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1,3,4-Oxadiazole derivatives are among the most studied anticancer drugs. Previous studies have analyzed the action of different 1,3,4-oxadiazole derivatives and their effects on cancer cells. This study investigated the characterization of two new compounds named 6 and 14 on HeLa and PC-3 cancer cell lines. Based on the previously obtained IC50, cell cycle effects were monitored by flow cytometry. RNA sequencing (RNAseq) was performed to identify differentially expressed genes, followed by functional annotation using gene ontology (GO), KEGG signaling pathway enrichment, and protein–protein interaction (PPI) network analyses. The tubulin polymerization assay was used to analyze the interaction of both compounds with tubulin. The results showed that 6 and 14 strongly inhibited the proliferation of cancer cells by arresting them in the G2/M phase of the cell cycle. Transcriptome analysis showed that exposure of HeLa and PC-3 cells to the compounds caused a marked reprograming of gene expression. Functional enrichment analysis indicated that differentially expressed genes were significantly enriched throughout the cell cycle and cancer-related biological processes. Furthermore, PPI network, hub gene, and CMap analyses revealed that compounds 14 and 6 shared target genes with established microtubule inhibitors, indicating points of similarity between the two molecules and microtubule inhibitors in terms of the mechanism of action. They were also able to influence the polymerization process of tubulin, suggesting the potential of these new compounds to be used as efficient chemotherapeutic agents.

Rhabdomyosarcoma (RMS) is a pediatric tumor, which arises from muscle precursor cells. Recently, it has been demonstrated that Hippo Pathway (Hpo), a pathway that regulates several physiological and biological features, is involved in RMS tumorigenesis. For instance, an upregulation of the Hpo downstream effector Yes-Associated Protein 1 (YAP) leads to the development of embryonal rhabdomyosarcoma (eRMS) in murine activated muscle satellite cells. On the other hand, the YAP paralog transcriptional co-activator with PDZ-binding motif (TAZ) is overexpressed in alveolar rhabdomyosarcoma (aRMS) patients with poor survival. YAP and TAZ exhibit both cytoplasmic and nuclear functions. In the nucleus, YAP binds TEADs (TEA domain family members) factors and together they constitute a complex that is able either to activate the transcription of several genes such as MYC, Tbx5 and PAX8 or to maintain the stability of others like p73. Due to the key role of YAP and TAZ in cancer, the identification and/or development of new compounds able to block their activity might be an effective antineoplastic strategy. Verteporfin (VP) is a molecule able to stop the formation of YAP/TEAD complex in the nucleus. The aim of this study is to evaluate the action of VP on RMS cell lines. This work shows that VP has an anti-proliferative activity on all RMS cell lines analyzed. Depending on RMS cell lines, VP affects cell cycle differently. Moreover, VP is able to decrease YAP protein levels, and to induce the activation of apoptosis mechanism through the cleavage of PARP-1. In addition, Annexin V assay showed the activation of apoptosis and necrosis after VP treatment. In summary, the ability of VP to disrupt RMS cell proliferation could be a novel and valuable strategy to improve the therapeutic approaches in treating rhabdomyosarcoma.

Heart failure of ischemic origin is caused by the presence of a large scar resulting from an acute myocardial infarction. Acute myocardial infarction generally occurs when blood supply to the heart is blocked. Regenerative strategies that limit infarct injury would be able to prevent adverse post-ischemic remodelling and maintain the structural support necessary for effective cardiomyocyte contraction. Our understanding of endogenous cardiac regeneration and its biology has exposed a variety of targets for therapeutic approaches, such as non-coding RNAs, DNA methylation, histone modifications, direct cardiac reprogramming, cell transplantation, stimulation of resident cardiomyocytes, proliferation, and inhibition of cardiomyocyte death. In this review, we address the epigenetic mechanisms underlying these strategies and the use of therapeutic epigenetic molecules or epidrugs.

