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Sample records for epigenetic enhancer silencing

  1. Epigenetic silencing in transgenic plants

    PubMed Central

    Rajeevkumar, Sarma; Anunanthini, Pushpanathan; Sathishkumar, Ramalingam

    2015-01-01

    Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review. PMID:26442010

  2. Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

    PubMed Central

    Harris, Robin E; Setiawan, Linda; Saul, Josh; Hariharan, Iswar K

    2016-01-01

    Many organisms lose the capacity to regenerate damaged tissues as they mature. Damaged Drosophila imaginal discs regenerate efficiently early in the third larval instar (L3) but progressively lose this ability. This correlates with reduced damage-responsive expression of multiple genes, including the WNT genes wingless (wg) and Wnt6. We demonstrate that damage-responsive expression of both genes requires a bipartite enhancer whose activity declines during L3. Within this enhancer, a damage-responsive module stays active throughout L3, while an adjacent silencing element nucleates increasing levels of epigenetic silencing restricted to this enhancer. Cas9-mediated deletion of the silencing element alleviates WNT repression, but is, in itself, insufficient to promote regeneration. However, directing Myc expression to the blastema overcomes repression of multiple genes, including wg, and restores cellular responses necessary for regeneration. Localized epigenetic silencing of damage-responsive enhancers can therefore restrict regenerative capacity in maturing organisms without compromising gene functions regulated by developmental signals. DOI: http://dx.doi.org/10.7554/eLife.11588.001 PMID:26840050

  3. Titration and hysteresis in epigenetic chromatin silencing

    NASA Astrophysics Data System (ADS)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

    Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs.

  4. Epigenetic chromatin silencing: bistability and front propagation

    NASA Astrophysics Data System (ADS)

    Sedighi, Mohammad; Sengupta, Anirvan M.

    2007-12-01

    The role of post-translational modification of histones in eukaryotic gene regulation is well recognized. Epigenetic silencing of genes via heritable chromatin modifications plays a major role in cell fate specification in higher organisms. We formulate a coarse-grained model of chromatin silencing in yeast and study the conditions under which the system becomes bistable, allowing for different epigenetic states. We also study the dynamics of the boundary between the two locally stable states of chromatin: silenced and unsilenced. The model could be of use in guiding the discussion on chromatin silencing in general. In the context of silencing in budding yeast, it helps us understand the phenotype of various mutants, some of which may be non-trivial to see without the help of a mathematical model. One such example is a mutation that reduces the rate of background acetylation of particular histone side chains that competes with the deacetylation by Sir2p. The resulting negative feedback due to a Sir protein depletion effect gives rise to interesting counter-intuitive consequences. Our mathematical analysis brings forth the different dynamical behaviors possible within the same molecular model and guides the formulation of more refined hypotheses that could be addressed experimentally.

  5. DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD.

    PubMed

    Lim, Jong-Won; Snider, Lauren; Yao, Zizhen; Tawil, Rabi; Van Der Maarel, Silvère M; Rigo, Frank; Bennett, C Frank; Filippova, Galina N; Tapscott, Stephen J

    2015-09-01

    Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD. PMID:26041815

  6. Epigenetic silencers are enriched in dormant desert frog muscle.

    PubMed

    Hudson, Nicholas J; Lonhienne, T G A; Franklin, Craig E; Harper, Gregory S; Lehnert, S A

    2008-08-01

    Green-striped burrowing frogs, Cyclorana alboguttata, survive droughts by entering a metabolic depression called aestivation, characterised by a reduction in resting oxygen consumption by 80%. Aestivation in C. alboguttata is manifest by transcriptional silencing of skeletal muscle bioenergetic genes, such as NADH ubiquinone oxidoreductase 1, ATP synthase and superoxide dismutase 2. In this study, we hypothesised that aestivation is associated with epigenetic change in frog muscle. We assessed mRNA transcript abundance of seven genes that code for proteins with established roles in epigenetically-mediated gene silencing [transcriptional co-repressor SIN3A, DNA (cytosine-5-) methyltransferase 1, methyl CpG binding protein 2, chromodomain helicase DNA binding protein 4, histone binding protein rbbp4, histone deacetylase 1 and nuclear receptor co-repressor 2] using qRT-PCR. These seven genes showed a modest (1.1-3.5-fold) but coordinated upregulation in 6-month aestivating muscle. This reached significance for SIN3A and DNA cytosine-5-methyltransferase 1 in standard pair-wise comparisons (p < 0.05), and the candidates as a whole when analysed by Fisher's combined probability test (p < 0.01). These data are consistent with the hypothesis that the transcriptional silencing and metabolic depression that occurs during seasonal dormancy are associated with chromatin remodelling, and present a novel example of an environmentally induced epigenetic modification in an adult vertebrate. PMID:18369641

  7. Epigenetic gene silencing in cancer: the DNA hypermethylome.

    PubMed

    Esteller, Manel

    2007-04-15

    Epigenetic gene inactivation in transformed cells involves many 'belts of silencing'. One of the best-known lesions of the malignant cell is the transcriptional repression of tumor-suppressor genes by promoter CpG island hypermethylation. We are in the process of completing the molecular dissection of the entire epigenetic machinery involved in methylation-associated silencing, such as DNA methyltransferases, methyl-CpG binding domain proteins, histone deacetylases, histone methyltransferases, histone demethylases and Polycomb proteins. The first indications are also starting to emerge about how the combination of cellular selection and targeted pathways leads to abnormal DNA methylation. One thing is certain already, promoter CpG island hypermethylation of tumor-suppressor genes is a common hallmark of all human cancers. It affects all cellular pathways with a tumor-type specific profile, and in addition to classical tumor-suppressor and DNA repair genes, it includes genes involved in premature aging and microRNAs with growth inhibitory functions. The importance of hypermethylation events is already in evidence at the bedside of cancer patients in the form of cancer detection markers and chemotherapy predictors, and in the approval of epigenetic drugs for the treatment of hematological malignancies. In the very near future, the synergy of candidate gene approaches and large-scale epigenomic technologies, such as methyl-DIP, will yield the complete DNA hypermethylome of cancer cells. PMID:17613547

  8. Epigenetic silencing of tumor suppressor genes: Paradigms, puzzles, and potential.

    PubMed

    Kazanets, Anna; Shorstova, Tatiana; Hilmi, Khalid; Marques, Maud; Witcher, Michael

    2016-04-01

    Cancer constitutes a set of diseases with heterogeneous molecular pathologies. However, there are a number of universal aberrations common to all cancers, one of these being the epigenetic silencing of tumor suppressor genes (TSGs). The silencing of TSGs is thought to be an early, driving event in the oncogenic process. With this in consideration, great efforts have been made to develop small molecules aimed at the restoration of TSGs in order to limit tumor cell proliferation and survival. However, the molecular forces that drive the broad epigenetic reprogramming and transcriptional repression of these genes remain ill-defined. Undoubtedly, understanding the molecular underpinnings of transcriptionally silenced TSGs will aid us in our ability to reactivate these key anti-cancer targets. Here, we describe what we consider to be the five most logical molecular mechanisms that may account for this widely observed phenomenon: 1) ablation of transcription factor binding, 2) overexpression of DNA methyltransferases, 3) disruption of CTCF binding, 4) elevation of EZH2 activity, 5) aberrant expression of long non-coding RNAs. The strengths and weaknesses of each proposed mechanism is highlighted, followed by an overview of clinical efforts to target these processes. PMID:27085853

  9. Epigenetic silencing of CYP24 in the tumor microenvironment

    PubMed Central

    Johnson, Candace S.; Chung, Ivy; Trump, Donald L.

    2010-01-01

    Calcitriol (1,25 dihydroxycholecalciferol) has significant antitumor activity in vitro and in vivo in a number of tumor model systems. We developed a system for isolation of fresh endothelial cells from tumors and Matrigel environments which demonstrate that CYP24, the catabolic enzyme involved in vitamin D signaling, is epigenetically silenced selectively in tumor-derived endothelial cells (TDEC). TDEC maintain phenotypic characteristics which are distinct from endothelial cells isolated from normal tissues and from Matrigel plugs (MDEC). In TDEC, calcitriol induces G0/G1 arrest, modulates p27 and p21, and induces apoptotic cell death and decreases P-Erk and P-Akt. In contrast, endothelial cells isolated from normal tissues and MDEC are unresponsive to calcitriol-mediated anti-proliferative effects despite intact signaling through the vitamin D receptor (VDR). In TDEC, which is sensitive to calcitriol, the CYP24 promoter is hypermethylated in two CpG island regions located at the 5′end; this hypermethylation may contribute to gene silencing of CYP24. The extent of methylation in these two regions is significantly less in MDEC. Lastly, treatment of TDEC with a DNA methyltransferase inhibitor restores calcitriol-mediated induction of CYP24 and resistance to calcitriol. These data suggest that epigenetic silencing of CYP24 modulates cellular responses to calcitriol. PMID:20304059

  10. Epigenetic silencing of AKAP12 in juvenile myelomonocytic leukemia.

    PubMed

    Wilhelm, Thomas; Lipka, Daniel B; Witte, Tania; Wierzbinska, Justyna A; Fluhr, Silvia; Helf, Monika; Mücke, Oliver; Claus, Rainer; Konermann, Carolin; Nöllke, Peter; Niemeyer, Charlotte M; Flotho, Christian; Plass, Christoph

    2016-02-01

    A-kinase anchor protein 12 (AKAP12) is a regulator of protein kinase A and protein kinase C signaling, acting downstream of RAS. Epigenetic silencing of AKAP12 has been demonstrated in different cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative high-resolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three AKAP12 promoters (i.e., α, β, and γ) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The AKAP12α promoter shows DNA hypermethylation in JMML samples, which is associated with decreased AKAP12α expression. Promoter methylation of AKAP12α correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. In silico screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the AKAP12α promoter revealed highly significant scores for GATA-2/-1 sequence motifs. Both transcription factors are known to be involved in the haematopoietic differentiation process. Methylation of a reporter construct containing this region resulted in strong suppression of AKAP12 promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the AKAP12 promoter in JMML. Exposure to DNMT- and HDAC-inhibitors reactivates AKAP12α expression in vitro, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of AKAP12α in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis. PMID:26891149

  11. Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana

    PubMed Central

    Bond, Donna M.; Baulcombe, David C.

    2015-01-01

    In plants, RNA-directed DNA methylation (RdDM), a mechanism where epigenetic modifiers are guided to target loci by small RNAs, plays a major role in silencing of transposable elements (TEs) to maintain genome integrity. So far, two RdDM pathways have been identified: RNA Polymerase IV (PolIV)-RdDM and RNA-dependent RNA Polymerase 6 (RDR6)-RdDM. PolIV-RdDM involves a self-reinforcing feedback mechanism that maintains TE silencing, but cannot explain how epigenetic silencing is first initiated. A function of RDR6-RdDM is to reestablish epigenetic silencing of active TEs, but it is unknown if this pathway can induce DNA methylation at naïve, non-TE loci. To investigate de novo establishment of RdDM, we have used virus-induced gene silencing (VIGS) of an active FLOWERING WAGENINGEN epiallele. Using genetic mutants we show that unlike PolIV-RdDM, but like RDR6-RdDM, establishment of VIGS-mediated RdDM requires PolV and DRM2 but not Dicer like-3 and other PolIV pathway components. DNA methylation in VIGS is likely initiated by a process guided by virus-derived small (s) RNAs that are 21/22-nt in length and reinforced or maintained by 24-nt sRNAs. We demonstrate that VIGS-RdDM as a tool for gene silencing can be enhanced by use of mutant plants with increased production of 24-nt sRNAs to reinforce the level of RdDM. PMID:25561534

  12. Epigenetic silencing of Th1 type chemokines shapes tumor immunity and immunotherapy

    PubMed Central

    Peng, Dongjun; Kryczek, Ilona; Nagarsheth, Nisha; Zhao, Lili; Wei, Shuang; Wang, Weimin; Sun, Yuqing; Zhao, Ende; Vatan, Linda; Szeliga, Wojciech; Kotarski, Jan; Tarkowski, Rafał; Dou, Yali; Cho, Kathleen; Hensley-Alford, Sharon; Munkarah, Adnan; Liu, Rebecca; Zou, Weiping

    2015-01-01

    Summary Epigenetic silencing including histone modifications and DNA methylation is an important tumorigenic mechanism1 However, its role in cancer immunopathology and immunotherapy is poorly understood. Using ovarian cancers as our model, we found that enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) and DNA methyltransferase (DNMT) 1-mediated DNA methylation repress the tumor production of Th1-type chemokines CXCL9 and CXCL10, and subsequently determine effector T cell trafficking to the tumor microenvironment. Treatment with epigenetic modulators removes the repression and increases effector T cell tumor infiltration, slows down tumor progression, and improves therapeutic efficacy of PD-L1 (B7-H1) checkpoint blockade2–4 and adoptive T cell transfusion5 in tumor bearing mice. Moreover, tumor EZH2 and DNMT1 are negatively associated with tumor infiltrating CD8+ T cells and patient outcome. Thus, epigenetic silencing of Th1-type chemokine is a novel tumor immune evasion mechanism. Selective epigenetic reprogramming alters T cell landscape6 in cancer and may enhance clinical efficacy of cancer therapy. PMID:26503055

  13. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

    PubMed

    Tchasovnikarova, Iva A; Timms, Richard T; Matheson, Nicholas J; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A; Lehner, Paul J

    2015-06-26

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing. PMID:26022416

  14. Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas.

    PubMed Central

    Cerutti, H; Johnson, A M; Gillham, N W; Boynton, J E

    1997-01-01

    The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants. In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s). Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells. Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I. Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila. By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation. PMID:9212467

  15. Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome.

    PubMed

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D; Jaffrey, Samie R

    2014-02-28

    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide-repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5' untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA. PMID:24578575

  16. Promoter-Bound Trinucleotide Repeat mRNA Drives Epigenetic Silencing in Fragile X Syndrome

    PubMed Central

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S.; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D.; Jaffrey, Samie R.

    2015-01-01

    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide–repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5′ untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA. PMID:24578575

  17. A role for DNA polymerase alpha in epigenetic control of transcriptional silencing in fission yeast.

    PubMed

    Nakayama Ji; Allshire, R C; Klar, A J; Grewal, S I

    2001-06-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase alpha (pol(alpha)) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that pol(alpha) mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polalpha and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Pol(alpha) in vitro. Moreover, silencing-defective mutant Pol(alpha) displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Pol(alpha), in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  18. Induction of stable epigenetic gene silencing in plants using a virus vector.

    PubMed

    Kanazawa, Akira; Kasai, Megumi

    2015-01-01

    Gene silencing through transcriptional repression can be induced by double-stranded RNA targeted to a gene promoter, a process known as RNA-mediated transcriptional gene silencing (TGS). This phenomenon is associated with epigenetic changes involving cytosine methylation of the promoter. Plant virus vectors have been used to induce RNA-mediated TGS. Here, we describe methods relevant to the induction of epigenetic changes and RNA-mediated TGS in plants using a virus vector, which include inoculation of recombinant virus, detection of short interfering RNAs, bisulfite sequencing analysis, and nuclear run-on transcription assay. PMID:25740361

  19. Polycomb PRC2 complex mediates epigenetic silencing of a critical osteogenic master regulator in the hippocampus.

    PubMed

    Aguilar, Rodrigo; Bustos, Fernando J; Saez, Mauricio; Rojas, Adriana; Allende, Miguel L; van Wijnen, Andre J; van Zundert, Brigitte; Montecino, Martin

    2016-08-01

    During hippocampal neuron differentiation, the expression of critical inducers of non-neuronal cell lineages must be efficiently silenced. Runx2 transcription factor is the master regulator of mesenchymal cells responsible for intramembranous osteoblast differentiation and formation of the craniofacial bone tissue that surrounds and protects the central nervous system (CNS) in mammalian embryos. The molecular mechanisms that mediate silencing of the Runx2 gene and its downstream target osteogenic-related genes in neuronal cells have not been explored. Here, we assess the epigenetic mechanisms that mediate silencing of osteoblast-specific genes in CNS neurons. In particular, we address the contribution of histone epigenetic marks and histone modifiers on the silencing of the Runx2/p57 bone-related isoform in rat hippocampal tissues at embryonic to adult stages. Our results indicate enrichment of repressive chromatin histone marks and of the Polycomb PRC2 complex at the Runx2/p57 promoter region. Knockdown of PRC2 H3K27-methyltransferases Ezh2 and Ezh1, or forced expression of the Trithorax/COMPASS subunit Wdr5 activates Runx2/p57 mRNA expression in both immature and mature hippocampal cells. Together these results indicate that complementary epigenetic mechanisms progressively and efficiently silence critical osteoblastic genes during hippocampal neuron differentiation. PMID:27216774

  20. Epigenetic Inheritance of Transcriptional Silencing and Switching Competence in Fission Yeast

    PubMed Central

    Thon, G.; Friis, T.

    1997-01-01

    Epigenetic events allow the inheritance of phenotypic changes that are not caused by an alteration in DNA sequence. Here we characterize an epigenetic phenomenon occuring in the mating-type region of fission yeast. Cells of fission yeast switch between the P and M mating-type by interconverting their expressed mating-type cassette between two allelic forms, mat1-P and mat1-M. The switch results from gene conversions of mat1 by two silent cassettes, mat2-P and mat3-M, which are linked to each other and to mat1. GREWAL and KLAR observed that the ability to both switch mat1 and repress transcription near mat2-P and mat3-M was maintained epigenetically in a strain with an 8-kb deletion between mat2 and mat3. Using a strain very similar to theirs, we determined that interconversions between the switching-and silencing-proficient state and the switching and silencing-deficient state occurred less frequently than once per 1000 cell divisions. Although transcriptional silencing was alleviated by the 8-kb deletion, it was not abolished. We performed a mutant search and obtained a class of trans-acting mutations that displayed a strong cumulative effect with the 8-kb deletion. These mutations allow to assess the extent to which silencing is affected by the deletion and provide new insights on the redundancy of the silencing mechanism. PMID:9055078

  1. Breaking an Epigenetic Chromatin Switch: Curious Features of Hysteresis in Saccharomyces cerevisiae Telomeric Silencing

    PubMed Central

    Nagaraj, Vijayalakshmi H.; Mukhopadhyay, Swagatam; Dayarian, Adel; Sengupta, Anirvan M.

    2014-01-01

    In addition to gene network switches, local epigenetic modifications to DNA and histones play an important role in all-or-none cellular decision-making. Here, we study the dynamical design of a well-characterized epigenetic chromatin switch: the yeast SIR system, in order to understand the origin of the stability of epigenetic states. We study hysteresis in this system by perturbing it with a histone deacetylase inhibitor. We find that SIR silencing has many characteristics of a non-linear bistable system, as observed in conventional genetic switches, which are based on activities of a few promoters affecting each other through the abundance of their gene products. Quite remarkably, our experiments in yeast telomeric silencing show a very distinctive pattern when it comes to the transition from bistability to monostability. In particular, the loss of the stable silenced state, upon increasing the inhibitor concentration, does not seem to show the expected saddle node behavior, instead looking like a supercritical pitchfork bifurcation. In other words, the ‘off’ state merges with the ‘on’ state at a threshold concentration leading to a single state, as opposed to the two states remaining distinct up to the threshold and exhibiting a discontinuous jump from the ‘off’ to the ‘on’ state. We argue that this is an inevitable consequence of silenced and active regions coexisting with dynamic domain boundaries. The experimental observations in our study therefore have broad implications for the understanding of chromatin silencing in yeast and beyond. PMID:25536038

  2. Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes.

    PubMed

    Onorati, Maria Cristina; Arancio, Walter; Cavalieri, Vincenzo; Ingrassia, Antonia M R; Pavesi, Giulio; Corona, Davide F V

    2015-08-01

    In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA's and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes. PMID:26292210

  3. Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes

    PubMed Central

    Cavalieri, Vincenzo; Ingrassia, Antonia M. R.; Pavesi, Giulio; Corona, Davide F. V.

    2015-01-01

    In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA’s and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes. PMID:26292210

  4. Cutting Edge: EZH2 Promotes Osteoclastogenesis by Epigenetic Silencing of the Negative Regulator IRF8.

    PubMed

    Fang, Celestia; Qiao, Yu; Mun, Se Hwan; Lee, Min Joon; Murata, Koichi; Bae, Seyeon; Zhao, Baohong; Park-Min, Kyung-Hyun; Ivashkiv, Lionel B

    2016-06-01

    Osteoclasts are resorptive cells that are important for homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role for epigenetic mechanisms in osteoclastogenesis. A recent study showed that epigenetic silencing of the negative regulator of osteoclastogenesis Irf8 by DNA methylation is required for osteoclast differentiation. In this study, we investigated the role of EZH2, which epigenetically silences gene expression by histone methylation, in osteoclastogenesis. Inhibition of EZH2 by the small molecule GSK126, or decreasing its expression using antisense oligonucleotides, impeded osteoclast differentiation. Mechanistically, EZH2 was recruited to the IRF8 promoter after RANKL stimulation to deposit the negative histone mark H3K27me3 and downregulate IRF8 expression. GSK126 attenuated bone loss in the ovariectomy mouse model of postmenopausal osteoporosis. Our findings provide evidence for an additional mechanism of epigenetic IRF8 silencing during osteoclastogenesis that likely works cooperatively with DNA methylation, further emphasizing the importance of IRF8 as a negative regulator of osteoclastogenesis. PMID:27183582

  5. Heat-Induced Release of Epigenetic Silencing Reveals the Concealed Role of an Imprinted Plant Gene

    PubMed Central

    Sanchez, Diego H.; Paszkowski, Jerzy

    2014-01-01

    Epigenetic mechanisms suppress the transcription of transposons and DNA repeats; however, this suppression can be transiently released under prolonged heat stress. Here we show that the Arabidopsis thaliana imprinted gene SDC, which is silent during vegetative growth due to DNA methylation, is activated by heat and contributes to recovery from stress. SDC activation seems to involve epigenetic mechanisms but not canonical heat-shock perception and signaling. The heat-mediated transcriptional induction of SDC occurs particularly in young developing leaves and is proportional to the level of stress. However, this occurs only above a certain window of absolute temperatures and, thus, resembles a thermal-sensing mechanism. In addition, the re-silencing kinetics during recovery can be entrained by repeated heat stress cycles, suggesting that epigenetic regulation in plants may conserve memory of stress experience. We further demonstrate that SDC contributes to the recovery of plant biomass after stress. We propose that transcriptional gene silencing, known to be involved in gene imprinting, is also co-opted in the specific tuning of SDC expression upon heat stress and subsequent recovery. It is therefore possible that dynamic properties of the epigenetic landscape associated with silenced or imprinted genes may contribute to regulation of their expression in response to environmental challenges. PMID:25411840

  6. Biological and clinical significance of epigenetic silencing of MARVELD1 gene in lung cancer

    PubMed Central

    Shi, Ming; Wang, Shan; Yao, Yuanfei; Li, Yiqun; Zhang, Hao; Han, Fang; Nie, Huan; Su, Jie; Wang, Zeyu; Yue, Lei; Cao, Jingyan; Li, Yu

    2014-01-01

    Epigenetic silence in cancer frequently altered signal-transduction pathways during the early stages of tumor development. Recent progress in the field of cancer epigenetics has led to new opportunities for diagnosis and treatment of cancer. We previously demonstrated that novel identified nuclear factor MARVELD1 was widely expressed in human tissues, but down-regulated by promoter methylation in multiple cancers. This study was carried out to determine the biological and clinical significance of MARVELD1 gene silencing in lung cancer. Here, we found the reduced MARVELD1 expression significantly correlated with diagnostic histopathology and malignant degree of lung cancers. DNA hypermethylation and histone deacetylation synergistically inactivated MARVELD1 gene in lung cancer cells. Moreover, MARVELD1 modulated the efficiency of nonsense-mediated mRNA decay (NMD) through interaction with NMD core factor SMG1. The decreased MARVELD1 level in lung cancer reduces NMD efficiency through diminishing the association between NMD complex component UPF1/SMG1 and premature termination codons containing mRNA (PTC-mRNA). The results suggested that MARVELD1 silencing is an appealing diagnostic biomarker for lung cancer and epigenetic silencing of MARVELD1 gene links with the regulatory mechanism of NMD pathway in lung cancer, which may be required for tumorigenesis. PMID:25520033

  7. LHP1, the Arabidopsis homologue of HETEROCHROMATIN PROTEIN1, is required for epigenetic silencing of FLC

    PubMed Central

    Mylne, Joshua S.; Barrett, Lynne; Tessadori, Federico; Mesnage, Stéphane; Johnson, Lianna; Bernatavichute, Yana V.; Jacobsen, Steven E.; Fransz, Paul; Dean, Caroline

    2006-01-01

    Vernalization is the acceleration of flowering by prolonged cold that aligns the onset of reproductive development with spring conditions. A key step of vernalization in Arabidopsis is the epigenetic silencing of FLOWERING LOCUS C (FLC), which encodes a repressor of flowering. The vernalization-induced epigenetic silencing of FLC is associated with histone deacetylation and H3K27me2 and H3K9me2 methylation mediated by VRN/VIN proteins. We have analyzed whether different histone methyltransferases and the chromodomain protein LIKE HETEROCHROMATIN PROTEIN (LHP)1 might play a role in vernalization. No single loss-of-function mutation in the histone methyltransferases studied disrupted the vernalization response; however, lhp1 mutants revealed a role for LHP1 in maintaining epigenetic silencing of FLC. Like LHP1, VRN1 functions in both flowering-time control and vernalization. We explored the localization of VRN1 and found it to be associated generally with Arabidopsis chromosomes but not the heterochromatic chromocenters. This association did not depend on vernalization or VRN2 function and was maintained during mitosis but was lost in meiotic chromosomes, suggesting that VRN1 may contribute to chromatin silencing that is not meiotically stable. PMID:16549797

  8. Epigenetic silencing of EYA2 in pancreatic adenocarcinomas promotes tumor growth

    PubMed Central

    Vincent, Audrey; Hong, Seung-Mo; Hu, Chaoxin; Omura, Noriyuki; Young, Angela; Kim, Haeryoung; Yu, Jun; Knight, Spencer; Ayars, Michael; Griffith, Margaret; Van Seuningen, Isabelle; Maitra, Anirban; Goggins, Michael

    2014-01-01

    To identify potentially important genes dysregulated in pancreatic cancer, we analyzed genome-wide transcriptional analysis of pancreatic cancers and normal pancreatic duct samples and identified the transcriptional coactivator, EYA2 (Drosophila Eyes Absent Homologue-2) as silenced in the majority of pancreatic cancers. We investigated the role of epigenetic mechanisms of EYA2 gene silencing in pancreatic cancers, performed in vitro and in vivo proliferation and migration assays to assess the effect of EYA2 silencing on tumor cell growth and metastasis formation, and expression analysis to identify genes transcriptionally regulated by EYA2. We found loss of tumoral Eya2 expression in 63% of pancreatic cancers (120/189 cases). Silencing of EYA2 expression in pancreatic cancer cell lines correlated with promoter methylation and histone deacetylation and was reversible with DNA methyltransferase and HDAC inhibitors. EYA2 knockdown in pancreatic cancer cell lines increased cell proliferation. Compared to parental pancreatic cancer cells, pancreatic cancers stably-expressing EYA2 grew more slowly and had fewer metastases in orthotopic models. The transcriptional changes after stable expression of EYA2 in pancreatic cancer cells included induction of genes in the TGFbeta pathway. Epigenetic silencing of EYA2 is a common event in pancreatic cancers and stable expression EYA2 limits the growth and metastases of pancreatic adenocarcinoma. PMID:24810906

  9. Sustained IL-6/STAT-3 Signaling in Cholangiocarcinoma Cells due to SOCS-3 Epigenetic Silencing

    PubMed Central

    Isomoto, Hajime; Mott, Justin L.; Kobayashi, Shogo; Werneburg, Nathan W.; Bronk, Steve F.; Haan, Serge; Gores, Gregory J.

    2008-01-01

    Background and aims IL-6 mediated STAT-3 phosphorylation (activation) is aberrantly sustained in cholangiocarcinoma cells resulting in enhanced Mcl-1 expression and resistance to apoptosis. Because SOCS-3 controls the IL-6/STAT-3 signaling pathway by a classic feedback loop, the aims of this study were to examine SOCS-3 regulation in human cholangiocarcinoma. Methods SOCS-3 expression was assessed in human cholangiocarcinoma tissue and the Mz-ChA-1 and CCLP1 human cholangiocarcinoma cell lines. Results An inverse correlation was observed between phospho-STAT-3 and SOCS-3 protein expression in cholangiocarcinoma. In those cancers failing to express SOCS-3, extensive methylation of the SOCS-3 promoter was demonstrated in tumor but not in paired non-tumor tissue. Likewise, methylation of the socs-3 promoter was also identified in two cholangiocarcinoma cell lines. Treatment with a demethylating agent, 5-aza-2′-deoxycytidine (DAC), restored IL-6 induction of SOCS-3, terminated the phospho-STAT-3 response, and reduced cellular levels of Mcl-1. Enforced expression of SOCS-3 also reduced IL-6 induction of phospho-STAT-3 and Mcl-1. Either DAC treatment or enforced SOCS-3 expression sensitized the cells to TRAIL-mediated apoptosis. Conclusion SOCS-3 epigenetic silencing is responsible for sustained IL-6/STAT-3 signaling and enhanced Mcl-1 expression in cholangiocarcinoma. PMID:17241887

  10. Epigenetic silencing of GDF1 disrupts SMAD signaling to reinforce gastric cancer development.

    PubMed

    Yang, W; Mok, M T S; Li, M S M; Kang, W; Wang, H; Chan, A W; Chou, J-L; Chen, J; Ng, E K W; To, K-F; Yu, J; Chan, M W Y; Chan, F K L; Sung, J J Y; Cheng, A S L

    2016-04-21

    Accumulating evidence reveals the effectiveness of epigenetic therapy in gastric cancer. However, the molecular mechanisms and targets underlying such therapeutic responses remain elusive. Herein, we report an aberrant yet therapeutically rectifiable epigenetic signaling in gastric carcinogenesis. Administration of DNA-demethylating drug 5-aza-2'-deoxycytidine (5-aza-dC) reduced gastric cancer incidence by ~74% (P<0.05) in N-nitroso-N-methylurea-treated mice. Through genome-wide methylation scanning, novel promoter hypermethylation-silenced and drug-targeted genes were identified in the resected murine stomach tumors and tissues. We uncovered that growth/differentiation factor 1 (Gdf1), a member of the transforming growth factor-β superfamily, was silenced by promoter hypermethylation in control tumor-bearing mice, but became reactivated in 5-aza-dC-treated mice (P<0.05). In parallel, the downregulated SMAD2/3 phosphorylation in gastric cancer was revived by 5-aza-dC in vivo. Such hypermethylation-dependent silencing and 5-aza-dC-mediated reactivation of GDF1-SMAD2/3 activity was conserved in human gastric cancer cells (P<0.05). Subsequent functional characterization further revealed the antiproliferative activity of GDF1, which was exerted through activation of SMAD2/3/4-mediated signaling, transcriptional controls on p15, p21 and c-Myc cell-cycle regulators and phosphorylation of retinoblastoma protein. Clinically, hypermethylation and loss of GDF1 was significantly associated with reduced phosphorylated-SMAD2/3 and poor survival in stomach cancer patients (P<0.05). Taken together, we demonstrated a causal relationship between DNA methylation and a tumor-suppressive pathway in gastric cancer. Epigenetic silencing of GDF1 abrogates the growth-inhibitory SMAD signaling and renders proliferation advantage to gastric epithelial cells during carcinogenesis. This study lends support to epigenetic therapy for gastric cancer chemoprevention and identifies a potential

  11. Arabidopsis DNA polymerase ϵ recruits components of Polycomb repressor complex to mediate epigenetic gene silencing.

    PubMed

    Del Olmo, Iván; López, Juan A; Vázquez, Jesús; Raynaud, Cécile; Piñeiro, Manuel; Jarillo, José A

    2016-07-01

    Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ϵ involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ϵ is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1 We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells. PMID:26980282

  12. Epigenetic silencing of ARRDC3 expression in basal-like breast cancer cells

    NASA Astrophysics Data System (ADS)

    Soung, Young Hwa; Pruitt, Kevin; Chung, Jun

    2014-01-01

    Arrestin domain-containing 3 (ARRDC3) is a tumor suppressor whose expression is either lost or suppressed in basal-like breast cancer (BLBC). However, the mechanism by which BLBC suppresses ARRDC3 expression is not established. Here, we show that expression of ARRDC3 in BLBC cells is suppressed at the transcriptional level. Suppression of ARRDC3 expression in BLBC cells involves epigenetic silencing as inhibitors of class III histone deacetylases (HDACs) significantly restores ARRDC3 levels in BLBC cells. SIRT2, among class III HDACs, plays a major role in epigenetic silencing of ARRDC3 in MDA-MB-231 cells. Acetylation levels of the ARRDC3 promoter in BLBC cells is significantly lower than that of other sub-types of BC cells. Chromatin immunopreciptitation analysis established SIRT2 binding at ARRDC3 promoter in BLBC cells. Our studies indicate that SIRT2 dependent epigenetic silencing of ARRDC3 is one of the important events that may contribute to the aggressive nature of BLBC cells.

  13. Arabidopsis DNA polymerase ϵ recruits components of Polycomb repressor complex to mediate epigenetic gene silencing

    PubMed Central

    del Olmo, Iván; López, Juan A.; Vázquez, Jesús; Raynaud, Cécile; Piñeiro, Manuel; Jarillo, José A.

    2016-01-01

    Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ϵ involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ϵ is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1. We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells. PMID:26980282

  14. Barriers and silencers: a theoretical toolkit for control and containment of nucleosome-based epigenetic states.

    PubMed

    Dodd, Ian B; Sneppen, Kim

    2011-12-01

    Positive feedback in nucleosome modification has been proposed to allow large chromatin regions to exist stably and heritably in distinct expression states. However, modeling has shown that such epigenetic bistability requires that modifying enzymes recruited by nucleosomes are active on distant nucleosomes, potentially allowing uncontrollable spreading of modification. By modeling the silencing of mating-type loci in Saccharomyces cerevisiae, we show that a modification reaction that combines a long-range component and a locally acting component can provide bistability and can be blocked by simple barriers that interrupt the nucleosome chain. We find that robust containment of the silenced region could be achieved by the presence of a number of weak simple barriers in the surrounding chromatin and a limited capacity of the positive feedback reaction. In addition, we show that the state of the silenced region can be regulated by silencer elements acting only on neighboring nucleosomes. Thus, a relatively simple set of nucleosome-modifying enzymes and recognition domains is all that is needed to make chromatin-based epigenetics useful and safe. PMID:22037584

  15. The C. elegans CSR-1 Argonaute pathway counteracts epigenetic silencing to promote germline gene expression

    PubMed Central

    Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C.

    2014-01-01

    SUMMARY Organisms can develop adaptive sequence-specific immunity by re-expressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piRNA pathway recruits RNA-dependent RNA polymerase RdRP to foreign sequences to amplify a trans-generational small RNA-induced epigenetic silencing signal (termed RNAe). Here we provide evidence that in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self mRNAs. We refer to this mechanism, which can prevent or reverse RNAe as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a trans-generational CSR-1 memory that recognizes and protects self mRNAs, allowing piRNAs to recognize foreign sequences innately, without need for prior exposure. PMID:24360782

  16. RASSF10 is epigenetically silenced and functions as a tumor suppressor in gastric cancer

    SciTech Connect

    Wei, Ziran; Chen, Xia; Chen, Ji; Wang, Weimin; Xu, Xudong; Cai, Qingping

    2013-03-22

    Highlights: ► Epigenetic silencing of RASSF10 gene expression in GC cells. ► RASSF10 overexpression inhibits cell growth in vitro and in vivo. ► RASSF10 induces apoptosis in GC cells. ► RASSF10 inhibits Wnt/β-catenin signaling pathway. -- Abstract: Ras association domain family (RASSF) proteins are encoded by several tumor suppressor genes that are frequently silenced in human cancers. In this study, we investigated RASSF10 as a target of epigenetic inactivation and examined its functions as a tumor suppressor in gastric cancer. RASSF10 was silenced in six out of eight gastric cancer cell lines. Loss or downregulation of RASSF10 expression was associated with promoter hypermethylation, and could be restored by a demethylating agent. Overexpression of RASSF10 in gastric cancer cell lines (JRST, BGC823) suppressed cell growth and colony formation, and induced apoptosis, whereas RASSF10 depletion promoted cell growth. In xenograft animal experiments, RASSF10 overexpression effectively repressed tumor growth. Mechanistic investigations revealed that RASSF10 inhibited tumor growth by blocking activation of β-catenin and its downstream targets including c-Myc, cyclinD1, cyclinE1, peroxisome proliferator-activated receptor δ, transcription factor 4, transcription factor 1 and CD44. In conclusion, the results of this study provide insight into the role of RASSF10 as a novel functional tumor suppressor in gastric cancer through inhibition of the Wnt/β-catenin signaling pathway.

  17. Reversal of epigenetic promoter silencing in Friedreich ataxia by a class I histone deacetylase inhibitor

    PubMed Central

    Chutake, Yogesh K.; Lam, Christina C.; Costello, Whitney N.; Anderson, Michael P.; Bidichandani, Sanjay I.

    2016-01-01

    Friedreich ataxia, the most prevalent inherited ataxia, is caused by an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene. Repressive chromatin spreads from the expanded GAA triplet-repeat sequence to cause epigenetic silencing of the FXN promoter via altered nucleosomal positioning and reduced chromatin accessibility. Indeed, deficient transcriptional initiation is the predominant cause of transcriptional deficiency in Friedreich ataxia. Treatment with 109, a class I histone deacetylase (HDAC) inhibitor, resulted in increased level of FXN transcript both upstream and downstream of the expanded GAA triplet-repeat sequence, without any change in transcript stability, suggesting that it acts via improvement of transcriptional initiation. Quantitative analysis of transcriptional initiation via metabolic labeling of nascent transcripts in patient-derived cells revealed a >3-fold increase (P < 0.05) in FXN promoter function. A concomitant 3-fold improvement (P < 0.001) in FXN promoter structure and chromatin accessibility was observed via Nucleosome Occupancy and Methylome Sequencing, a high-resolution in vivo footprint assay for detecting nucleosome occupancy in individual chromatin fibers. No such improvement in FXN promoter function or structure was observed upon treatment with a chemically-related inactive compound (966). Thus epigenetic promoter silencing in Friedreich ataxia is reversible, and the results implicate class I HDACs in repeat-mediated promoter silencing. PMID:26896803

  18. A role for DNA polymerase α in epigenetic control of transcriptional silencing in fission yeast

    PubMed Central

    Nakayama, Jun-ichi; Allshire, Robin C.; Klar, Amar J.S.; Grewal, Shiv I.S.

    2001-01-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase α (polα) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that polα mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polα and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Polα in vitro. Moreover, silencing-defective mutant Polα displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Polα, in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  19. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells*

    PubMed Central

    Matheson, Nicholas J.; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A.; Lehner, Paul J.

    2015-01-01

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A non-lethal forward genetic screen in near-haploid KBM7 cells identified the Human Silencing Hub (HUSH), a complex of three poorly-characterised proteins, TASOR, MPP8, and periphilin, which is absent from Drosophila but conserved from fish to humans. Loss of HUSH subunits resulted in decreased H3K9me3 at both endogenous genomic loci and retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing. PMID:26022416

  20. Epigenetic Switch from Posttranscriptional to Transcriptional Silencing Is Correlated with Promoter Hypermethylation1

    PubMed Central

    Fojtova, Miloslava; Van Houdt, Helena; Depicker, Anna; Kovarik, Ales

    2003-01-01

    Changes in the distribution of methylcytosine residues along a transgene locus of tobacco (Nicotiana tabacum) in relation to the type of gene silencing were studied in parental plant leaves, calli, and regenerated plants derived thereof. Parental-silenced HeLo1 (hemizygous for locus 1) plants show posttranscriptional silencing of the residing nptII (neomycin phosphotransferase II) transgene and cytosine methylation restricted to the 3′ end and center part of the transcribed region. Here, we report that with an increasing number of cell cycles, DNA methylation changes gradually, and methylation is introduced into the promoter during cell culture and more slowly in vegetatively propagated plants. After 24 months of callus in vitro cultivation, an epigenetic variant, designated locus 1E, was obtained in which cytosine methylation of symmetrical (CG and CNG) sites was almost complete within the 5′ end of the nptII-transcribed region and the 35S promoter. Further, methylation of nonsymmetrical sites appeared de novo in the promoter, whereas this type of methylation was significantly reduced in the 3′ end of the transcribed region when compared with locus 1. The newly established epigenetic patterns were stably transmitted from calli into regenerated plants and their progeny. The protein and steady-state RNA levels remained low in locus 1E, whereas with nuclear run-on assays, no detectable amounts of primary transcripts were found along the nptII gene, indicating that the methylated promoter became inactivated. The results suggest that a switch between posttranscriptional and transcriptional gene silencing could be a mechanism leading to irrevocable shut down of gene expression within a finite number of generations. PMID:14551338

  1. DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis

    PubMed Central

    Yelina, Nataliya E.; Lambing, Christophe; Hardcastle, Thomas J.; Zhao, Xiaohui; Santos, Bruno; Henderson, Ian R.

    2015-01-01

    During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes. PMID:26494791

  2. Genome-wide unmasking of epigenetically silenced genes in lung adenocarcinoma from smokers and never smokers

    PubMed Central

    Yingling, Christin M.; Liu, Yushi; Tellez, Carmen S.; Van Neste, Leander; Baylin, Stephen S.; Belinsky, Steven A.

    2014-01-01

    Lung cancer in never smokers (NS) shows striking demographic, clinicopathological and molecular distinctions from the disease in smokers (S). Studies on selected genetic and epigenetic alterations in lung cancer identified that the frequency and profile of some abnormalities significantly differ by smoking status. This study compared the transcriptome of lung adenocarcinoma cell lines derived from S (n = 3) and NS (n = 3) each treated with vehicle (control), histone deacetylation inhibitor (trichostatin A) or DNA methylation inhibitor (5-aza-2′-deoxycytidine). Among 122 genes reexpressed following 5-aza-2′-deoxycytidine but not trichostatin A treatment in two or more cell lines (including 32 genes in S-only and 12 NS-only), methylation was validated for 80% (98/122 genes). After methylation analysis of 20 normal tissue samples and 14 additional non–small cell lung cancer cell lines (total 20), 39 genes frequently methylated in normal (>20%, 4/20) and 21 genes rarely methylated in non–small cell lung cancer (≤10%, 2/20) were excluded. The prevalence for methylation of the remaining 38 genes in lung adenocarcinomas from S (n = 97) and NS (n = 75) ranged from 8–89% and significantly differs between S and NS for CPEB1, CST6, EMILIN2, LAYN and MARVELD3 (P < 0.05). Furthermore, methylation of EMILIN2, ROBO3 and IGDCC4 was more prevalent in advanced (Stage II–IV, n = 61) than early (Stage I, n = 110) tumors. Knockdown of MARVELD3, one of the novel epigenetically silenced genes, by small interfering RNA significantly reduced anchorage-independent growth of lung cancer cells (P < 0.001). Collectively, this study has identified multiple, novel, epigenetically silenced genes in lung cancer and provides invaluable resources for the development of diagnostic and prognostic biomarkers. PMID:24398667

  3. Epigenetic silencing of tumor suppressor genes during in vitro Epstein–Barr virus infection

    PubMed Central

    Saha, Abhik; Jha, Hem C.; Upadhyay, Santosh K.; Robertson, Erle S.

    2015-01-01

    DNA-methylation at CpG islands is one of the prevalent epigenetic alterations regulating gene-expression patterns in mammalian cells. Hypo- or hypermethylation-mediated oncogene activation, or tumor suppressor gene (TSG) silencing mechanisms, widely contribute to the development of multiple human cancers. Furthermore, oncogenic viruses, including Epstein–Barr virus (EBV)-associated human cancers, were also shown to be influenced by epigenetic modifications on the viral and cellular genomes in the infected cells. We investigated EBV infection of resting B lymphocytes, which leads to continuously proliferating lymphoblastoid cell lines through examination of the expression pattern of a comprehensive panel of TSGs and the epigenetic modifications, particularly methylation of their regulatory sequences. EBV infection of primary B lymphocytes resulted in global transcriptional repression of TSGs through engagement of hypermethylation. Therefore, CpG methylation profiles of TSGs may be used as a prognostic marker as well as development of potential therapeutic strategies for controlling acute infection and EBV-associated B-cell lymphomas. PMID:26324942

  4. A key role for EZH2 in epigenetic silencing of HOX genes in mantle cell lymphoma

    PubMed Central

    Kanduri, Meena; Sander, Birgitta; Ntoufa, Stavroula; Papakonstantinou, Nikos; Sutton, Lesley-Ann; Stamatopoulos, Kostas; Kanduri, Chandrasekhar; Rosenquist, Richard

    2013-01-01

    The chromatin modifier EZH2 is overexpressed and associated with inferior outcome in mantle cell lymphoma (MCL). Recently, we demonstrated preferential DNA methylation of HOX genes in MCL compared with chronic lymphocytic leukemia (CLL), despite these genes not being expressed in either entity. Since EZH2 has been shown to regulate HOX gene expression, to gain further insight into its possible role in differential silencing of HOX genes in MCL vs. CLL, we performed detailed epigenetic characterization using representative cell lines and primary samples. We observed significant overexpression of EZH2 in MCL vs. CLL. Chromatin immune precipitation (ChIP) assays revealed that EZH2 catalyzed repressive H3 lysine 27 trimethylation (H3K27me3), which was sufficient to silence HOX genes in CLL, whereas in MCL H3K27me3 is accompanied by DNA methylation for a more stable repression. More importantly, hypermethylation of the HOX genes in MCL resulted from EZH2 overexpression and subsequent recruitment of the DNA methylation machinery onto HOX gene promoters. The importance of EZH2 upregulation in this process was further underscored by siRNA transfection and EZH2 inhibitor experiments. Altogether, these observations implicate EZH2 in the long-term silencing of HOX genes in MCL, and allude to its potential as a therapeutic target with clinical impact. PMID:24107828

  5. AUGMENTATION OF EFFECTS OF INTERFERON-STIMULATED GENES BY REVERSAL OF EPIGENETIC SILENCING: POTENTIAL APPLICATION TO MELANOMA

    PubMed Central

    Borden, Ernest C.

    2009-01-01

    Increased expression of genes, silenced by methylation of their promoters, could have relevance for increasing effects of not only interferons (IFNs) but also APO2L/TRAIL, cytotoxics and immunotherapeutics for melanoma and other malignancies. A resistant melanoma cell line, A375, lacked APO2L/TRAIL or apoptosis induction by either IFN-α2 or IFN-β. However, apoptosis was induced by IFNs in A375 cells by 5-aza, 2′deoxycytidine, evaluated based upon the postulate that promoter methylation might be silencing pro-apopoptotic IFN-stimulated genes (ISGs). RASSF1A, commonly methylated at high frequency in many tumors including melanoma, which we discovered to be also an IFN-regulated gene, was increased by 5-Aza-dC. RASSF1A was important in enhancing apoptotic effects of not only IFNs and APO2L/TRAIL but also cisplatin. Unraveling epigenetic regulatory mechanisms, as yet only partially identified, will result in new biological insights and improved strategies for therapeutic use of IFNs or ISGs such as APO2L/TRAIL. PMID:17689283

  6. Epigenetic silencing of monoallelically methylated miRNA loci in precancerous colorectal lesions

    PubMed Central

    Menigatti, M; Staiano, T; Manser, C N; Bauerfeind, P; Komljenovic, A; Robinson, M; Jiricny, J; Buffoli, F; Marra, G

    2013-01-01

    Epigenetic silencing of protein-encoding genes is common in early-stage colorectal tumorigenesis. Less is known about the methylation-mediated silencing of genes encoding microRNAs (miRNAs), which are also important epigenetic modulators of gene expression. Using quantitative PCR, we identified 56 miRNAs that were expressed in normal colorectal mucosa and in HT29 colorectal cancer cells treated with demethylating agents but not in untreated HT29 cells, suggesting that they probably undergo methylation-induced silencing during colorectal tumorigenesis. One of these, miR-195, had recently been reported to be underexpressed in colorectal cancers and to exert tumor-suppressor effects in colorectal cancer cells. We identified the transcription start site (TSS) for primary miRNA (pri-miR)-497/195, the primary precursor that yields miR-195 and another candidate on our list, miR-497, and a single CpG island upstream to the TSS, which controls expression of both miRNAs. Combined bisulfite restriction analysis and bisulfite genomic sequencing studies revealed monoallelic methylation of this island in normal colorectal mucosa (50/50 samples) and full methylation in most colorectal adenomas (38/50; 76%). The hypermethylated precancerous lesions displayed significantly downregulated expression of both miRNAs. Similar methylation patterns were observed at two known imprinted genes, MEG3 and GNAS-AS1, which encode several of the 56 miRNAs on our list. Imprinting at these loci was lost in over half the adenomas (62% at MEG3 and 52% at GNAS-AS1). Copy-number alterations at MEG3, GNAS-AS1 and pri-miR-497/195, which are frequent in colorectal cancers, were less common in adenomas and confined to tumors displaying differential methylation at the involved locus. Our data show that somatically acquired, epigenetic changes at monoallelically methylated regions encoding miRNAs are relatively frequent in sporadic colorectal adenomas and might contribute to the onset and progression of

  7. Epigenetic silencing of monoallelically methylated miRNA loci in precancerous colorectal lesions.

    PubMed

    Menigatti, M; Staiano, T; Manser, C N; Bauerfeind, P; Komljenovic, A; Robinson, M; Jiricny, J; Buffoli, F; Marra, G

    2013-01-01

    Epigenetic silencing of protein-encoding genes is common in early-stage colorectal tumorigenesis. Less is known about the methylation-mediated silencing of genes encoding microRNAs (miRNAs), which are also important epigenetic modulators of gene expression. Using quantitative PCR, we identified 56 miRNAs that were expressed in normal colorectal mucosa and in HT29 colorectal cancer cells treated with demethylating agents but not in untreated HT29 cells, suggesting that they probably undergo methylation-induced silencing during colorectal tumorigenesis. One of these, miR-195, had recently been reported to be underexpressed in colorectal cancers and to exert tumor-suppressor effects in colorectal cancer cells. We identified the transcription start site (TSS) for primary miRNA (pri-miR)-497/195, the primary precursor that yields miR-195 and another candidate on our list, miR-497, and a single CpG island upstream to the TSS, which controls expression of both miRNAs. Combined bisulfite restriction analysis and bisulfite genomic sequencing studies revealed monoallelic methylation of this island in normal colorectal mucosa (50/50 samples) and full methylation in most colorectal adenomas (38/50; 76%). The hypermethylated precancerous lesions displayed significantly downregulated expression of both miRNAs. Similar methylation patterns were observed at two known imprinted genes, MEG3 and GNAS-AS1, which encode several of the 56 miRNAs on our list. Imprinting at these loci was lost in over half the adenomas (62% at MEG3 and 52% at GNAS-AS1). Copy-number alterations at MEG3, GNAS-AS1 and pri-miR-497/195, which are frequent in colorectal cancers, were less common in adenomas and confined to tumors displaying differential methylation at the involved locus. Our data show that somatically acquired, epigenetic changes at monoallelically methylated regions encoding miRNAs are relatively frequent in sporadic colorectal adenomas and might contribute to the onset and progression of

  8. MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms.

    PubMed

    Heair, Hannah M; Kemper, Austin G; Roy, Bhaskar; Lopes, Helena B; Rashid, Harunur; Clarke, John C; Afreen, Lubana K; Ferraz, Emanuela P; Kim, Eddy; Javed, Amjad; Beloti, Marcio M; MacDougall, Mary; Hassan, Mohammad Q

    2015-09-01

    Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis. PMID:26124283

  9. MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms

    PubMed Central

    Heair, Hannah M.; Kemper, Austin G.; Roy, Bhaskar; Lopes, Helena B.; Rashid, Harunur; Clarke, John C.; Afreen, Lubana K.; Ferraz, Emanuela P.; Kim, Eddy; Javed, Amjad; Beloti, Marcio M.; MacDougall, Mary

    2015-01-01

    Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis. PMID:26124283

  10. Epigenetic mediated silencing of EYA4 contributes to tumorigenesis in oral dysplastic cells.

    PubMed

    Towle, Rebecca; Truong, Danielle; Garnis, Cathie

    2016-07-01

    Five-year survival rates for oral squamous cell carcinoma (OSCC) have remained at a dismal 50% for the past several decades. Molecular analyses of premalignant tissues are a key means of identifying early foundational drivers of disease, which may be exploitable as biomarkers or therapeutic targets for improving disease outcomes. We previously identified EYA4 as frequently hypermethylated and silenced in premalignant disease based on an analysis of lesion-adjacent normal, dysplasia, and carcinoma in situ/squamous cell carcinoma tissues from the oral cavity. Herein, we further evaluate the role of this putative tumor suppressor gene in transformation of oral tissues and OSCC. By an initial assessment, EYA4 promoter hypermethylation was found in 24/32 (75%) of paired tumor samples in The Cancer Genome Atlas oral cancer data set, with significant correlation noted between methylation status and relative gene expression. To assess the impact of EYA4 in oral tumorigenesis, we overexpressed EYA4 in two oral dysplasia cell lines. Expression of EYA4 caused an increase in cell proliferation, DNA damage repair capabilities, and increased the level of apoptosis. Taken together, we find evidence that EYA4 is a novel tumor suppressor in oral cancer, which becomes methylated and silenced at the premalignant stage and appears to be epigenetically regulated. Further studies are warranted to investigate its role as a marker for progression in oral cancer. © 2016 Wiley Periodicals, Inc. PMID:27015871

  11. Epigenetic silencing of the XAF1 gene is mediated by the loss of CTCF binding

    PubMed Central

    Victoria-Acosta, Georgina; Vazquez-Santillan, Karla; Jimenez-Hernandez, Luis; Muñoz-Galindo, Laura; Maldonado, Vilma; Martinez-Ruiz, Gustavo Ulises; Melendez-Zajgla, Jorge

    2015-01-01

    XAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding. PMID:26443201

  12. miR-196b Is Epigenetically Silenced during the Premalignant Stage of Lung Carcinogenesis.

    PubMed

    Tellez, Carmen S; Juri, Daniel E; Do, Kieu; Picchi, Maria A; Wang, Teresa; Liu, Gang; Spira, Avrum; Belinsky, Steven A

    2016-08-15

    miRNA silencing by promoter hypermethylation may represent a mechanism by which lung cancer develops and progresses, but the miRNAs involved during malignant transformation are unknown. We previously established a model of premalignant lung cancer wherein we treated human bronchial epithelial cells (HBEC) with low doses of tobacco carcinogens. Here, we demonstrate that next-generation sequencing of carcinogen-transformed HBECs treated with the demethylating agent 5-aza-2'deoxycytidine revealed miR-196b and miR-34c-5p to be epigenetic targets. Bisulfite sequencing confirmed dense promoter hypermethylation indicative of silencing in multiple malignant cell lines and primary tumors. Chromatin immunoprecipitation studies further demonstrated an enrichment in repressive histone marks on the miR-196b promoter during HBEC transformation. Restoration of miR-196b expression by transfecting transformed HBECs with specific mimics led to cell-cycle arrest mediated in part through transcriptional regulation of the FOS oncogene, and miR-196b reexpression also significantly reduced the growth of tumor xenografts. Luciferase assays demonstrated that forced expression of miR-196b inhibited the FOS promoter and AP-1 reporter activity. Finally, a case-control study revealed that methylation of miR-196b in sputum was strongly associated with lung cancer (OR = 4.7, P < 0.001). Collectively, these studies highlight miR-196b as a tumor suppressor whose silencing early in lung carcinogenesis may provide a selective growth advantage to premalignant cells. Targeted delivery of miR-196b could therefore serve as a preventive or therapeutic strategy for the management of lung cancer. Cancer Res; 76(16); 4741-51. ©2016 AACR. PMID:27302168

  13. Consolidation of the cancer genome into domains of repressive chromatin by long range epigenetic silencing (LRES) reduces transcriptional plasticity

    PubMed Central

    Coolen, Marcel W.; Stirzaker, Clare; Song, Jenny Z.; Statham, Aaron L.; Kassir, Zena; Moreno, Carlos S.; Young, Andrew N.; Varma, Vijay; Speed, Terence P.; Cowley, Mark; Lacaze, Paul; Kaplan, Warren; Robinson, Mark D.; Clark, Susan J.

    2011-01-01

    SUMMARY Silencing of individual genes can occur by genetic and epigenetic processes during carcinogenesis, but the underlying mechanisms remain unclear. By creating an integrated prostate cancer epigenome map using tiling arrays, we show that contiguous regions of gene suppression commonly occur due to Long Range Epigenetic Silencing (LRES). We identified 47 novel LRES regions in prostate cancer, typically spanning ~2 Mb and harbouring ~12 genes, with a prevalence of tumour suppressor genes and miRNAs. Our data reveal that LRES is associated with regional histone deacetylation combined with sub-domains of different epigenetic remodelling patterns, that include re-enforcement, gain or exchange of repressive histone and DNA methylation marks. The transcriptional and epigenetic state of genes in normal prostate epithelial and human embryonic stem cells can play a critical role in defining the mode of cancer-associated epigenetic remodelling. We propose that a consolidation or effective reduction of the cancer genome commonly occurs in domains, due to a combination of LRES and LOH or genomic deletion, resulting in reduced transcriptional plasticity within these regions. PMID:20173741

  14. Cooperative stabilization of the SIR complex provides robust epigenetic memory in a model of SIR silencing in Saccharomyces cerevisiae

    PubMed Central

    Sneppen, Kim; Dodd, Ian B

    2015-01-01

    How alternative chromatin-based regulatory states can be made stable and heritable in order to provide robust epigenetic memory is poorly understood. Here, we develop a stochastic model of the silencing system in Saccharomyces cerevisiae that incorporates cooperative binding of the repressive SIR complex and antisilencing histone modifications, in addition to positive feedback in Sir2 recruitment. The model was able to reproduce key features of SIR regulation of an HM locus, including heritable bistability, dependence on the silencer elements, and sensitivity to SIR dosage. We found that antisilencing methylation of H3K79 by Dot1 was not needed to generate these features, but acted to reduce spreading of SIR binding, consistent with its proposed role in containment of silencing. In contrast, cooperative inter-nucleosome interactions mediated by the SIR complex were critical for concentrating SIR binding around the silencers in the absence of barriers, and for providing bistability in SIR binding. SIR-SIR interactions magnify the cooperativity in the Sir2-histone deacetylation positive feedback reaction and complete a double-negative feedback circuit involving antisilencing modifications. Thus, our modeling underscores the potential importance of cooperative interactions between nucleosome-bound complexes both in the SIR system and in other chromatin-based complexes in epigenetic regulation. PMID:25830651

  15. Cooperative stabilization of the SIR complex provides robust epigenetic memory in a model of SIR silencing in Saccharomyces cerevisiae.

    PubMed

    Sneppen, Kim; Dodd, Ian B

    2015-01-01

    How alternative chromatin-based regulatory states can be made stable and heritable in order to provide robust epigenetic memory is poorly understood. Here, we develop a stochastic model of the silencing system in Saccharomyces cerevisiae that incorporates cooperative binding of the repressive SIR complex and antisilencing histone modifications, in addition to positive feedback in Sir2 recruitment. The model was able to reproduce key features of SIR regulation of an HM locus, including heritable bistability, dependence on the silencer elements, and sensitivity to SIR dosage. We found that antisilencing methylation of H3K79 by Dot1 was not needed to generate these features, but acted to reduce spreading of SIR binding, consistent with its proposed role in containment of silencing. In contrast, cooperative inter-nucleosome interactions mediated by the SIR complex were critical for concentrating SIR binding around the silencers in the absence of barriers, and for providing bistability in SIR binding. SIR-SIR interactions magnify the cooperativity in the Sir2-histone deacetylation positive feedback reaction and complete a double-negative feedback circuit involving antisilencing modifications. Thus, our modeling underscores the potential importance of cooperative interactions between nucleosome-bound complexes both in the SIR system and in other chromatin-based complexes in epigenetic regulation. PMID:25830651

  16. Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development

    PubMed Central

    Hayward, Jane D.; Kannan, Athilakshmi; Mould, Tim; West, James; Zikan, Michal; Cibula, David; Fiegl, Heidi; Lee, Shih-Han; Wik, Elisabeth; Hadwin, Richard; Arora, Rupali; Lemech, Charlotte; Turunen, Henna; Pakarinen, Päivi; Jacobs, Ian J.; Salvesen, Helga B.; Bagchi, Milan K.; Bagchi, Indrani C.; Widschwendter, Martin

    2013-01-01

    Background Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development. Methods and Findings Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to

  17. Re-expression of CXCL14, a common target for epigenetic silencing in lung cancer, induces tumor necrosis

    PubMed Central

    Tessema, Mathewos; Klinge, Donna M.; Yingling, Christin M.; Do, Kieu; Van Neste, Leander; Belinsky, Steven A.

    2010-01-01

    Chemokines are important regulators of directional cell migration and tumor metastasis. A genome-wide transcriptome array designed to uncover novel genes silenced by methylation in lung cancer identified the CXC-subfamily of chemokines. Expression of eleven of the sixteen known human CXC-chemokines was increased in lung adenocarcinoma cell lines after treatment with 5-aza-2deoxycytidine (DAC). Tumor-specific methylation leading to silencing of CXCL5, 12 and 14 was found in over 75% of primary lung adenocarcinomas and DAC treatment restored expression of each silenced gene. Forced expression of CXCL14 in H23 cells where this gene is silenced by methylation increased cell death in vitro and dramatically reduced in vivo growth of lung tumor xenografts through necrosis of up to 90% of the tumor mass. CXCL14 re-expression had a profound effect on the genome altering the transcription of over 1,000 genes, including increased expression of 30 cell cycle inhibitor and pro-apoptosis genes. In addition, CXCL14 methylation in sputum from asymptomatic early stage lung cancer cases was associated with a 2.9-fold elevated risk for this disease compared to controls, substantiating its potential as a biomarker for early detection of lung cancer. Together these findings identify CXCL14 as an important tumor suppressor gene epigenetically silenced during lung carcinogenesis. PMID:20562917

  18. Arabidopsis AGO3 predominantly recruits 24-nt small RNAs to regulate epigenetic silencing.

    PubMed

    Zhang, Zhonghui; Liu, Xiuying; Guo, Xinwei; Wang, Xiu-Jie; Zhang, Xiuren

    2016-01-01

    Argonaute (AGO) proteins recruit 21-24-nucleotide (nt) small RNAs (sRNAs) to constitute RNA-induced silencing complexes (RISCs) to regulate gene expression at transcriptional or posttranscriptional levels(1-3). Arabidopsis encodes nine functional AGO proteins. These proteins are classified into three clusters, AGO1/5/10, AGO2/3/7 and AGO4/6/9, based on their sequence similarity, functional redundancy, as well as species and features of AGO-bound sRNAs(4-7). Although most Arabidopsis AGO proteins have been studied well, AGO3-bound sRNAs and their basic function remain unknown. Here we observed that AGO3 could not complement the signature function of AGO2, the closest genetic paralog of AGO3, in host antiviral defence. We also found, surprisingly, that AGO3 predominantly bound 24-nt sRNAs with 5'-terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2, further indicating their functional divergence. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4, and over 60% of AGO3-associated 24-nt sRNA-enriched loci were identical to those of AGO4. Moreover, the redundancy of AGO3- and AGO4-bound sRNAs is much more than that of AGO6- and AGO4-recruited sRNAs. In addition, expression of AGO3 driven by the AGO4 promoter partially complemented AGO4 function and rescued a DNA methylation defect in the ago4-1 background. Together, our results indicated that AGO3, similarly to AGO4, is a component in the epigenetic pathway. PMID:27243648

  19. EZH2 mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3 and NGFR

    PubMed Central

    Wang, Chunxi; Liu, Zhihui; Woo, Chan-Wook; Li, Zhijie; Wang, Lifeng; Wei, Jun S.; Marquez, Victor E.; Bates, Susan E.; Jin, Qihuang; Khan, Javed; Ge, Kai; Thiele, Carol J.

    2012-01-01

    Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb complex histone methytransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNAi-mediated knockdown or pharmacological inhibiton with 3-deazaneplanocin A (DZNep) increased CASZ1 expression, inhibited NB cell growth and induced neurite extension. Similarly, EZH2−/− mouse embryonic fibroblasts (MEFs) displayed 3-fold higher levels of CASZ1 mRNA compared to EZH2+/+ MEFs. In cells with increased expression of CASZ1, treatment with HDAC inhibitors decreased expression of EZH2 and the Polycomb complex component SUZ12. Under steady-state conditions H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors. PMID:22068036

  20. Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors12

    PubMed Central

    Calmon, Marilia F; Rodrigues, Rodrigo V; Kaneto, Carla M; Moura, Ricardo P; Silva, Sabrina D; Mota, Louise Danielle C; Pinheiro, Daniel G; Torres, Cesar; de Carvalho, Alex F; Cury, Patrícia M; Nunes, Fabio D; Nishimoto, Ines Nobuko; Soares, Fernando A; da Silva, Adriana MA; Kowalski, Luis P; Brentani, Helena; Zanelli, Cleslei F; Silva, Wilson A; Rahal, Paula; Tajara, Eloiza H; Carraro, Dirce M; Camargo, Anamaria A; Valentini, Sandro R

    2009-01-01

    Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2′-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5′ region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, ≥3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients. PMID:20019841

  1. Aberrant JAK/STAT Signaling Suppresses TFF1 and TFF2 through Epigenetic Silencing of GATA6 in Gastric Cancer.

    PubMed

    Wu, Cheng-Shyong; Wei, Kuo-Liang; Chou, Jian-Liang; Lu, Chung-Kuang; Hsieh, Ching-Chuan; Lin, Jora M J; Deng, Yi-Fang; Hsu, Wan-Ting; Wang, Hui-Min David; Leung, Chung-Hang; Ma, Dik-Lung; Li, Chin; Chan, Michael W Y

    2016-01-01

    Aberrant Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling is crucial to the development of gastric cancer. In this study, we examined the role of STAT3 in the expression and methylation of its targets in gastric cancer patients. Results from RNA sequencing identified an inverse correlation between the expression of STAT3 and GATA6 in 23 pairs of gastric cancer patient samples. We discovered that the expression of GATA6 is epigenetically silenced through promoter methylation in gastric cancer cell lines. Interestingly, the inhibition of STAT3 using a novel STAT3 inhibitor restored the expression of GATA6 and its targets, trefoil factors 1 and 2 (TFF1/2). Moreover, disruption of STAT3 binding to GATA6 promoter by small hairpin RNA restored GATA6 expression in AGS cells. A clinically significant correlation was also observed between the expression of GATA6 and TFF1/2 among tissue samples from 60 gastric cancer patients. Finally, bisulfite pyrosequencing revealed GATA6 methylation in 65% (39/60) of the patients, and those with higher GATA6 methylation tended to have shorter overall survival. In conclusion, we demonstrated that aberrant JAK/STAT signaling suppresses TFF1/2 partially through the epigenetic silencing of GATA6. Therapeutic intervention of STAT3 in reversing the epigenetic status of GATA6 could benefit the treatment of gastric cancer and is worthy of further investigation. PMID:27598141

  2. A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe

    PubMed Central

    Tucker, James Franklin; Ohle, Corina; Schermann, Géza; Bendrin, Katja; Zhang, Wei; Fischer, Tamás; Zhang, Ke

    2016-01-01

    Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3’-5’ exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5’-3’ exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome. PMID:26889830

  3. A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5'-3' Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe.

    PubMed

    Tucker, James Franklin; Ohle, Corina; Schermann, Géza; Bendrin, Katja; Zhang, Wei; Fischer, Tamás; Zhang, Ke

    2016-02-01

    Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3'-5' exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5'-3' exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome. PMID:26889830

  4. Epigenetic silencing of the kinase tumor suppressor WNK2 is tumor-type and tumor-grade specific

    PubMed Central

    Jun, Peter; Hong, Chibo; Lal, Anita; Wong, Judith M.; McDermott, Michael W.; Bollen, Andrew W.; Plass, Christoph; Held, William A.; Smiraglia, Dominic J.; Costello, Joseph F.

    2009-01-01

    Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genomewide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5′ CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate epidermal growth factor receptor signaling via inhibition of MEK1 (mitogen-activated protein kinase kinase 1), and point mutations have been reported in WNK1, WNK2, WNK3, and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of grade II and III meningiomas, respectively, but rarely in a total of 209 tumors from 13 other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation. PMID:19001526

  5. A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers.

    PubMed

    Vrba, Lukas; Garbe, James C; Stampfer, Martha R; Futscher, Bernard W

    2015-01-01

    Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis. PMID

  6. A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

    DOE PAGESBeta

    Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2015-10-19

    Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the genemore » expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'- deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. In conclusion, the epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human

  7. A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

    SciTech Connect

    Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2015-10-19

    Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'- deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. In conclusion, the epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human

  8. A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

    PubMed Central

    Vrba, Lukas; Garbe, James C; Stampfer, Martha R; Futscher, Bernard W

    2015-01-01

    Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2′-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis. PMID

  9. Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

    SciTech Connect

    Vaid, Mudit; Prasad, Ram; Singh, Tripti; Jones, Virginia; Katiyar, Santosh K.

    2012-08-15

    Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16{sup INK4a} and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans. -- Highlights: ►Epigenetic modulations have been shown to have a role in cancer risk. ►Proanthocyanidins decrease the levels of DNA methylation and histone deacetylation. ►Proanthocyanidins inhibit histone deacetylase activity in skin cancer cells. ►Proanthocyanidins reactivate tumor suppressor genes in skin

  10. The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition

    PubMed Central

    Tan, E-Jean; Kahata, Kaoru; Idås, Oskar; Thuault, Sylvie; Heldin, Carl-Henrik; Moustakas, Aristidis

    2015-01-01

    The loss of the tumour suppressor E-cadherin (Cdh1) is a key event during tumourigenesis and epithelial–mesenchymal transition (EMT). Transforming growth factor-β (TGFβ) triggers EMT by inducing the expression of non-histone chromatin protein High Mobility Group A2 (HMGA2). We have previously shown that HMGA2, together with Smads, regulate a network of EMT-transcription factors (EMT-TFs) like Snail1, Snail2, ZEB1, ZEB2 and Twist1, most of which are well-known repressors of the Cdh1 gene. In this study, we show that the Cdh1 promoter is hypermethylated and epigenetically silenced in our constitutive EMT cell model, whereby HMGA2 is ectopically expressed in mammary epithelial NMuMG cells and these cells are highly motile and invasive. Furthermore, HMGA2 remodels the chromatin to favour binding of de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter. E-cadherin expression could be restored after treatment with the DNA de-methylating agent 5-aza-2′-deoxycytidine. Here, we describe a new epigenetic role for HMGA2, which follows the actions that HMGA2 initiates via the EMT-TFs, thus achieving sustained silencing of E-cadherin expression and promoting tumour cell invasion. PMID:25492890

  11. A genomic screen for long noncoding RNA genes epigenetically silenced by aberrant DNA methylation in colorectal cancer.

    PubMed

    Kumegawa, Kohei; Maruyama, Reo; Yamamoto, Eiichiro; Ashida, Masami; Kitajima, Hiroshi; Tsuyada, Akihiro; Niinuma, Takeshi; Kai, Masahiro; Yamano, Hiro-O; Sugai, Tamotsu; Tokino, Takashi; Shinomura, Yasuhisa; Imai, Kohzoh; Suzuki, Hiromu

    2016-01-01

    Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced adenomas (87.8%), suggesting that this methylation is an early event during colorectal tumorigenesis. Methylation of ZNF582-AS1 is associated with poor survival of CRC patients, and ectopic expression of ZNF582-AS1 suppressed colony formation by CRC cells. Our findings offer insight into the association between epigenetic alterations and lncRNA dysregulation in cancer and suggest that ZNF582-AS1 may be a novel tumor-suppressive lncRNA. PMID:27215978

  12. A genomic screen for long noncoding RNA genes epigenetically silenced by aberrant DNA methylation in colorectal cancer

    PubMed Central

    Kumegawa, Kohei; Maruyama, Reo; Yamamoto, Eiichiro; Ashida, Masami; Kitajima, Hiroshi; Tsuyada, Akihiro; Niinuma, Takeshi; Kai, Masahiro; Yamano, Hiro-o; Sugai, Tamotsu; Tokino, Takashi; Shinomura, Yasuhisa; Imai, Kohzoh; Suzuki, Hiromu

    2016-01-01

    Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced adenomas (87.8%), suggesting that this methylation is an early event during colorectal tumorigenesis. Methylation of ZNF582-AS1 is associated with poor survival of CRC patients, and ectopic expression of ZNF582-AS1 suppressed colony formation by CRC cells. Our findings offer insight into the association between epigenetic alterations and lncRNA dysregulation in cancer and suggest that ZNF582-AS1 may be a novel tumor-suppressive lncRNA. PMID:27215978

  13. Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

    PubMed Central

    Kumpatla, S P; Teng, W; Buchholz, W G; Hall, T C

    1997-01-01

    Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa L.) that displayed aberrant segregation in R1 progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R1 plants had a DNA hybridization profile identical to that of the resistant R0 parent and R1 siblings. Genomic DNA analysis revealed substantial methylation of the Ubi1/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R2 progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences. PMID:9342860

  14. Epigenetic silencing of p21 by long non-coding RNA HOTAIR is involved in the cell cycle disorder induced by cigarette smoke extract.

    PubMed

    Liu, Yi; Wang, Bairu; Liu, Xinlu; Lu, Lu; Luo, Fei; Lu, Xiaolin; Shi, Le; Xu, Wenchao; Liu, Qizhan

    2016-01-01

    Long noncoding RNAs (lncRNAs), which are epigenetic regulators, are involved in human malignancies. Little is known, however, about the molecular mechanisms for lncRNA regulation of genes induced by cigarette smoke. We recently found that, in human bronchial epithelial (HBE) cells, the lncRNA, Hox transcript antisense intergenic RNA (HOTAIR), is associated with changes in the cell cycle caused by cigarette smoke extract (CSE). In the present study, we report that increased expression of HOTAIR and enhancer of zeste homolog 2 (EZH2), and tri-methylation of Lys 27 of histone H3 (H3K27me3), affect cell cycle progression during CSE-induced transformation of HBE cells. Inhibition of HOTAIR and EZH2 by siRNAs attenuated CSE-induced decreases of p21 levels. Further, ChIP assays verified that HOTAIR and EZH2 were needed to maintain the interaction of H3K27me3 with the promoter regions of p21; combined use of a HOTAIR plasmid and EZH2 siRNA supported this observation. Thus, HOTAIR epigenetic silencing of p21 via EZH2-mediated H3K27 trimethylation contributes to changes in the cell cycle induced by CSE. These observations provide further understanding of the regulation of CSE-induced lung carcinogenesis and identify new therapeutic targets. PMID:26506537

  15. Epigenetic silencing of JMJD5 promotes the proliferation of hepatocellular carcinoma cells by down-regulating the transcription of CDKN1A

    PubMed Central

    Fang, Jia-Zhu; Wu, Chong-Chao; Huang, Li-Yu; Wang, Lan; Han, Ze-Guang

    2016-01-01

    Proteins that contain jumonji C (JmjC) domains have recently been identified as major contributors to various malignant human cancers through epigenetic remodeling. However, the roles of these family members in the pathogenesis of hepatocellular carcinoma (HCC) are obscure. By mining public databases, we found that the HCC patients with lower JmjC domain-containing protein 5 (JMJD5) expression exhibited shorter survival time. We then confirmed that JMJD5 expression was indeed decreased in HCC specimens, which was caused by the altered epigenetic histone modifications, the decreased H3K9ac, H3K27ac and H3K4me2/3 together with the increased trimethylation of H3K27 and H3K9 on the JMJD5 promoter. Functional experiments revealed that JMJD5 knockdown promoted HCC cell proliferation and in vivo tumorigenicity by accelerating the G1/S transition of the cell cycle; in contrast, ectopic JMJD5 expression had the opposite effects. At molecular mechanism, we found that, in HCC cell lines including TP53-null Hep3B, JMJD5 knockdown led to the down-regulation of CDKN1A and ectopic expression of JMJD5 not only increased but also rescued CDKN1A transcription. Moreover, CDKN1A knockdown could abrogate the effect of JMJD5 knockdown or overexpression on cell proliferation, suggesting that JMJD5 inhibits HCC cell proliferation mainly by activating CDKN1A expression. We further revealed that JMJD5 directly enhances CDKN1A transcription by binding to CDKN1A's promoter independent of H3K36me2 demethylase activity. In short, we first prove that JMJD5 is a tumor suppressor gene in HCC pathogenesis, and the epigenetic silencing of JMJD5 promotes HCC cell proliferation by directly down-regulating CDKN1A transcription. PMID:26760772

  16. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner

    PubMed Central

    Cano-Rodriguez, David; Gjaltema, Rutger A F.; Jilderda, Laura J; Jellema, Pytrick; Dokter-Fokkens, Jelleke; Ruiters, Marcel H J.; Rots, Marianne G

    2016-01-01

    Histone modifications reflect gene activity, but the relationship between cause and consequence of transcriptional control is heavily debated. Recent developments in rewriting local histone codes of endogenous genes elucidated instructiveness of certain marks in regulating gene expression. Maintenance of such repressive epigenome editing is controversial, while stable reactivation is still largely unexplored. Here we demonstrate sustained gene re-expression using two types of engineered DNA-binding domains fused to a H3K4 methyltransferase. Local induction of H3K4me3 is sufficient to allow re-expression of silenced target genes in various cell types. Maintenance of the re-expression is achieved, but strongly depends on the chromatin microenvironment (that is, DNA methylation status). We further identify H3K79me to be essential in allowing stable gene re-expression, confirming its role in epigenetic crosstalk for stable reactivation. Our approach uncovers potent epigenetic modifications to be directly written onto genomic loci to stably activate any given gene. PMID:27506838

  17. Epigenetic Silencing of SPINT2 promotes Cancer Cell Motility via HGF-MET Pathway Activation in Melanoma

    PubMed Central

    Hwang, Soonyean; Kim, Hye-Eun; Min, Michelle; Raghunathan, Rekha; Panova, Izabela P.; Munshi, Ruchi; Ryu, Byungwoo

    2015-01-01

    Aberrant HGF-MET signaling activation via interactions with surrounding stromal cells in tumor microenvironment plays significant roles in malignant tumor progression. However, extracellular proteolytic regulation of HGF activation which is influenced by the tumor microenvironment and its consequential effects on melanoma malignancy remain uncharacterized. In this study we identified SPINT2: a proteolytic inhibitor of hepatocyte growth factor activator (HGFA), which plays a significant role in the suppression of the HGF-MET pathway and malignant melanoma progression. SPINT2 expression is significantly lower in metastatic melanoma tissues compared to those in early stage primary melanomas which also corresponded with DNA methylation levels isolated from tissue samples. Treatment with the DNA hypomethylating agent decitabine in cultured melanoma cells induced transcriptional reactivation of SPINT2, suggesting that this gene is epigenetically silenced in malignant melanomas. Furthermore, we show that ectopically expressed SPINT2 in melanoma cells inhibits HGF induced MET-AKT signaling pathway and decreases malignant phenotype potential such as cell motility, and invasive growth of melanoma cells. These results suggest that SPINT2 is associated with tumor suppressive functions in melanoma by inhibiting an extracellular signal regulator of HGF which is typically activated by tumor-stromal interactions. These findings indicate that epigenetic impairment of the tightly regulated cytokine-receptor communications in tumor microenvironment may contribute to malignant tumor progression. PMID:25910030

  18. Chromatin inactivation precedes de novo dna methylation during the progressive epigenetic silencing of the rassf1a promoter

    SciTech Connect

    Strunnikova Maria; Schagdarsurengin, Undraga; Kehlen, Astrid; Garbe, James C.; Stampfer, Martha R.; Dammann, Reinhard

    2005-02-23

    Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1 A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.

  19. G9a Is Essential for Epigenetic Silencing of K+ Channel Genes in Acute-to-Chronic Pain Transition

    PubMed Central

    Laumet, Geoffroy; Garriga, Judit; Chen, Shao-Rui; Zhang, Yuhao; Li, De-Pei; Smith, Trevor M.; Dong, Yingchun; Jelinek, Jaroslav; Cesaroni, Matteo; Issa, Jean-Pierre; Pan, Hui-Lin

    2015-01-01

    Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K+ channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. Here we show that nerve injury increased H3K9me2 occupancy at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect DNA methylation levels of these genes in DRGs. Nerve injury increased activity of G9a, histone deacetylases and EZH2, but only G9a inhibition consistently restored K+ channel expression. Selective G9a knockout in DRG neurons completely blocked K+ channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced K+ channel genes but also normalized 638 genes down- or up-regulated by nerve injury. Thus G9a plays a dominant role in transcriptional repression of K+ channels and in acute-to-chronic pain transition after nerve injury. PMID:26551542

  20. ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo

    PubMed Central

    De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M.

    2015-01-01

    A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition. PMID:25926359

  1. Epigenetics

    PubMed Central

    Tollervey, James; Lunyak, Victoria V.

    2012-01-01

    Emerging evidence is shedding light on a large and complex network of epigenetic modifications at play in human stem cells. This “epigenetic landscape” governs the fine-tuning and precision of gene expression programs that define the molecular basis of stem cell pluripotency, differentiation and reprogramming. This review will focus on recent progress in our understanding of the processes that govern this landscape in stem cells, such as histone modification, DNA methylation, alterations of chromatin structure due to chromatin remodeling and non-coding RNA activity. Further investigation into stem cell epigenetics promises to provide novel advances in the diagnosis and treatment of a wide array of human diseases. PMID:22805743

  2. Reversible epigenetic fingerprint-mediated glutathione-S-transferase P1 gene silencing in human leukemia cell lines.

    PubMed

    Karius, Tommy; Schnekenburger, Michael; Ghelfi, Jenny; Walter, Jörn; Dicato, Mario; Diederich, Marc

    2011-06-01

    Glutathione-S-transferase P1 (GSTP1) gene is commonly silenced by CpG island promoter hypermethylation in prostate, breast, and liver cancers. However, mechanisms leading to GSTP1 repression by promoter hypermethylation in leukemia and its relationship with pathological alterations of the chromatin structure remain poorly understood. A panel of leukemia cell lines was analyzed for their GSTP1 expression, revealing cell lines with high, moderate or no detectable GSTP1 expression. Bisulfite sequencing, methylation-specific PCR and combined bisulfite restriction analysis revealed that GSTP1 promoter was completely methylated in transcriptionally inactive RAJI and MEG-01 cell lines. In contrast, cell lines expressing GSTP1 exhibited an unmethylated and transcriptionally active promoter. Furthermore, histone marks and effector proteins associated with transcriptional activity were detected by chromatin immunoprecipitation in the GSTP1 expressing hypomethylated K-562 cell line. However, repressive chromatin marks and the recruitment of silencing protein complexes were found in the non-expressing hypermethylated RAJI and MEG-01 cell lines. Finally, we provide evidence that treatment of RAJI and MEG-01 cells with the DNA demethylating agent, 5-aza-2'-deoxycytidine, resulted in GSTP1 promoter demethylation, drastic changes of histone modifications and promoter associated proteins and GSTP1 gene activation. In contrast, treatments with HDAC inhibitors failed to demethylate and reactivate the GSTP1 gene. Our study extends the knowledge on leukemia-specific epigenetic alterations of GSTP1 gene. Furthermore, we are showing the correlation of DNA methylation and histone modifications with the positive/negative GSTP1 transcriptional expression state. Finally, these data support the concept of the dominance of DNA methylation over HDAC inhibitor-sensitive histone deacetylation in gene silencing. PMID:21453686

  3. SOX30, a novel epigenetic silenced tumor suppressor, promotes tumor cell apoptosis by transcriptional activating p53 in lung cancer

    PubMed Central

    Han, F; Liu, W; Jiang, X; Shi, X; Yin, L; Ao, L; Cui, Z; Li, Y; Huang, C; Cao, J; Liu, J

    2015-01-01

    Although members of SOX family have been well documented for their essential roles in embryonic development, cell proliferation and disease, the functional role and molecular mechanism of SOX30 in cancer are largely unexplored. Here, we first identified SRY-box containing gene 30 (SOX30) as a novel preferentially methylated gene using genome-wide methylation screening. SOX30 hypermethylation was detected in 100% of lung cancer cell lines (9/9) and 70.83% (85/120) of primary lung tumor tissues compared with none (0/20) of normal and 8.0% (2/25) of peri-tumoral lung tissues (P<0.01). SOX30 was expressed in normal and peri-tumoral lung tissues in which SOX30 was unmethylated, but was silenced or downregulated in lung cancer cell lines and primary lung tumor tissues harboring a hypermethylated SOX30. De-methylation experiments further confirmed that silence of SOX30 was regulated by its hypermethylation. Ectopic expression of SOX30 induces cancer cell apoptosis with inhibiting proliferation in vitro and represses tumor formation in vivo, whereas knockdown of SOX30 demonstrates a reversed effect both in vitro and in vivo. At the molecular level, the antitumorigenic effect of SOX30 is mediated by directly binding to CACTTTG (+115 to +121) of p53 promoter region and activating p53 transcription, suggesting that SOX30 is a novel transcriptional activating factor of p53. Indeed, blockade of p53 attenuates the tumor inhibition of SOX30. Overall, these findings demonstrate that SOX30 is a novel epigenetic silenced tumor suppressor acting through direct regulation of p53 transcription and expression. This study provides novel insights on the mechanism of tumorigenesis in lung cancer. PMID:25435374

  4. Epigenetic silencing of myogenic gene program by Myb-binding protein 1a suppresses myogenesis

    PubMed Central

    Yang, Chang-Ching; Liu, Hsuan; Chen, Shen Liang; Wang, Tzu-Hao; Hsieh, Chia-Ling; Huang, Yi; Chen, Shu-Jen; Chen, Hua-Chien; Yung, Benjamin Yat-Ming; Chin-Ming Tan, Bertrand

    2012-01-01

    Skeletal myogenesis involves highly coordinated steps that integrate developmental cues at the chromatin of muscle progenitors. Here, we identify Myb-binding protein 1a (Mybbp1a) as a novel negative regulator of muscle-specific gene expression and myoblast differentiation. The mode of action of Mybbp1a was linked to promoter regulation as illustrated by its interaction with MyoD at the genomic regions of silent muscle-specific genes as well as its negative effect on MyoD-mediated transcriptional activity. We propose that Mybbp1a exerts its repressive role by inducing a less permissible chromatin structure following recruitment of negative epigenetic modifiers such as HDAC1/2 and Suv39h1. At the onset of differentiation, Mybbp1a undergoes a promoter disengagement that may be due to the differentiation-responsive, miR-546-mediated downregulation of Mybbp1a expression. Moreover, such alteration gave rise to promoter enrichment of activators and histone acetylation, an epigenetic status amenable to gene activation. Together, these findings unveil a hitherto unrecognized transcriptional co-repressor role of Mybbp1a in proliferating muscle progenitor cells, and highlight an epigenetic mechanism by which Mybbp1a and miR-546 interplay to control myoblast differentiation transition. PMID:22333916

  5. Maternal diet and aging alter the epigenetic control of a promoter–enhancer interaction at the Hnf4a gene in rat pancreatic islets

    PubMed Central

    Sandovici, Ionel; Smith, Noel H.; Nitert, Marloes Dekker; Ackers-Johnson, Matthew; Uribe-Lewis, Santiago; Ito, Yoko; Jones, R. Huw; Marquez, Victor E.; Cairns, William; Tadayyon, Mohammed; O’Neill, Laura P.; Murrell, Adele; Ling, Charlotte; Constância, Miguel; Ozanne, Susan E.

    2011-01-01

    Environmental factors interact with the genome throughout life to determine gene expression and, consequently, tissue function and disease risk. One such factor that is known to play an important role in determining long-term metabolic health is diet during critical periods of development. Epigenetic regulation of gene expression has been implicated in mediating these programming effects of early diet. The precise epigenetic mechanisms that underlie these effects remain largely unknown. Here, we show that the transcription factor Hnf4a, which has been implicated in the etiology of type 2 diabetes (T2D), is epigenetically regulated by maternal diet and aging in rat islets. Transcriptional activity of Hnf4a in islets is restricted to the distal P2 promoter through its open chromatin configuration and an islet-specific interaction between the P2 promoter and a downstream enhancer. Exposure to suboptimal nutrition during early development leads to epigenetic silencing at the enhancer region, which weakens the P2 promoter–enhancer interaction and results in a permanent reduction in Hnf4a expression. Aging leads to progressive epigenetic silencing of the entire Hnf4a locus in islets, an effect that is more pronounced in rats exposed to a poor maternal diet. Our findings provide evidence for environmentally induced epigenetic changes at the Hnf4a enhancer that alter its interaction with the P2 promoter, and consequently determine T2D risk. We therefore propose that environmentally induced changes in promoter-enhancer interactions represent a fundamental epigenetic mechanism by which nutrition and aging can influence long-term health. PMID:21385945

  6. Reversal of epigenetic silencing of MHC class I chain-related protein A and B improves immune recognition of Merkel cell carcinoma

    PubMed Central

    Ritter, Cathrin; Fan, Kaiji; Paulson, Kelly G.; Nghiem, Paul; Schrama, David; Becker, Jürgen C.

    2016-01-01

    Merkel cell carcinoma (MCC) is a virally associated cancer characterized by its aggressive behavior and strong immunogenicity. Both viral infection and malignant transformation induce expression of MHC class I chain-related protein (MIC) A and B, which signal stress to cells of the immune system via Natural Killer group 2D (NKG2D) resulting in elimination of target cells. However, despite transformation and the continued presence of virally-encoded proteins, MICs are only expressed in a minority of MCC tumors in situ and are completely absent on MCC cell lines in vitro. This lack of MIC expression was due to epigenetic silencing via MIC promoter hypo-acetylation; indeed, MIC expression was re-induced by pharmacological inhibition of histone deacetylases (HDACs) both in vitro and in vivo. This re-induction of MICs rendered MCC cells more sensitive to immune-mediated lysis. Thus, epigenetic silencing of MICs is an important immune escape mechanism of MCCs. PMID:26902929

  7. Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia

    PubMed Central

    Huang, Sai; Jiang, Meng-Meng; Chen, Guo-Feng; Qian, Kun; Gao, Hong-Hao; Guan, Wei; Shi, Jin-Long; Liu, An-Qi; Liu, Jing; Wang, Bian-Hong; Li, Yong-Hui; Yu, Li

    2016-01-01

    Background: The acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML. Methods: Qualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay. Results: EYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively. Conclusions: Our study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target

  8. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway.

    PubMed

    Foda, Bardees M; Singh, Upinder

    2015-08-21

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

  9. Epigenetic Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Contexts in Human Cells.

    PubMed

    Mitsuda, Sho-Hei; Shimizu, Noriaki

    2016-01-01

    A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a "DNA methylation-core and chromatin-spread" model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model. PMID:27525955

  10. Epigenetic Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Contexts in Human Cells

    PubMed Central

    Mitsuda, Sho-hei; Shimizu, Noriaki

    2016-01-01

    A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a “DNA methylation-core and chromatin-spread” model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model. PMID:27525955

  11. Cbx7 is epigenetically silenced in glioblastoma and inhibits cell migration by targeting YAP/TAZ-dependent transcription.

    PubMed

    Nawaz, Zahid; Patil, Vikas; Arora, Anjali; Hegde, Alangar S; Arivazhagan, Arimappamagan; Santosh, Vani; Somasundaram, Kumaravel

    2016-01-01

    Glioblastomas (GBM) are the most malignant form of astrocytomas which are difficult to treat and portend a grave clinical course and poor prognosis. In this study, we identified Chromobox homolog 7 (Cbx7), a member of Polycomb Repressive Complex 1 (PRC1), as a downregulated gene in GBM owing to its promoter hypermethylation. Bisulphite sequencing and methylation inhibitor treatment established the hypermethylation of Cbx7 in GBM. Exogenous overexpression of Cbx7 induced cell death, inhibited cell proliferation, colony formation and migration/invasion of the glioma cells. GSEA of Cbx7 regulated genes identified Cbx7 as a repressor of transcription co-activators YAP/TAZ, the inhibitory targets of the Hippo signalling pathway. In good correlation, the exogenous expression of Cbx7 repressed the YAP/TAZ-dependent transcription and downregulated CTGF, a bonafide YAP/TAZ target. We also observed reduced levels of phospho-JNK in Cbx7 expressing cells. Additionally, CTGF silencing and pharmacological inhibition of JNK also inhibited glioma cell migration. Further, Cbx7 failed to inhibit cell migration significantly in the presence of exogenously overexpressed CTGF or constitutively active JNK. Thus, our study identifies Cbx7 as an inhibitor of glioma cell migration through its inhibitory effect on YAP/TAZ-CTGF-JNK signalling axis and underscores the importance of epigenetic inactivation of Cbx7 in gliomagenesis. PMID:27291091

  12. Transcriptional read-through is not sufficient to induce an epigenetic switch in the silencing activity of Polycomb response elements

    PubMed Central

    Erokhin, Maksim; Elizar’ev, Pavel; Parshikov, Aleksander; Schedl, Paul; Georgiev, Pavel; Chetverina, Darya

    2015-01-01

    In Drosophila, Polycomb (PcG) and Trithorax (TrxG) group proteins are assembled on Polycomb response elements (PREs) to maintain tissue and stage-specific patterns of gene expression. Critical to coordinating gene expression with the process of differentiation, the activity of PREs can be switched “on” and “off.” When on, the PRE imposes a silenced state on the genes in the same domain that is stably inherited through multiple rounds of cell division. When the PRE is switched off, the domain is in a state permissive for gene expression that can be stably inherited. Previous studies have suggested that a burst of transcription through a PRE sequence displaces PcG proteins and provides a universal mechanism for inducing a heritable switch in PRE activity from on to off; however, the evidence favoring this model is indirect. Here, we have directly tested the transcriptional read-through mechanism. Contrary to previous suggestions, we show that transcription through the PRE is not sufficient for inducing an epigenetic switch in PRE activity. In fact, even high levels of continuous transcription through a PRE fails to dislodge the PcG proteins, nor does it remove repressive histone marks. Our results indicate that other mechanisms involving adjacent DNA regulatory elements must be implicated in heritable switch of PRE activity. PMID:26504232

  13. Cbx7 is epigenetically silenced in glioblastoma and inhibits cell migration by targeting YAP/TAZ-dependent transcription

    PubMed Central

    Nawaz, Zahid; Patil, Vikas; Arora, Anjali; Hegde, Alangar S.; Arivazhagan, Arimappamagan; Santosh, Vani; Somasundaram, Kumaravel

    2016-01-01

    Glioblastomas (GBM) are the most malignant form of astrocytomas which are difficult to treat and portend a grave clinical course and poor prognosis. In this study, we identified Chromobox homolog 7 (Cbx7), a member of Polycomb Repressive Complex 1 (PRC1), as a downregulated gene in GBM owing to its promoter hypermethylation. Bisulphite sequencing and methylation inhibitor treatment established the hypermethylation of Cbx7 in GBM. Exogenous overexpression of Cbx7 induced cell death, inhibited cell proliferation, colony formation and migration/invasion of the glioma cells. GSEA of Cbx7 regulated genes identified Cbx7 as a repressor of transcription co-activators YAP/TAZ, the inhibitory targets of the Hippo signalling pathway. In good correlation, the exogenous expression of Cbx7 repressed the YAP/TAZ-dependent transcription and downregulated CTGF, a bonafide YAP/TAZ target. We also observed reduced levels of phospho-JNK in Cbx7 expressing cells. Additionally, CTGF silencing and pharmacological inhibition of JNK also inhibited glioma cell migration. Further, Cbx7 failed to inhibit cell migration significantly in the presence of exogenously overexpressed CTGF or constitutively active JNK. Thus, our study identifies Cbx7 as an inhibitor of glioma cell migration through its inhibitory effect on YAP/TAZ-CTGF-JNK signalling axis and underscores the importance of epigenetic inactivation of Cbx7 in gliomagenesis. PMID:27291091

  14. Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes.

    PubMed

    Matsuoka, Yuji; Bando, Tetsuya; Watanabe, Takahito; Ishimaru, Yoshiyasu; Noji, Sumihare; Popadić, Aleksandar; Mito, Taro

    2015-01-01

    In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that an intriguing transition of the PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes. PMID:25948756

  15. Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes

    PubMed Central

    Matsuoka, Yuji; Bando, Tetsuya; Watanabe, Takahito; Ishimaru, Yoshiyasu; Noji, Sumihare; Popadić, Aleksandar; Mito, Taro

    2015-01-01

    In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that an intriguing transition of the PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes. PMID:25948756

  16. Epigenetic silencing of NKD2, a major component of Wnt signaling, promotes breast cancer growth

    PubMed Central

    Dong, Yan; Cao, Baoping; Zhang, Meiying; Han, Weidong; Herman, James G.; Fuks, François; Zhao, Yali; Guo, Mingzhou

    2015-01-01

    Naked cuticle homolog 2 (NKD2) has been reported to antagonize Wnt signaling in zebrafish, mouse and mammals. The aim of this study is to investigate the epigenetic changes and mechanisms of NKD2 in human breast cancer development. Six breast cancer cell lines (MCF-7, ZR75-1, MDA-MB-468, MDA-MB-231, T47D and BT474) and 68 cases of primary human breast cancer were studied using methylation specific PCR, immunohistochemistry, western blot, flow cytometry techniques and a xenograft mouse model. The expression of NKD1 and NKD2 was regulated by promoter region methylation in breast cancer cells. No NKD1 methylation was found in primary human breast cancer. NKD2 was methylated in 51.4% (35/68) of human primary breast cancer samples. NKD2 methylation was significantly associated with reduction of NKD2 expression, and tumor stage (p < 0.05). NKD2 suppressed breast cancer cell proliferation both in vitro and in vivo. NKD2 induced G1/S arrest and inhibited Wnt signaling in breast cancer cells. In conclusion, NKD2 is frequently methylated in human breast cancer, and the expression of NKD2 is regulated by promoter region methylation. NKD2 suppresses breast cancer proliferation by inhibiting Wnt signaling. PMID:26124080

  17. Epigenetically silenced GNG4 inhibits SDF1α/CXCR4 signaling in mesenchymal glioblastoma

    PubMed Central

    Pal, Jagriti; Patil, Vikas; Mondal, Baisakhi; Shukla, Sudhanshu; Hegde, Alangar S.; Arivazhagan, Arimappamagan; Santosh, Vani; Somasundaram, Kumaravel

    2016-01-01

    The most common and aggressive form of primary brain tumor in adults is glioblastoma (GBM). From the global DNA methylation profiling study, previously published from our laboratory, we identified Guanine Nucleotide binding-protein Gamma subunit 4 (GNG4) to be one of the most hyper methylated and down regulated genes in GBM. GBM derived cell lines showed reduced GNG4 transcript levels, which could be reversed by methylation inhibitor treatment. Bisulphite sequencing confirmed the methylation status in glioblastoma tumor tissue and GBM derived cell lines. Overexpression of GNG4 was found to inhibit proliferation and colony formation of GBM cell lines and in vitro transformation of immortalized human astrocytes, thus suggesting a potential tumor suppressor role of GNG4 in GBM. Correlation of GNG4 transcript levels with that of all GPCRs from TCGA data revealed chemokine receptors as the potential target of GNG4. Furthermore, exogenous over expression of GNG4 inhibited SDF1α/CXCR4-dependent chemokine signaling as seen by reduced pERK and pJNK and GBM cell migration. The inhibitory association between GNG4 and SDF1α/CXCR4 was more evident in mesenchymal subtype of GBM. Thus, this study identifies GNG4 as an inhibitor of SDF1α/CXCR4-dependent signaling and emphasizes the significance of epigenetic inactivation of GNG4 in glioblastoma, especially in mesenchymal subtype. PMID:27382437

  18. Loss of epigenetic silencing of the DUX4 transcription factor gene in facioscapulohumeral muscular dystrophy.

    PubMed

    Hewitt, Jane E

    2015-10-15

    Current genetic and molecular evidence best supports an epigenetic mechanism for facioscapulohumeral muscular dystrophy (FSHD), whereby de-repression of the D4Z4 macrosatellite array leads to aberrant expression of the DUX4 transcription factor in skeletal muscle. This de-repression is triggered by either array contraction or (more rarely) by mutation of the SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) gene. Activation of DUX4 targets, including germline genes and several mammalian retrotransposons, then drives pathogenesis. A direct role for DUX4 mRNA in suppression of nonsense-mediated decay pathways has recently been demonstrated and may also contribute to muscle pathology. Loss of D4Z4 repression in FSHD is observed as hypomethylation of the array accompanied by loss of repressive chromatin marks. The molecular mechanisms of D4Z4 repression are poorly understood, but recent data have identified an Argonaute (AGO)-dependent siRNA pathway. Targeting this pathway by exogenous siRNAs could be a therapeutic strategy for FSHD. PMID:26113644

  19. Epigenetic silencing of S100A2 in bladder and head and neck cancers

    PubMed Central

    Lee, Juna; Wysocki, Piotr T.; Topaloglu, Ozlem; Maldonado, Leonel; Brait, Mariana; Begum, Shahnaz; Moon, David; Kim, Myoung Sook; Califano, Joseph A.; Sidransky, David; Hoque, Mohammad O.; Moon, Chulso

    2015-01-01

    S100A2, a member of the S100 protein family, is known to be downregulated in a number of human cancers, leading to its designation as a potential tumor suppressor gene. Here, we investigated the expression and methylation status of S100A2 in head&neck and bladder cancer. Reduced mRNA and protein expression was observed in 8 head&neck and bladder cancer cell lines. To explore the mechanism responsible for the downregulation of S100A2, we treated six cell lines with 5-aza-2′-deoxycytidine. We found S100A2 is silenced in association with aberrant promoter-region methylation and its expression is restored with 5-aza-2′-deoxycytidine treatment. Of 31 primary head&neck cancer cases and 31 bladder cancer cases, promoter methylation was detected in 90% and 80% of cases, respectively. Interestingly, only 1/9 of normal head&neck tissues and 2/6 of normal bladder tissues showed promoter methylation. S100A2 promoter methylation can be detected in urine and is more frequent in bladder cancer patients than in healthy subjects (96% vs 48% respectively). Moreover, increased methylation of S100A2 is linked to the progression of the tumor in bladder cancer (p<0.01). Together, this data shows that methylation-associated inactivation of S100A2 is frequent and may be an important event in the tumorigenesis of head&neck and bladder cancer. PMID:26097874

  20. Epigenetic silencing of Bim transcription by Spi-1/PU.1 promotes apoptosis resistance in leukaemia.

    PubMed

    Ridinger-Saison, M; Evanno, E; Gallais, I; Rimmelé, P; Selimoglu-Buet, D; Sapharikas, E; Moreau-Gachelin, F; Guillouf, C

    2013-09-01

    Deregulation of transcriptional networks contributes to haematopoietic malignancies. The transcription factor Spi-1/PU.1 is a master regulator of haematopoiesis and its alteration leads to leukaemia. Spi-1 overexpression inhibits differentiation and promotes resistance to apoptosis in erythroleukaemia. Here, we show that Spi-1 inhibits mitochondrial apoptosis in vitro and in vivo through the transcriptional repression of Bim, a proapoptotic factor. BIM interacts with MCL-1 that behaves as a major player in the survival of the preleukaemic cells. The repression of BIM expression reduces the amount of BIM-MCL-1 complexes, thus increasing the fraction of potentially active antiapoptotic MCL-1. We then demonstrate that Spi-1 represses Bim transcription by binding to the Bim promoter and by promoting the trimethylation of histone 3 on lysine 27 (H3K27me3, a repressive histone mark) on the Bim promoter. The PRC2 repressive complex of Polycomb is directly responsible for the deposit of H3K27me3 mark at the Bim promoter. SUZ12 and the histone methyltransferase EZH2, two PRC2 subunits bind to the Bim promoter at the same location than H3K27me3, distinct of the Spi-1 DNA binding site. As Spi-1 interacts with SUZ12 and EZH2, these results indicate that Spi-1 modulates the activity of PRC2 without directly recruiting the complex to the site of its activity on the chromatin. Our results identify a new mechanism whereby Spi-1 represses transcription and provide mechanistic insights on the antiapoptotic function of a transcription factor mediated by the epigenetic control of gene expression. PMID:23852375

  1. Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development.

    PubMed

    Lewandowski, Sara L; Janardhan, Harish P; Trivedi, Chinmay M

    2015-11-01

    About two-thirds of human congenital heart disease involves second heart field-derived structures. Histone-modifying enzymes, histone deacetylases (HDACs), regulate the epigenome; however, their functions within the second heart field remain elusive. Here we demonstrate that histone deacetylase 3 (HDAC3) orchestrates epigenetic silencing of Tgf-β1, a causative factor in congenital heart disease pathogenesis, in a deacetylase-independent manner to regulate development of second heart field-derived structures. In murine embryos lacking HDAC3 in the second heart field, increased TGF-β1 bioavailability is associated with ascending aortic dilatation, outflow tract malrotation, overriding aorta, double outlet right ventricle, aberrant semilunar valve development, bicuspid aortic valve, ventricular septal defects, and embryonic lethality. Activation of TGF-β signaling causes aberrant endothelial-to-mesenchymal transition and altered extracellular matrix homeostasis in HDAC3-null outflow tracts and semilunar valves, and pharmacological inhibition of TGF-β rescues these defects. HDAC3 recruits components of the PRC2 complex, methyltransferase EZH2, EED, and SUZ12, to the NCOR complex to enrich trimethylation of Lys-27 on histone H3 at the Tgf-β1 regulatory region and thereby maintains epigenetic silencing of Tgf-β1 specifically within the second heart field-derived mesenchyme. Wild-type HDAC3 or catalytically inactive HDAC3 expression rescues aberrant endothelial-to-mesenchymal transition and epigenetic silencing of Tgf-β1 in HDAC3-null outflow tracts and semilunar valves. These findings reveal that epigenetic dysregulation within the second heart field is a predisposing factor for congenital heart disease. PMID:26420484

  2. RARβ Promoter Methylation as an Epigenetic Mechanism of Gene Silencing in Non-small Cell Lung Cancer.

    PubMed

    Dutkowska, A; Antczak, A; Pastuszak-Lewandoska, D; Migdalska-Sek, M; Czarnecka, K H; Górski, P; Kordiak, J; Nawrot, E; Brzeziańska-Lasota, E

    2016-01-01

    The retinoid acid receptor-p (RARβ) gene is one of the tumor suppressor genes (TSGs), which is frequently deleted or epigenetically silenced at an early stage of tumor progression. In this study we investigated the promoter methylation and expression status of the RARβ gene in 60 surgically resected non-small cell lung cancer (NSCLC) tissue samples and 60 corresponding unchanged lung tissue samples, using methylation-specific PCR and real-time-polymerase chain reaction (qPCR) techniques. We correlated the results with the pathological features of tumors and clinical characteristics of patients. qPCR analysis detected a significantly lower RARβ expression in the patients with adenocarcinoma (AC) and large cell carcinoma (LCC) than in those with squamous cell carcinoma (SCC) (AC vs. SCC, p = 0.032; AC and LCC vs. SCC, p = 0.0 13). Additionally, significantly lower expression of the RARβ gene was revealed in the patients with non-squamous cell cancer with a history of smoking assessed as pack-years (PY < 40 vs. PY ≥ 40, p = 0.045). Regarding RARβ promoter methylation, we found significant differences in the methylation index in the SCC group when considering pTNM staging; with higher index values in T1a + T1b compared with T2a + T2b and T3 + T4 groups (p = 0.024). There was no correlation between the methylation status and expression level of the RARβ gene, which suggests that other molecular mechanisms influence the RARβ expression in NSCLC patients. In conclusion, different expression of the RARβ gene in SCC and NSCC makes the RARβ gene a valuable diagnostic marker for differentiating the NSCLC subtypes. PMID:26453065

  3. Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber, Apostichopus japonicus (Selenka), during aestivation

    NASA Astrophysics Data System (ADS)

    Wang, Tianming; Yang, Hongsheng; Zhao, Huan; Chen, Muyan; Wang, Bing

    2011-11-01

    The sea cucumber, Apostichopus japonicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling (DNA (cytosine-5)-methyltransferase 1, Methyl-CpG-binding domain protein 2), and Chromodomain-helicase-DNA-binding protein 5) was significantly higher during aestivation (Days 20 and 40). Similarly, we observed an increase in the expression of genes involved in histone acetylation (Histone deacetylase 3) and Histone-binding protein RBBP4) during the early (Days 5 and 10) and late phases (Days 20 and 40) of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers (Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5) was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

  4. Epigenetic silencing of the NR4A3 tumor suppressor, by aberrant JAK/STAT signaling, predicts prognosis in gastric cancer

    PubMed Central

    Yeh, Chung-Min; Chang, Liang-Yu; Lin, Shu-Hui; Chou, Jian-Liang; Hsieh, Hsiao-Yen; Zeng, Li-Han; Chuang, Sheng-Yu; Wang, Hsiao-Wen; Dittner, Claudia; Lin, Cheng-Yu; Lin, Jora M. J.; Huang, Yao-Ting; Ng, Enders K. W.; Cheng, Alfred S. L.; Wu, Shu-Fen; Lin, Jiayuh; Yeh, Kun-Tu; Chan, Michael W. Y.

    2016-01-01

    While aberrant JAK/STAT signaling is crucial to the development of gastric cancer (GC), its effects on epigenetic alterations of its transcriptional targets remains unclear. In this study, by expression microarrays coupled with bioinformatic analyses, we identified a putative STAT3 target gene, NR4A3 that was downregulated in MKN28 GC daughter cells overexpressing a constitutively activated STAT3 mutant (S16), as compared to an empty vector control (C9). Bisulphite pyrosequencing and demethylation treatment showed that NR4A3 was epigenetically silenced by promoter DNA methylation in S16 and other GC cell lines including AGS cells, showing constitutive activation of STAT3. Subsequent experiments revealed that NR4A3 promoter binding by STAT3 might repress its transcription. Long-term depletion of STAT3 derepressed NR4A3 expression, by promoter demethylation, in AGS GC cells. NR4A3 re-expression in GC cell lines sensitized the cells to cisplatin, and inhibited tumor growth in vitro and in vivo, in an animal model. Clinically, GC patients with high NR4A3 methylation, or lower NR4A3 protein expression, had significantly shorter overall survival. Intriguingly, STAT3 activation significantly associated only with NR4A3 methylation in low-stage patient samples. Taken together, aberrant JAK/STAT3 signaling epigenetically silences a potential tumor suppressor, NR4A3, in gastric cancer, plausibly representing a reliable biomarker for gastric cancer prognosis. PMID:27528092

  5. Epigenetic silencing of the NR4A3 tumor suppressor, by aberrant JAK/STAT signaling, predicts prognosis in gastric cancer.

    PubMed

    Yeh, Chung-Min; Chang, Liang-Yu; Lin, Shu-Hui; Chou, Jian-Liang; Hsieh, Hsiao-Yen; Zeng, Li-Han; Chuang, Sheng-Yu; Wang, Hsiao-Wen; Dittner, Claudia; Lin, Cheng-Yu; Lin, Jora M J; Huang, Yao-Ting; Ng, Enders K W; Cheng, Alfred S L; Wu, Shu-Fen; Lin, Jiayuh; Yeh, Kun-Tu; Chan, Michael W Y

    2016-01-01

    While aberrant JAK/STAT signaling is crucial to the development of gastric cancer (GC), its effects on epigenetic alterations of its transcriptional targets remains unclear. In this study, by expression microarrays coupled with bioinformatic analyses, we identified a putative STAT3 target gene, NR4A3 that was downregulated in MKN28 GC daughter cells overexpressing a constitutively activated STAT3 mutant (S16), as compared to an empty vector control (C9). Bisulphite pyrosequencing and demethylation treatment showed that NR4A3 was epigenetically silenced by promoter DNA methylation in S16 and other GC cell lines including AGS cells, showing constitutive activation of STAT3. Subsequent experiments revealed that NR4A3 promoter binding by STAT3 might repress its transcription. Long-term depletion of STAT3 derepressed NR4A3 expression, by promoter demethylation, in AGS GC cells. NR4A3 re-expression in GC cell lines sensitized the cells to cisplatin, and inhibited tumor growth in vitro and in vivo, in an animal model. Clinically, GC patients with high NR4A3 methylation, or lower NR4A3 protein expression, had significantly shorter overall survival. Intriguingly, STAT3 activation significantly associated only with NR4A3 methylation in low-stage patient samples. Taken together, aberrant JAK/STAT3 signaling epigenetically silences a potential tumor suppressor, NR4A3, in gastric cancer, plausibly representing a reliable biomarker for gastric cancer prognosis. PMID:27528092

  6. High-Throughput Screening Using iPSC-Derived Neuronal Progenitors to Identify Compounds Counteracting Epigenetic Gene Silencing in Fragile X Syndrome.

    PubMed

    Kaufmann, Markus; Schuffenhauer, Ansgar; Fruh, Isabelle; Klein, Jessica; Thiemeyer, Anke; Rigo, Pierre; Gomez-Mancilla, Baltazar; Heidinger-Millot, Valerie; Bouwmeester, Tewis; Schopfer, Ulrich; Mueller, Matthias; Fodor, Barna D; Cobos-Correa, Amanda

    2015-10-01

    Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused in most of cases by epigenetic silencing of the Fmr1 gene. Today, no specific therapy exists for FXS, and current treatments are only directed to improve behavioral symptoms. Neuronal progenitors derived from FXS patient induced pluripotent stem cells (iPSCs) represent a unique model to study the disease and develop assays for large-scale drug discovery screens since they conserve the Fmr1 gene silenced within the disease context. We have established a high-content imaging assay to run a large-scale phenotypic screen aimed to identify compounds that reactivate the silenced Fmr1 gene. A set of 50,000 compounds was tested, including modulators of several epigenetic targets. We describe an integrated drug discovery model comprising iPSC generation, culture scale-up, and quality control and screening with a very sensitive high-content imaging assay assisted by single-cell image analysis and multiparametric data analysis based on machine learning algorithms. The screening identified several compounds that induced a weak expression of fragile X mental retardation protein (FMRP) and thus sets the basis for further large-scale screens to find candidate drugs or targets tackling the underlying mechanism of FXS with potential for therapeutic intervention. PMID:26024946

  7. DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

    PubMed Central

    Norregaard, Kamilla; Andersson, Magnus; Sneppen, Kim; Nielsen, Peter Eigil; Brown, Stanley; Oddershede, Lene B.

    2013-01-01

    Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch regulation is understood. Using single molecule analysis, we directly examined the stability of the CI-operator structure in its natural, supercoiled state. We marked positions adjacent to the λ operators with peptide nucleic acids and monitored their movement by tethered particle tracking. Compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient. PMID:24101469

  8. DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch.

    PubMed

    Norregaard, Kamilla; Andersson, Magnus; Sneppen, Kim; Nielsen, Peter Eigil; Brown, Stanley; Oddershede, Lene B

    2013-10-22

    Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch regulation is understood. Using single molecule analysis, we directly examined the stability of the CI-operator structure in its natural, supercoiled state. We marked positions adjacent to the λ operators with peptide nucleic acids and monitored their movement by tethered particle tracking. Compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient. PMID:24101469

  9. DNA methylome analysis identifies epigenetic silencing of FHIT as a determining factor for radiosensitivity in oral cancer: an outcome-predicting and treatment-implicating study

    PubMed Central

    Lin, Hon-Yi; Hung, Shih-Kai; Lee, Moon-Sing; Chiou, Wen-Yen; Huang, Tze-Ta; Tseng, Chih-En; Shih, Liang-Yu; Lin, Ru-Inn; Lin, Jora M.J.; Lai, Yi-Hui; Chang, Chia-Bin; Hsu, Feng-Chun; Chen, Liang-Cheng; Tsai, Shiang-Jiun; Su, Yu-Chieh; Li, Szu-Chi; Lai, Hung-Chih; Hsu, Wen-Lin; Liu, Dai-Wei; Tai, Chien-Kuo; Wu, Shu-Fen; Chan, Michael W.Y.

    2015-01-01

    Radioresistance is still an emerging problem for radiotherapy of oral cancer. Aberrant epigenetic alterations play an important role in cancer development, yet the role of such alterations in radioresistance of oral cancer is not fully explored. Using a methylation microarray, we identified promoter hypermethylation of FHIT (fragile histidine triad) in radioresistant OML1-R cells, established from hypo-fractionated irradiation of parental OML1 radiosensitive oral cancer cells. Further analysis confirmed that transcriptional repression of FHIT was due to promoter hypermethylation, H3K27me3 and overexpression of methyltransferase EZH2 in OML1-R cells. Epigenetic interventions or depletion of EZH2 restored FHIT expression. Ectopic expression of FHIT inhibited tumor growth in both in vitro and in vivo models, while also resensitizing radioresistant cancer cells to irradiation, by restoring Chk2 phosphorylation and G2/M arrest. Clinically, promoter hypermethylation of FHIT inversely correlated with its expression and independently predicted both locoregional control and overall survival in 40 match-paired oral cancer patient samples. Further in vivo therapeutic experiments confirmed that inhibition of DNA methylation significantly resensitized radioresistant oral cancer cell xenograft tumors. These results show that epigenetic silencing of FHIT contributes partially to radioresistance and predicts clinical outcomes in irradiated oral cancer. The radiosensitizing effect of epigenetic interventions warrants further clinical investigation. PMID:25460508

  10. Epigenetic silencing of the p16(INK4a) tumor suppressor is associated with loss of CTCF binding and a chromatin boundary.

    PubMed

    Witcher, Michael; Emerson, Beverly M

    2009-05-15

    The p16(INK4a) tumor suppressor gene is a frequent target of epigenetic inactivation in human cancers, which is an early event in breast carcinogenesis. We describe the existence of a chromatin boundary upstream of the p16 gene that is lost when this gene is aberrantly silenced. We show that the multifunctional protein CTCF associates in the vicinity of this boundary and absence of binding strongly coincides with p16 silencing in multiple types of cancer cells. CTCF binding also correlates with RASSF1A and CDH1 gene activation, and CTCF interaction is absent when these genes are methylated and silenced. Interestingly, defective poly(ADP-ribosyl)ation of CTCF and dissociation from the molecular chaperone Nucleolin occur in p16-silenced cells, abrogating its proper function. Thus, destabilization of specific chromosomal boundaries through aberrant crosstalk between CTCF, poly(ADP-ribosyl)ation, and DNA methylation may be a general mechanism to inactivate tumor suppressor genes and initiate tumorigenesis in numerous forms of human cancers. PMID:19450526

  11. Three SRA-Domain Methylcytosine-Binding Proteins Cooperate to Maintain Global CpG Methylation and Epigenetic Silencing in Arabidopsis

    PubMed Central

    Woo, Hye Ryun; Dittmer, Travis A.; Richards, Eric J.

    2008-01-01

    Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5′ region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing. PMID:18704160

  12. Chromatin insulators: regulatory mechanisms and epigenetic inheritance

    PubMed Central

    Bushey, Ashley M.; Dorman, Elizabeth R.; Corces, Victor G.

    2008-01-01

    Enhancer-blocking insulators are DNA elements that disrupt the communication between a regulatory sequence, such as an enhancer or a silencer, and a promoter. Insulators participate in both transcriptional regulation and global nuclear organization, two features of chromatin that are thought to be maintained from one generation to the next through epigenetic mechanisms. Furthermore, there are many regulatory mechanisms in place that enhance or hinder insulator activity. These modes of regulation could be used to establish cell-type specific insulator activity that is epigenetically inherited along a cell and/or organismal lineage. This review will discuss the evidence for epigenetic inheritance and regulation of insulator function. PMID:18851828

  13. Epigenetic silencing of genes and microRNAs within the imprinted Dlk1-Dio3 region at human chromosome 14.32 in giant cell tumor of bone

    PubMed Central

    2014-01-01

    Background Growing evidence exists that the neoplastic stromal cell population (GCTSC) within giant cell tumors (GCT) originates from mesenchymal stem cells (MSC). In a previous study we identified a microRNA signature that differentiates between these cell types. Five differentially expressed microRNAs are located within the Dlk1-Dio3 region on chromosome 14. Aberrant regulation within this region is known to influence cell growth, differentiation and the development of cancer. The aim of this study was to elucidate the involvement of deregulations within the Dlk1-Dio3 region in GCT pathogenesis. Methods Quantitative gene and microRNA expression analyses were performed on GCTSCs and MSCs with or without treatment with epigenetic modifiers. Methylation analysis of differentially methylated regions was performed by bisulfite sequencing. Results In addition to microRNA silencing we detected a significant downregulation of Dlk1, Meg3 and Meg8 in GCTSCs compared to MSCs. DNA methylation analyses of the Meg3-DMR and IG-DMR revealed a frequent hypermethylation within the IG-DMR in GCTs. Epigenetic modification could restore expression of some but not all analyzed genes and microRNAs suggesting further regulatory mechanisms. Conclusion Epigenetic silencing of genes and microRNAs within the Dlk1-Dio3 region is a common event in GCTSCs, in part mediated by hypermethylation within the IG-DMR. The identified genes, micro RNAs and microRNA target genes might be valuable targets for the development of improved strategies for GCT diagnosis and therapy. PMID:25005035

  14. Long noncoding RNA PVT1 promotes cervical cancer progression through epigenetically silencing miR-200b.

    PubMed

    Zhang, Shaorong; Zhang, Guanli; Liu, Jingying

    2016-08-01

    Long noncoding RNA PVT1 has been reported to be dysregulated and play vital roles in a variety of cancers. However, the functions and molecular mechanisms of PVT1 in cervical cancer remain unclear. The objective of this study was to investigate the expression, clinical significance, biological roles, and underlying functional mechanisms of PVT1 in cervical cancer. Our results revealed that PVT1 is upregulated in cervical cancer tissues. Enhanced expression of PVT1 is associated with larger tumor size, advanced International Federation of Gynecology and Obstetrics stage, and poor prognosis of cervical cancer patients. Using gain-of-function and loss-of-function approaches, we demonstrated that overexpression of PVT1 promotes cervical cancer cells proliferation, cell cycle progression and migration, and depletion of PVT1 inhibits cervical cancer cell proliferation, cell cycle progression, and migration. Mechanistically, we verified that PVT1 binds to EZH2, recruits EZH2 to the miR-200b promoter, increases histone H3K27 trimethylation level on the miR-200b promoter, and inhibits miR-200b expression. Furthermore, the effects of PVT1 on cervical cell proliferation and migration depend upon silencing of miR-200b. Taken together, our findings confirmed that PVT1 functions as an oncogene in cervical cancer and indicated that PVT1 is not only an important prognostic marker, but also a potential therapy target for cervical cancer. PMID:27272214

  15. Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation

    PubMed Central

    Imagawa, Kei; de Andrés, María C; Hashimoto, Ko; Itoi, Eiji; Otero, Miguel; Roach, Helmtrud I; Goldring, Mary B; Oreffo, Richard O C

    2014-01-01

    Objective To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter. Methods Expression levels were determined using quantitative reverse transcription–polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation. Results Although COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter. Conclusion This first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment. PMID:25048791

  16. Epigenetic silencing and unstable inheritance of MuDR activity monitored at four bz2-mu alleles in maize (Zea mays L.).

    PubMed

    Takumi, Shigeo; Walbot, Virginia

    2007-10-01

    Maize MuDR/Mu elements are one of the most active Class II transposons and are widely used for transposon tagging for gene cloning. The autonomous MuDR encodes a transposase, while diverse non-autonomous elements share similarity to MuDR only within their ~215 bp terminal inverted repeats (TIRs). Four independent Mu-induced mutable alleles of the anthocyanin pigment pathway Bronze2 (Bz2) locus have been sequenced; bz2-mu1, bz2-mu2, and bz2-mu3 contain Mu1 element insertions while bz2-mu4 contains a MuDR insertion. Somatic excision activity can be monitored for each allele as a purple spotted phenotype on the otherwise beige epidermal layer of the kernel. To study epigenetic silencing of Mu elements, we investigated inheritance of somatic transposition of these four reporter alleles, and using samples from leaves just below the ear and the tassel, DNA methylation status and mudrA expression were quantified through three consecutive generations. Percentages of spotted kernels when crossed into bz2 tester as pollen parent were lower than those when crossed with bz2 tester as ear parent in all bz2-mu alleles. The propensity for silencing in kernels with different frequencies of spotting was investigated. In the inactive lines, both MuDR and Mu1 elements were de novo methylated. The transposition frequency was negatively correlated with the level of Mu methylation and positively correlated with the level of mudrA transcript. The most reliable indicator of incipient silencing was a decrease in mudrA transcript levels in the leaf below the tassel, and this transcriptional silencing could precede methylation of Mu elements. PMID:17991994

  17. BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

    PubMed Central

    Gong, C; Yao, S; Gomes, A R; Man, E P S; Lee, H J; Gong, G; Chang, S; Kim, S-B; Fujino, K; Kim, S-W; Park, S K; Lee, J W; Lee, M H; Khoo, U S; Lam, E W-F

    2016-01-01

    BRCA1 mutation or depletion correlates with basal-like phenotype and poor prognosis in breast cancer but the underlying reason remains elusive. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with downregulation of the expression of the pleiotropic tumour suppressor FOXO3. Knockdown of BRCA1 by small interfering RNA (siRNA) resulted in downregulation of FOXO3 expression in the BRCA1-competent MCF-7, whereas expression of BRCA1 restored FOXO3 expression in BRCA1-defective HCC70 and MDA-MB-468 cells, suggesting a role of BRCA1 in the control of FOXO3 expression. Treatment of HCC70 and MDA-MB-468 cells with either the DNA methylation inhibitor 5-aza-2'-deoxycitydine, the N-methyltransferase enhancer of zeste homologue 2 (EZH2) inhibitor GSK126 or EZH2 siRNA induced FOXO3 mRNA and protein expression, but had no effect on the BRCA1-competent MCF-7 cells. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNMT1/3a/b and histone H3 lysine 27 trimethylation (H3K27me3) are recruited to the endogenous FOXO3 promoter, further advocating that these proteins interact to modulate FOXO3 methylation and expression. In addition, ChIP results also revealed that BRCA1 depletion promoted the recruitment of the DNA methyltransferases DNMT1/3a/3b and the enrichment of the EZH2-mediated transcriptional repressive epigenetic marks H3K27me3 on the FOXO3 promoter. Methylated DNA immunoprecipitation assays also confirmed increased CpG methylation of the FOXO3 gene on BRCA1 depletion. Analysis of the global gene methylation profiles of a cohort of 33 familial breast tumours revealed that FOXO3 promoter methylation is significantly associated with BRCA1 mutation. Furthermore, immunohistochemistry further suggested that FOXO3 expression was significantly associated with BRCA1 status in EZH2-positive breast cancer. Consistently, high FOXO3 and EZH2 mRNA levels were significantly associated with good and poor

  18. Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways

    PubMed Central

    Yu, Weishi; McIntosh, Carl; Lister, Ryan; Zhu, Iris; Han, Yixing; Ren, Jianke; Landsman, David; Lee, Eunice; Briones, Victorino; Terashima, Minoru; Leighty, Robert; Ecker, Joseph R.

    2014-01-01

    Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation. PMID:25170028

  19. Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses.

    PubMed

    White, Clayton A; Pone, Egest J; Lam, Tonika; Tat, Connie; Hayama, Ken L; Li, Guideng; Zan, Hong; Casali, Paolo

    2014-12-15

    Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require activation-induced cytidine deaminase (AID), and plasma cell differentiation, which requires B lymphocyte-induced maturation protein-1 (Blimp-1), are critical for the generation of class-switched and hypermutated (mature) Ab and autoantibody responses. We show that histone deacetylase inhibitors valproic acid and butyrate dampened AICDA/Aicda (AID) and PRDM1/Prdm1 (Blimp-1) mRNAs by upregulating miR-155, miR-181b, and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a, and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1, and X-box binding protein 1, thereby inhibiting CSR, SHM, and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of histone deacetylase inhibitor-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2, or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a, and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell-intrinsic epigenetic mechanisms, valproic acid blunted class-switched and hypermutated T-dependent and T-independent Ab responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease, and extended survival in lupus MRL/Fas(lpr/lpr) mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and X-box binding protein 1) that are critical to the B cell differentiation processes that underpin Ab and autoantibody responses. They also provide therapeutic proof-of-principle in autoantibody-mediated autoimmunity. PMID:25392531

  20. Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    PubMed Central

    Zylicz, Jan J; Dietmann, Sabine; Günesdogan, Ufuk; Hackett, Jamie A; Cougot, Delphine; Lee, Caroline; Surani, M Azim

    2015-01-01

    Early mouse development is accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2), which is essential for embryonic development. Here we show that genome-wide accumulation of H3K9me2 is crucial for postimplantation development, and coincides with redistribution of enhancer of zeste homolog 2 (EZH2)-dependent histone H3 lysine 27 trimethylation (H3K27me3). Loss of G9a or EZH2 results in upregulation of distinct gene sets involved in cell cycle regulation, germline development and embryogenesis. Notably, the H3K9me2 modification extends to active enhancer elements where it promotes developmentally-linked gene silencing and directly marks promoters and gene bodies. This epigenetic mechanism is important for priming gene regulatory networks for critical cell fate decisions in rapidly proliferating postimplantation epiblast cells. DOI: http://dx.doi.org/10.7554/eLife.09571.001 PMID:26551560

  1. The silencing of Kierkegaard in Habermas' critique of genetic enhancement.

    PubMed

    Christiansen, Karin

    2009-06-01

    The main purpose of this paper is to draw attention to an important part of Habermas' critique of genetic enhancement, which has been largely ignored in the discussion; namely his use of Kierkegaard's reflections on the existential conditions for becoming one-self from Either/or and the Sickness unto Death. It will be argued that, although Habermas presents some valuable and highly significant perspectives on the effect of genetic enhancement on the individual's self-understanding and ability to experience him- or herself as a free and equal individual, he does not succeed in working out a consistent argument. The claim is that he fails to explain how the existential analysis is related to his reflections on the sociological and psychological impacts of genetic enhancement in the realm of communicative action. It is this lack of theoretical clarity, which seems to render Habermas vulnerable to some of the critique which has been raised against his theory from a number of different scientific disciplines and areas of research. Hence, the first part of the paper provides some examples of the nature and variety of this critique, the second part presents Habermas' own critique of genetic enhancement in the context of a dispute between so-called 'liberal' and 'conservative' arguments, and finally, the third part discusses the limits and possibilities of his position in a future debate about genetic enhancement. PMID:19234761

  2. piggyBac transposon plus insulators overcome epigenetic silencing to provide for stable signaling pathway reporter cell lines.

    PubMed

    Mossine, Valeri V; Waters, James K; Hannink, Mark; Mawhinney, Thomas P

    2013-01-01

    Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other "transgene unfriendly" cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5'- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular. PMID:24376882

  3. piggyBac Transposon plus Insulators Overcome Epigenetic Silencing to Provide for Stable Signaling Pathway Reporter Cell Lines

    PubMed Central

    Mossine, Valeri V.; Waters, James K.; Hannink, Mark; Mawhinney, Thomas P.

    2013-01-01

    Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other “transgene unfriendly” cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5’- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular. PMID:24376882

  4. MicroRNA-212 functions as an epigenetic-silenced tumor suppressor involving in tumor metastasis and invasion of gastric cancer through down-regulating PXN expression

    PubMed Central

    Li, Daojiang; Li, Zhengrong; Xiong, Jianbo; Gong, Binbin; Zhang, Guoyang; Cao, Chao; Jie, Zhigang; Liu, Yi; Cao, Yi; Yan, Yufeng; Xiong, Hong; Qiu, Lingyu; Yang, Miantian; Chen, Hongping; Jiang, Shuping; Yang, Xiongwen; Chen, Heping

    2015-01-01

    Altered expression of paxillin (PXN) is closely linked to the pathogenesis progression, metastasis and prognosis of different malignancies including gastric cancer (GC). Epigenetic silencing of tumor-suppressive microRNAs (miRNAs) is a crucial component of the mechanism underlying activation of oncogenes in tumor. To screen for epigenetically silenced miRNAs which target PXN in GC, we performed bioinformatics algorithms and real-time PCR analysis, and identified miR-212 as the optimum candidate gene. A luciferase reporter gene assay validated that miR-212 directly targets the 3’UTR region of PXN. Importantly, miR-212 levels were inversely correlated with PXN expression in GC cell lines and clinical tumor tissues. The use of miR-212 minics decrease PXN mRNA and protein level in GC cell lines. Moreover, low expression of miR-212 and its promoter hypermethylation were causally related and were associated with aggressive tumor phenotype and adverse prognosis in GC. Restoring mir-212 expression by exogenous mirprecursor molecules transfection or reexpression of endogenous miR-212 treated by 5-aza-2’-deoxycytidine (5-aza) can exert similar effect that reduce GC cells invasion and metastasis abilities in vitro by interacting PXN gene. In addition, 5-aza-induced PXN reduction could be partically blocked by miR-212 inhibitor, resulting in a reversal of weankening cell migration and invasion ability of 5-aza. A rescue experiment and a loss-of-function experiment in vitro and vivo showed that PXN restoration rescues migration and invasion phenotype in miR-212 overexpressed GC cell lines and PXN knockdown blocks GC cells migration and invasion in the presence miR-212 inhibitors. Taken together, our results clearly show that overexpression of PXN induced by methylationsuppressed miR-212 promotes tumor metastasis and invasion, and regulation of miR-212 expression may be a novel therapeutic strategy for gastric cancer. PMID:26693054

  5. The insulation of genes from external enhancers and silencing chromatin

    PubMed Central

    Burgess-Beusse, Bonnie; Farrell, Catherine; Gaszner, Miklos; Litt, Michael; Mutskov, Vesco; Recillas-Targa, Felix; Simpson, Melanie; West, Adam; Felsenfeld, Gary

    2002-01-01

    Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5′ end of the chicken β-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore. PMID:12154228

  6. Role of microRNAs in epigenetic silencing of the CHD5 tumor suppressor gene in neuroblastomas

    PubMed Central

    Naraparaju, Koumudi; Kolla, Venkatadri; Zhuang, Tiangang; Higashi, Mayumi; Iyer, Radhika; Kolla, Sriharsha; Okawa, Erin R.; Blobel, Gerd A.; Brodeur, Garrett M.

    2016-01-01

    Neuroblastoma (NB), a tumor of the sympathetic nervous system, is the most common extracranial solid tumor of childhood. We and others have identified distinct patterns of genomic change that underlie diverse clinical behaviors, from spontaneous regression to relentless progression. We first identified CHD5 as a tumor suppressor gene that is frequently deleted in NBs. Mutation of the remaining CHD5 allele is rare in these tumors, yet expression is very low or absent, so expression is likely regulated by epigenetic mechanisms. In order to understand the potential role of miRNA regulation of CHD5 protein expression in NBs, we examined all miRNAs that are predicted to target the 3′-UTR using miRanda, TargetScan and other algorithms. We identified 18 miRNAs that were predicted by 2 or more programs: miR-204, -211, -216b, -17, -19ab, -20ab, -93, -106ab, -130ab, -301ab, -454, -519d, -3666. We then performed transient transfections in two NB cell lines, NLF (MYCN amplified) and SY5Y (MYCN non-amplified), with the reporter plasmid and miRNA mimic, as well as appropriate controls. We found seven miRNAs that significantly downregulated CHD5 expression in NB: miR-211, 17, -93, -20b, -106b, -204, and -3666. Interestingly, MYCN upregulates several of the candidates we identified: miR-17, -93, -106b & -20b. This suggests that miRNAs driven by MYCN and other genes represent a potential epigenetic mechanism to regulate CHD5 expression. PMID:26895110

  7. Inhibition of human esophageal squamous cell carcinomas by targeted silencing of tumor enhancer genes: an overview

    PubMed Central

    Islamian, Jalil Pirayesh; Mohammadi, Mohsen; Baradaran, Behzad

    2014-01-01

    Esophageal cancer has been reported as the ninth most common malignancy and ranks as the sixth most frequent cause of death worldwide. Esophageal cancer treatment involves surgery, chemotherapy, radiation therapy, or combination therapy. Novel strategies are needed to boost the oncologic outcome. Recent advances in the molecular biology of esophageal cancer have documented the role of genetic alterations in tumorigenesis. Oncogenes serve a pivotal function in tumorigenesis. Targeted therapies are directed at the unique molecular signature of cancer cells for enhanced efficacy with low toxicity. RNA interference (RNAi) technology is a powerful tool for silencing endogenous or exogenous genes in mammalian cells. Related results have shown that targeting oncogenes with siRNAs, specifically the mRNA, effectively reduces tumor cell proliferation and induces apoptotic cell death. This article will briefly review studies on silencing tumor enhancer genes related to the induction of esophageal cancer. PMID:25009749

  8. LRIG1, a 3p tumor suppressor, represses EGFR signaling and is a novel epigenetic silenced gene in colorectal cancer

    SciTech Connect

    Kou, Changhua; Zhou, Tian; Han, Xilin; Zhuang, Huijie; Qian, Haixin

    2015-08-21

    Downregulation of LRIG1 was found in many types of cancer. However, data concerning the possible mechanism of LRIG1 reduction in cancers were not reported yet. To analyze the regulation and function of LRIG1 in colorectal cancer (CRC), 6 cell lines, 46 paired tissues from primary CRC cases were employed in this study. In CRC cell lines, under-expression of LRIG1 was correlated with promoter region hypermethylation, and restoration of LRIG1 was induced by 5-Aza-2'-deoxyazacytidine treatment. Subsequently, we ectopically expressed LRIG1 in LRIG1 low-expressing HCT-116 cells and suppressed LRIG1 in LRIG1 high-expressing LoVo cells. We found that over-expression of LRIG1 inhibits cell proliferation and colony formation and tumor growth, while knockdown of LRIG1 promotes cell proliferation and colony formation. Decreased and increased EGFR/AKT signaling pathway may partially explain the lower and higher rates of proliferation in CRC cells transfected with LRIG1 cDNA or shRNA. In clinical samples, we compared the methylation, mRNA and protein expression of LRIG1 in samples of CRC tissues. A significant increase in LRIG1 methylation was identified in CRC specimens compared to adjacent normal tissues and that it was negatively correlated with its mRNA and protein expression. In conclusion, LRIG1 is frequently methylated in human CRC and consequent mRNA and protein downregulation may contribute to tumor growth by activating EGFR/AKT signaling. - Highlights: • Promoter methylation of LRIG1 occurred in colorectal cancer cells and tumors. • Restoration of LRIG1 inhibits tumor growth in vitro and in vivo. • Overexpression or knockdown of LRIG1 regulates EGFR/AKT and downstream apoptosis. • Methylation of LRIG1 correlates with its mRNA and protein downregulation. • LRIG1 was firstly identified as an epigenetic target in cancer.

  9. Tolerance of Sir1p/origin recognition complex-dependent silencing for enhanced origin firing at HMRa.

    PubMed

    McConnell, Kristopher H; Müller, Philipp; Fox, Catherine A

    2006-03-01

    The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site. PMID:16479013

  10. Ku-deficient yeast strains exhibit alternative states of silencing competence.

    PubMed

    Maillet, L; Gaden, F; Brevet, V; Fourel, G; Martin, S G; Dubrana, K; Gasser, S M; Gilson, E

    2001-03-01

    In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control. PMID:11266361

  11. Mutations in ash1 and trx enhance P-element-dependent silencing in Drosophila melanogaster.

    PubMed

    McCracken, Allen; Locke, John

    2016-08-01

    In Drosophila melanogaster, the mini-w(+) transgene in Pci is normally expressed throughout the adult eye; however, when other P or KP elements are present, a variegated-eye phenotype results, indicating random w(+) silencing during development called P-element-dependent silencing (PDS). Mutant Su(var)205 and Su(var)3-7 alleles act as haplo-suppressors/triplo-enhancers of this variegated phenotype, indicating that these heterochromatic modifiers act dose dependently in PDS. Previously, we recovered a spontaneous mutation of P{lacW}ci(Dplac) called P{lacW}ci(DplacE1) (E1) that variegated in the absence of P elements, presumably due to the insertion of an adjacent gypsy element. From a screen for genetic modifiers of E1 variegation, we describe here the isolation of five mutations in ash1 and three in trx that enhance the E1 variegated phenotype in a dose-dependent and cumulative manner. These mutant alleles enhance PDS at E1, and in E1/P{lacW}ci(Dplac), but suppress position effect variegation (PEV) at In(1)w(m)(4). This opposite action is consistent with a model where ASH1 and TRX mark transcriptionally active chromatin domains. If ASH1 or TRX function is lost or reduced, heterochromatin can spread into these domains creating a sink that diverts heterochromatic proteins from other variegating locations, which then may express a suppressed phenotype. PMID:27373142

  12. CLDN14 is epigenetically silenced by EZH2-mediated H3K27ME3 and is a novel prognostic biomarker in hepatocellular carcinoma.

    PubMed

    Li, Chang-Peng; Cai, Mu-Yan; Jiang, Li-Juan; Mai, Shi-Juan; Chen, Jie-Wei; Wang, Feng-Wei; Liao, Yi-Ji; Chen, Wen-Hui; Jin, Xiao-Han; Pei, Xiao-Qing; Guan, Xin-Yuan; Zeng, Mu-Sheng; Xie, Dan

    2016-06-01

    Trimethylation of lysine 27 on histone H3 (H3K27ME3) is a transcription-suppressive histone mark mediated by enhancer of zeste homolog 2 (EZH2). We have previously suggested that EZH2-mediated H3K27ME3 plays a critical oncogenic role in human hepatocellular carcinoma (HCC) aggressiveness. However, the direct downstream targets of EZH2-H3K27ME3 and the molecular mechanisms by which regulates HCC pathogenesis remain unclear. In this study, we used chromatin immunoprecipitation together with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis to assess genome-wide chromatin occupancy of H3K27ME3 in HCC cells. We identified that claudin14 (CLDN14) is a potentially direct target for EZH2-mediated H3K27ME3 in HCC. In a large cohort of clinical HCC tissues, we found that low expression of CLDN14 was significantly associated with advanced tumor stage and determined to be an independent predictor of shortened survival of HCC patients. Next, functional experiment demonstrated that depletion of CLDN14 substantially restored EZH2-silenced HCC cells motility and invasive capacities and supported cell epithelial-mesenchymal transition (EMT). Furthermore, downregulation of CLDN14 dramatically re-enhanced the wnt/β-catenin signaling activity in EZH2-silenced HCC cells by increasing the levels of active β-catenin and promoting the nuclear localization of β-catenin. These results, collectively, uncover that CLDN14 is a novel direct target of EZH2-mediated H3K27ME3, and provide an explanation for the aggressive nature of HCC with downregulation of CLDN14 and the underling mechanism that links the tumor suppressor CLDN14 to the wnt/β-catenin signaling pathway. PMID:27207647

  13. Epigenetic priming of enhancers predicts developmental competence of hESC-derived endodermal lineage intermediates

    PubMed Central

    Wang, Allen; Yue, Feng; Li, Yan; Xie, Ruiyu; Harper, Thomas; Patel, Nisha A.; Muth, Kayla; Palmer, Jeffrey; Qiu, Yunjiang; Wang, Jinzhao; Lam, Dieter K.; Raum, Jeffrey C.; Stoffers, Doris A.; Ren, Bing; Sander, Maike

    2015-01-01

    Summary Embryonic development relies on the capacity of progenitor cells to appropriately respond to inductive cues; a cellular property known as developmental competence. Here we report that epigenetic priming of enhancers signifies developmental competence during endodermal lineage diversification. Chromatin mapping during pancreatic and hepatic differentiation of human embryonic stem cells revealed the en masse acquisition of a poised chromatin state at enhancers specific to endoderm-derived cell lineages in gut tube intermediates. Experimentally, the acquisition of this poised enhancer state predicts the ability of endodermal intermediates to respond to inductive signals. Furthermore, these enhancers are first recognized by the pioneer transcription factors FOXA1 and FOXA2 when competence is acquired, while subsequent recruitment of lineage-inductive transcription factors, such as PDX1, leads to enhancer and target gene activation. Together, our results identify the acquisition of a poised chromatin state at enhancers as a mechanism by which progenitor cells acquire developmental competence. PMID:25842977

  14. Enhancer of zeste homolog 2 silencing inhibits tumor growth and lung metastasis in osteosarcoma

    PubMed Central

    Lv, Yang-Fan; Yan, Guang-Ning; Meng, Gang; Zhang, Xi; Guo, Qiao-Nan

    2015-01-01

    The enhancer of zeste homolog 2 (EZH2) methyltransferase is the catalytic subunit of polycomb repressive complex 2 (PRC2), which acts as a transcription repressor via the trimethylation of lysine 27 of histone 3 (H3K27me3). EZH2 has been recognised as an oncogene in several types of tumors; however, its role in osteosarcoma has not been fully elucidated. Herein, we show that EZH2 silencing inhibits tumor growth and lung metastasis in osteosarcoma by facilitating re-expression of the imprinting gene tumor-suppressing STF cDNA 3 (TSSC3). Our previous study showed that TSSC3 acts as a tumor suppressor in osteosarcoma. In this study, we found that EZH2 was abnormally elevated in osteosarcoma, and its overexpression was associated with poor prognosis in osteosarcoma. Silencing of EZH2 resulted in tumor growth inhibition, apoptosis and chemosensitivity enhancement. Moreover, suppression of EZH2 markedly inhibited tumor growth and lung metastasis in vivo. Furthermore, EZH2 knockdown facilitated the re-expression of TSSC3 by reducing H3K27me3 in the promoter region. