathway enrichment. We find that rapidly induced genes are enriched for both estrogen receptor alpha (ERα) and FOXA1 binding in their proximal promoter regions.

HDAC inhibitors (HDACi) are widely used in the clinic to sensitize tumorigenic cells for treatment with other anticancer compounds. The major drawback of HDACi is the broad inhibition of the plethora of HDAC-containing complexes. In acute promyelocytic leukemia (APL), repression by the PML-RARα oncofusion protein is mediated by an HDAC-containing complex that can be dissociated by pharmacologic doses of all trans retinoic acid (ATRA) inducing differentiation and cell death at the expense of side effects and recurrence. We hypothesized that the context-specific close physical proximity of a retinoid and HDACi-binding protein in the repressive PML-RARα-HDAC complex may permit selective targeting by a hybrid molecule of ATRA with a 2-aminoanilide tail of the HDAC inhibitor MS-275, yielding MC2392. We show that MC2392 elicits weak ATRA and essentially no HDACi activity in vitro or in vivo. Genome-wide epigenetic analyses revealed that in NB4 cells expressing PML-RARα, MC2392 induces changes in H3 acetylation at a small subset of PML-RARα-binding sites. RNA-seq reveals that MC2392 alters expression of a number of stress-responsive and apoptotic genes. Concordantly, MC2392 induced rapid and massive, caspase-8-dependent cell death accompanied by RIP1 induction and ROS production. Solid and leukemic tumors are not affected by MC2392, but expression of PML-RARα conveys efficient MC2392-induced cell death. Our data suggest a model in which MC2392 binds to the RARα moiety and selectively inhibits the HDACs resident in the repressive complex responsible for the transcriptional impairment in APLs. Our findings provide proof-of-principle of the concept of a context-dependent targeted therapy., (©2014 AACR.)

The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methyltransferase that cooperates with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESCs), but its function in these cells is unknown. Through genome-wide analysis of Dnmt3L knockdown in ESCs, we found that Dnmt3L is a positive regulator of methylation at the gene bodies of housekeeping genes and, more surprisingly, is also a negative regulator of methylation at promoters of bivalent genes. Dnmt3L is required for the differentiation of ESCs into primordial germ cells (PGCs) through the activation of the homeotic gene Rhox5. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low methylation levels at the H3K27me3 regions. Thus, in ESCs, Dnmt3L counteracts the activity of de novo DNA methylases to maintain hypomethylation at promoters of bivalent developmental genes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The use of two kinase inhibitors (2i) enables derivation of mouse embryonic stem cells (ESCs) in the pluripotent ground state. Using whole-genome bisulfite sequencing (WGBS), we show that male 2i ESCs are globally hypomethylated compared to conventional ESCs maintained in serum. In serum, female ESCs are hypomethyated similarly to male ESCs in 2i, and DNA methylation is further reduced in 2i. Regions with elevated DNA methylation in 2i strongly correlate with the presence of H3K9me3 on endogenous retroviruses (ERVs) and imprinted loci. The methylome of male ESCs in serum parallels postimplantation blastocyst cells, while 2i stalls ESCs in a hypomethylated, ICM-like state. WGBS analysis during adaptation of 2i ESCs to serum suggests that deposition of DNA methylation is largely random, while loss of DNA methylation during reversion to 2i occurs passively, initiating at TET1 binding sites. Together, our analysis provides insight into DNA methylation dynamics in cultured ESCs paralleling early developmental processes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Grape (Vitis vinifera L.) flavour management in the vineyard requires knowledge of the derivation of individual flavour and aroma characteristics. Some of the most prevalent wine grape aroma constituents are terpenoids and this study represents a wide report about grape terpene synthase (TPS) gene transcript profiling in different tissues of two aromatic grapevine varieties, particularly flowers and developing berries, correlated with the accumulation patterns of free aroma compounds. All investigated genes belonging to the TPS-a and TPS-b subfamilies reached the highest expression in accordance with the peak of accumulation of the respective compounds. In the TPS-g subfamily, only one of the genes characterised for linalool synthases showed major transcript abundance in ripening berries, whereas the only geraniol synthase had a peak of expression in green berries and at the beginning of ripening, when geraniol concentration started to increase and overcome the linalool concentration. The genes identified in this study as being mainly responsible for linalool and geraniol synthesis during berry development, and the phenological phases in which they are mostly expressed, should be of interest to viticulturists and wine makers to improve decision making along the chain of production.

Identification of responsive genes to an extra-cellular cue enables characterization of pathophysiologically crucial biological processes. Deep sequencing technologies provide a powerful means to identify responsive genes, which creates a need for computational methods able to analyze dynamic and multi-level deep sequencing data. To answer this need we introduce here a data-driven algorithm, SPINLONG, which is designed to search for genes that match the user-defined hypotheses or models. SPINLONG is applicable to various experimental setups measuring several molecular markers in parallel. To demonstrate the SPINLONG approach, we analyzed ChIP-seq data reporting PolII, estrogen receptor α (ERα), H3K4me3 and H2A.Z occupancy at five time points in the MCF-7 breast cancer cell line after estradiol stimulus. We obtained 777 ERa early responsive genes and compared the biological functions of the genes having ERα binding within 20 kb of the transcription start site (TSS) to genes without such binding site. Our results show that the non-genomic action of ERα via the MAPK pathway, instead of direct ERa binding, may be responsible for early cell responses to ERα activation. Our results also indicate that the ERα responsive genes triggered by the genomic pathway are transcribed faster than those without ERα binding sites. The survival analysis of the 777 ERα responsive genes with 150 primary breast cancer tumors and in two independent validation cohorts indicated the ATAD3B gene, which does not have ERα binding site within 20 kb of its TSS, to be significantly associated with poor patient survival.

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.

Cross-talk between DNA methylation and histone modifications drives the establishment of composite epigenetic signatures and is traditionally studied using correlative rather than direct approaches. Here, we present sequential ChIP-bisulfite-sequencing (ChIP-BS-seq) as an approach to quantitatively assess DNA methylation patterns associated with chromatin modifications or chromatin-associated factors directly. A chromatin-immunoprecipitation (ChIP)-capturing step is used to obtain a restricted representation of the genome occupied by the epigenetic feature of interest, for which a single-base resolution DNA methylation map is then generated. When applied to H3 lysine 27 trimethylation (H3K27me3), we found that H3K27me3 and DNA methylation are compatible throughout most of the genome, except for CpG islands, where these two marks are mutually exclusive. Further ChIP-BS-seq-based analysis in Dnmt triple-knockout (TKO) embryonic stem cells revealed that total loss of CpG methylation is associated with alteration of H3K27me3 levels throughout the genome: H3K27me3 in localized peaks is decreased while broad local enrichments (BLOCs) of H3K27me3 are formed. At an even broader scale, these BLOCs correspond to regions of high DNA methylation in wild-type ES cells, suggesting that DNA methylation prevents H3K27me3 deposition locally and at a megabase scale. Our strategy provides a unique way of investigating global interdependencies between DNA methylation and other chromatin features.

Fusarium head blight (FHB) is a re-emerging wheat disease that causes extensive damage through direct losses in yield and quality due to the presence of damaged Fusarium kernels and their associated mycotoxins such as the trichothecene deoxynivalenol (DON). Biological control, including the treatment of crop residues with antagonists, in order to reduce pathogen inoculum of FHB, holds considerable promise. Ten Trichoderma isolates, previously selected for their ability to grow in the presence of DON, were preliminarily investigated as potential antagonists against Fusarium culmorum and F. graminearum mycotoxigenic strains in plate confrontation assays. The three Trichoderma isolates showing antibiosis and mycoparasitism were evaluated for their capacity to inhibit DON production by F. graminearum and F. culmorum on two natural substrates. The expression of some chitinase-encoding genes by the two best resulting Trichoderma strains, during interaction with F. culmorum and F. graminearum, was monitored. All investigated genes from chitinase subgroups A, B and the new subgroup C responded to mycoparasitic conditions and were upregulated before contact and/or when in contact with the host. T. gamsii 6085, the best antagonist, was finally used in a competition test against F. culmorum and F. graminearum on natural substrates, using a qPCR approach to evaluate its effect on the pathogen's growth and DON production in haulms and rice. This test confirmed the ability of T. gamsii 6085 to antagonize the pathogens on rice. On wheat haulms, an extreme oligotrophic environment, T. gamsii 6085 seemed to develop very poorly and the growth of both the pathogens was unaffected by the presence of the antagonist.

The discovery of the Ten-Eleven-Translocation (TET) oxygenases that catalyze the hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) has triggered an avalanche of studies aiming to resolve the role of 5hmC in gene regulation if any. Hitherto, TET1 is reported to bind to CpG-island (CGI) and bivalent promoters in mouse embryonic stem cells, whereas binding at DNAseI hypersensitive sites (HS) had escaped previous analysis. Significant enrichment/accumulation of 5hmC but not 5mC can indeed be detected at bivalent promoters and at DNaseI-HS. Surprisingly, however, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1. Our meta-analysis of DNA methylation profiling points to potential issues with regard to the various methodologies that are part of the toolbox used to detect 5mC and 5hmC. Discrepancies between published studies and technical limitations prevent an unambiguous assignment of 5hmC as a 'true' epigenetic mark, that is, read and interpreted by other factors and/or as a transiently accumulating intermediary product of the conversion of 5mC to unmodified cytosines.

Deregulation of the epigenome is recognized as cause of cancer and epigenetic factors are receiving major attention as therapeutic targets; yet, the molecular mode of action of existing epi-drugs is largely elusive. Here, we report on the decryption of the mechanism of action of UVI5008, a novel epigenetic modifier, that inhibits histone deacetylases, sirtuins, and DNA methyltransferases. UVI5008 highly efficiently induces cancer cell-selective death in a variety of models and exerts its activities in several human tumor xenografts and genetic mouse models of human breast cancer in vivo. Its anticancer activity involves independent activation of death receptors and reactive oxygen species production. Importantly, UVI5008 action is not critically dependent on p53, Bcl-2 modifying factor, and/or TNF-related apoptosis-inducing ligand as cell death is efficiently induced in cells mutated or deficient for these factors limiting the risk of drug resistance development and maximizing its application spectrum. The simultaneous modulation of multiple (epigenetic) targets promises to open new avenues with unanticipated potential against cancer.

For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21--pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program.

Fungi have a plethora of chitinases, which can be phylogenetically divided into three subgroups (A, B and C). Subgroup C (sgC) chitinases are especially interesting due to their multiple carbohydrate-binding modules, but they have not been investigated in detail yet. In this study, we analyzed sgC chitinases in the mycoparasites Trichoderma atroviride and Trichoderma virens. The expression of sgC chitinase genes in T. atroviride was induced during mycoparasitism of the fungal prey Botrytis cinerea, but not Rhizoctonia solani and correspondingly only by fungal cell walls of the former. Interestingly, only few sgC chitinase genes were inducible by chitin, suggesting that non-chitinous cell wall components can act as inducers. In contrast, the transcriptional profile of the most abundantly expressed sgC chitinase gene tac6 indicated a role of the protein in hyphal network formation. This shows that sgC chitinases have diverse functions and are not only involved in the mycoparasitic attack. However, sequence analysis and 3D modelling revealed that TAC6 and also its ortholog in T. virens have potentially detrimental deletions in the substrate-binding site and are thus probably not catalytically active enzymes. Genomic analysis showed that the genes neighboring sgC chitinases often encode proteins that are solely composed of multiple LysM modules, which were induced by similar stimuli as their neighboring sgC chitinase genes. This study provides first insights into fungal sgC chitinases and their associated LysM proteins.

Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these marks by high-accuracy, quantitative mass spectrometry. These chromatin "readers" are assigned to complexes by interaction proteomics of full-length BAC-GFP-tagged proteins. ChIP-Seq profiling identifies their genomic binding sites, revealing functional properties. Among the main findings, the human SAGA complex binds to H3K4me3 via a double Tudor-domain in the C terminus of Sgf29, and the PWWP domain is identified as a putative H3K36me3 binding motif. The ORC complex, including LRWD1, binds to the three most prominent transcriptional repressive lysine methylation sites. Our data reveal a highly adapted interplay between chromatin marks and their associated protein complexes. Reading specific trimethyl-lysine sites by specialized complexes appears to be a widespread mechanism to mediate gene expression., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17beta-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones.

Estrogens are direct mitogens for hormone-responsive human breast cancercells, where they promote cell cycle progression and induce transcriptional activation of "immediate early" and cyclin genes. Nongenomic signaling by estrogens, including rapid changes of mitogen-activated protein(MAP) kinase and other signal-transduction-cascades activity, has been proposed to be essential for the mitogenic actions of these hormones and their nuclear receptors. Because regulation of gene transcription is considered a key step in cell cycle control by mitogenic protein kinase cascades, here we investigated the possibility that estrogen might induce the activation of extracellular signal-regulated kinase (Erk) 1/2-, c-Jun NH(2)-terminal kinase-, p38- or protein kinase A-responsive transcription factors in the cell nucleus during stimulation of early G(1) progression, a timing coincident with the maximum effects of these hormones on such enzyme activity. No significant changes in protein kinase-mediated transcription factor activity could be detected here after estrogen stimulation of either MCF-7 or ZR-75.1 cells. Furthermore, these steroids were able to induce activation of the human CCND1 gene promoter, accumulation of cyclin D1 and pRb phosphorylation, all key events in cell cycle stimulation by mitogens, even in the presence of Erk1/2 activation blockade by a MAP kinase-activating kinase (Mek)1/2 inhibitor. Thus, estrogens do not appear to convey significant protein kinase-dependent signaling to the cell nucleus during the early phases of human breast cancer cell stimulation. Furthermore, hormonal regulation of G(1) gene transcription can occur even without additional activation of the Mek-Erk1/2 pathway by estrogen receptors.