Squamous cell carcinomas have a range of histopathological manifestations. The parameters that determine this clinically observed heterogeneity are not fully understood. Here, we report the generation of a cell culture model that reflects part of this heterogeneity. We have used the catalytic subunit of human telomerase hTERT and large T to immortalize primary UV-unexposed keratinocytes. Then, mutant HRAS G12V has been introduced to transform these immortal keratinocytes. When injected into immunosuppressed mice, transformed cells grew as xenografts with distinct histopathological characteristics. We observed three major tissue architectures: solid, sarcomatoid and cystic growth types, which were primarily composed of pleomorphic and basaloid cells but in some cases displayed focal apocrine differentiation. We demonstrate that the cells generated represent different stages of skin cancerogenesis and as such can be used to identify novel tumor-promoting alterations such as the overexpression of the PADI2 oncogene in solid-type SCC. Importantly, the cultured cells maintain the characteristics from the xenograft they were derived from while being amenable to manipulation and analysis. The availability of cell lines representing different clinical manifestations opens a new tool to study the stochastic and deterministic factors that cause case-to-case heterogeneity despite departing from the same set of oncogenes and the same genetic background.

Primate-specific NBL2 macrosatellite is hypomethylated in several types of tumors, yet the consequences of this DNA hypomethylation remain unknown. We show that NBL2 conserved repeats are close to the centromeres of most acrocentric chromosomes. NBL2 associates with the perinucleolar region and undergoes severe demethylation in a subset of colorectal cancer (CRC). Upon DNA hypomethylation and histone acetylation, NBL2 repeats are transcribed in tumor cell lines and primary CRCs. NBL2 monomers exhibit promoter activity, and are contained within novel, non-polyA antisense lncRNAs, which we designated TNBL (Tumor-associated NBL2 transcript). TNBL is stable throughout the mitotic cycle, and in interphase nuclei preferentially forms a perinucleolar aggregate in the proximity of a subset of NBL2 loci. TNBL aggregates interact with the SAM68 perinucleolar body in a mirror-image cancer specific perinucleolar structure. TNBL binds with high affinity to several proteins involved in nuclear functions and RNA metabolism, such as CELF1 and NPM1. Our data unveil novel DNA and RNA structural features of a non-coding macrosatellite frequently altered in cancer.

Chromatin remodeling by the SWI/SNF complex is required to activate the transcription of myogenic-specific genes. Our work addressed the details of how SWI/SNF is recruited to myogenic regulatory regions in response to differentiation signals. Surprisingly, the muscle determination factor MyoD and the SWI/SNF subunit BAF60c form a complex on the regulatory elements of MyoD-targeted genes in myogenic precursor cells. This Brg1-devoid MyoD-BAF60c complex flags the chromatin of myogenic-differentiation genes before transcription is activated. On differentiation, BAF60c phosphorylation on a conserved threonine by p38 α kinase promotes the incorporation of MyoD-BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Downregulation of BAF60c expression prevents MyoD access to the chromatin and the proper loading of an active myogenic transcriptosome preventing the expression of hundreds of myogenic genes. Our data support an unprecedented two-step model by which (1) pre-assembled BAF60c-MyoD complex poises the chromatin of myogenic genes for rapid transcription; (2) chromatin-bound BAF60c “senses” the myogenic differentiation cues and recruits an active SWI/SNF complex to remodel the chromatin allowing transcriptional activation.

Tissue-specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodelling complex. However, the molecular mechanism that regulates SWI/SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD-target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal-dependent recruitment of the SWI/SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation-activated p38α kinase is the signal that promotes incorporation of MyoD–BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Our data support an unprecedented two-step model by which pre-assembled BAF60c–MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues.

3′ Repair exonuclease (Trex1) is the most abundant mammalian 3′→5′ DNA exonuclease with specificity for ssDNA. Trex1 deficiency has been linked to the development of autoimmune disease in mice and humans, causing Aicardi-Goutières syndrome in the latter. In addition, polymorphisms in Trex1 are associated with systemic lupus erythematosus. On the basis of all these observations, it has been hypothesized that Trex1 acts by digesting an endogenous DNA substrate. In this study, we report that Trex1 is regulated by IFN-γ during the activation of primary macrophages. IFN-γ upregulates Trex1 with the time course of an early gene, and this induction occurs at the transcription level. The half-life of mRNA is relatively short (half-life of 70 min). The coding sequence of Trex1 has only one exon and an intron of 260 bp in the promoter in the nontranslated mRNA. Three transcription start sites were detected, the one at −580 bp being the most important. In transient transfection experiments using the Trex1 promoter, we have found that two IFN-γ activation site boxes, as well as an adaptor protein complex 1 box, were required for the IFN-γ–dependent induction. By using EMSA assays and chromatin immune precipitation assays, we determined that STAT1 binds to the IFN-γ activation site boxes. The requirement of STAT1 for Trex1 induction was confirmed using macrophages from Stat1 knockout mice. We also establish that c-Jun protein, but not c-Fos, jun-B, or CREB, bound to the adaptor protein complex 1 box. Therefore, our results indicate that IFN-γ induces the expression of the Trex1 exonuclease through STAT1 and c-Jun.

Summary How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified an inflammation-activated signaling in muscle stem (satellite) cells, by which the polycomb repressive complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38α kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EZH2, the enzymatic subunit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. TNF-α antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signaling—p38α and EZH2—invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signaling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signaling to Pax7 and satellite cell decision to proliferate or differentiate.

Myogenin is the dominant transcriptional regulator of embryonic and fetal muscle differentiation and during maturation is profoundly down-regulated. We show that a highly conserved 17-bp DNA cis-acting sequence element located upstream of the myogenin promoter (myogHCE) is essential for postnatal repression of myogenin in transgenic animals. We present multiple lines of evidence supporting the idea that repression is mediated by the Y-box protein MSY-3. Electroporation in vivo shows that myogHCE and MSY-3 are required for postnatal repression. We further show that, in the C2C12 cell culture system, ectopic MSY-3 can repress differentiation, while reduced MSY-3 promotes premature differentiation. MSY-3 binds myogHCE simultaneously with the homeodomain protein Pbx in postnatal innervated muscle. We therefore propose a model in which the myogHCE motif operates as a switch by specifying opposing functions; one that was shown previously is regulated by MyoD and Pbx and it specifies a chromatin opening, gene-activating function at the time myoblasts begin to differentiate; the other includes MYS-3 and Pbx, and it specifies a repression function that operates during and after postnatal muscle maturation in vivo and in myoblasts before they begin to differentiate.

Squamous cell carcinomas have a range of histopathological manifestations. The parameters that determine this clinically observed heterogeneity are not fully understood. Here, we report the generation of a cell culture model that reflects part of this heterogeneity. We have used the catalytic subunit of human telomerase hTERT and large T to immortalize primary UV-unexposed keratinocytes. Then, mutant HRAS G12V has been introduced to transform these immortal keratinocytes. When injected into immunosuppressed mice, transformed cells grew as xenografts with distinct histopathological characteristics. We observed three major tissue architectures: solid, sarcomatoid and cystic growth types, which were primarily composed of pleomorphic and basaloid cells but in some cases displayed focal apocrine differentiation. We demonstrate that the cells generated represent different stages of skin cancerogenesis and as such can be used to identify novel tumor-promoting alterations such as the overexpression of the PADI2 oncogene in solid-type SCC. Importantly, the cultured cells maintain the characteristics from the xenograft they were derived from while being amenable to manipulation and analysis. The availability of cell lines representing different clinical manifestations opens a new tool to study the stochastic and deterministic factors that cause case-to-case heterogeneity despite departing from the same set of oncogenes and the same genetic background.

The Second German-Catalan Workshop on Epigenetics and Cancer was held in Barcelona on November 19–21, 2014. The workshop brought together, for the second time, scientists from 2 German and 2 Catalan research institutions: the DKFZ, from Heidelberg, the CRCME, from Freiburg, and the IMPPC and PEBC/IDIBELL, both from Barcelona. The German-Catalan Workshops are intended to establish the framework for building a Research School to foster collaborations between researchers from the different institutions. Exchange programs for graduate students are among the activities of the future School. The topics presented and discussed in 33 talks were diverse and included work on DNA methylation, histone modifications, chromatin biology, characterization of imprinted regions in human tissues, non-coding RNAs, and epigenetic drug discovery. Among novel developments from the previous Workshop are the report of the epigenetics angle of the Warburg effect and the long-range trans-acting interaction of DNA methylation and of nucleosome remodeling. A shift in the view on DNA methylation became apparent by the realization of the intertwined interplay between hyper- and hypo-methylation in differentiation and cancer.

Transcriptional and posttranscriptional processes regulate expression of genetic networks in response to environmental cues. The extracellular signal-activated p38 MAP kinase (p38) pathway plays a fundamental role in conversion of myoblasts to differentiated myocytes. p38 phosphorylates specific transcription factors and chromatin-associated proteins promoting assembly of the myogenic transcriptome. Here, we demonstrate that p38 alpha and beta isoforms also control muscle-gene expression posttranscriptionally, by stabilizing critical myogenic transcripts. KSRP, an important factor for AU-rich element (ARE)-directed mRNA decay, undergoes p38-dependent phosphorylation during muscle differentiation. KSRP phosphorylated by p38 displays compromised binding to ARE-containing transcripts and fails to promote their rapid decay, although it retains the ability to interact with the mRNA degradation machinery. Overexpression of KSRP selectively impairs induction of ARE-containing early myogenic transcripts, without affecting p38-mediated transcriptional responses. Our results uncover an unanticipated role for KSRP in establishing a biochemical link between differentiation-activated p38 signaling and turnover of myogenic mRNAs.

During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatin-modifying enzymes to muscle-specific loci. The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38 alpha/beta repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38 alpha-beta in vitro, and forced activation of p38 alpha/beta in myoblasts by expression of a constitutively active MKK6 (refs. 5,6,7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements.

Jean-Michel Terme et al., In mammals, the linker histone H1, involved in DNA packaging into chromatin, is represented by a family of variants. H1 tails undergo post-translational modifications (PTMs) that can be detected by mass spectrometry. We developed antibodies to analyze several of these as yet unexplored PTMs including the combination of H1.4 K26 acetylation or trimethylation and S27 phosphorylation. H1.2-T165 phosphorylation was detected at S and G2/M phases of the cell cycle and was dispensable for chromatin binding and cell proliferation; while the H1.4-K26 residue was essential for proper cell cycle progression. We conclude that histone H1 PTMs are dynamic over the cell cycle and that the recognition of modified lysines may be affected by phosphorylation of adjacent residues. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., This work was supported by the Spanish Ministry of Science and Innovation (MICINN) and European Regional Development Fund (Grant BFU2011-23057 to A.J., and Grant BFU2008-00460 to P.S.), and by the Regional Government of Catalonia (Generalitat de Catalunya; Grant 2009-SGR-1222 to A.J.). J.-M.T. received a JAE-Doc contract from the Spanish National Research Council (CSIC)-MICINN; R.M. a TA contract from CSIC-MICINN; and L.M.-A. an FPU predoctoral fellowship from MICINN

Recently, numerous interdisciplinary approaches that include integration of bioinformatics, epigenetics, and molecular and cellular biology have started to shed light on the complex dynamics that regulate cellular processes such as cell “stemness,” reprogramming, and differentiation. From the sequencing of the first complete genomes to the recent high-throughput genomic and proteomic approaches, a huge amount of information has been generated to improve our understanding of how the eukaryotic genome works to activate and coordinate specific programs of gene expression. The focus of this review is to provide an overview of the epigenetic regulatory mechanisms controlling crucial cellular fate decisions, in particular during differentiation and reprogramming. A special emphasis has been given to skeletal myogenesis, as it provides an amenable model for in vitro and in vivo studies. Y. Ito et al. in “A system approach and skeletal myogenesis” provide an overview of the most common approaches currently used to address transcriptional regulation. They discuss deep sequencing and microarray-based methods, cell-based high throughput assays, and the generation of in situ gene expression databases as potent tools to dissect complex networks of gene expression. Using skeletal muscle as a paradigm, they highlight the main discoveries obtained through the different methods and underline the advantages of an integrated approach to obtain a full picture of cellular differentiation. An increasing number of studies suggest an epigenetic component in the development of many human diseases. In the review article “Epigenetic alterations in muscular disorders,” Dr. C. Lanzuolo discusses how altered epigenetic mechanisms contribute to human pathology and in particular to the development and progression of neuromuscular disorders. Muscular dystrophies arise from either mutations of proteins that play a role in the integrity of the sarcolemma—the plasma membrane that wraps the myofibers in skeletal muscles—or are due to a dysfunction within the cell nucleus. Dr. C. Lanzuolo focuses on the so-called nuclear muscular dystrophies and she analyzes the possible contribution of an important group of epigenetic modifiers, the polycomb-group (PcG) complexes, to the pathogenesis of these diseases. The dissection of the aberrant epigenetic profiles associated to several human pathologies has contributed to the design of experimental therapies with epigenetic drugs. The review “Tackling skeletal muscle cells epigenome in the next-generation sequencing era” by R. Fittipaldi and G. Caretti points to the significance of genome-wide study of the epigenetic modifications and the transcription factors that are activated during skeletal muscle differentiation. The authors discuss on the new genome-wide technologies to monitor the epigenetic landscape that characterizes myoblast-to-myotube transition in order to obtain innovative information on molecular mechanisms orchestrating biological processes, such myogenic differentiation. The fine regulation of a myogenic gene such as myogenin is discussed in “Turning on myogenin in muscle: A paradigm for understanding mechanisms of tissue-specific gene expression” by H. Faralli and F. J. Dilworth. Transcription factors, cofactors, and epigenetic modifications contribute to regulate muscle-specific gene expression during development. In particular, the authors highlight the fact that a tissue-specific factors such as MyoD cooperate with more ubiquitously expressed proteins (Six4, Mef2, and Pbx1) to establish a transcriptionally permissive environment to ensure proper spatiotemporal myogenin expression. The current challenge of epigenetics is how to analyse and extract biological knowledge from the large volumes of data produced with new high-throughput technologies. In this regard, the paper of S. Althammer et al., “Predictive models of gene regulation from high-throughput epigenomics data” describes a computational framework using integrative tools and machine learning algorithms that facilitate the systematic analysis of high-throughput sequencing epigenetic data for integrative studies. Their analysis shows a different epigenetic code of expression for intron-less and intron-containing genes, with more prominent differences for genes with low GC content (LGC) in their promoter, showing for instance that at the promoter regions of LGC intron-less genes, H3K36me3 and H3K4me1 are the most informative marks while in polyadenylation sites of expressed genes, the H3K36me3 signal is much weaker than in intron-containing genes. Based on epigenetic data, their model predicts very strongly gene expression between two conditions; therefore, this approach can be applied to study gene expression in different developmental stages, disease states, or treatments. Of tremendous relevance for its therapeutic potential, is the possibility to manipulate cell identity. In the review titled “The stability of the induced epigenetic programs”, M. J. Barrero discusses the epigenetic changes that occur during differentiation and the way back upon cellular reprogramming emphasizing on the thin regulation of pluripotent and lineage-specific genes expression to ensure a correct differentiation during development and in adult life. “The epigenetic regulation of B lymphocyte differentiation, transdifferentiation, and reprogramming” by B. Barneda-Zahonero et al. reviews the hierarchical network of transcription factors that mediate the epigenetic signature regulating the transcriptome during B cell development. B lymphocytes are highly specialized terminally differentiated cells responsible for generating high-affinity antibodies, providing the humoral immunity against pathogens. Every day the human body generates millions of B lymphocytes from precursor hematopoietic stem cells by a differentiation process that is a tightly regulated. This multistep differentiation process, vital to immunity, combines the successive expression of specific transcriptional factors and associated epigenetic changes to restrict the developmental potential of lymphoid progenitors to the B cell pathway by repressing B-lineage-inappropriate genes, while simultaneously promoting B cell development by activating B-lymphoid-specific genes. Lucia Latella Daniela Palacios Sonia Forcales Pier Lorenzo Puri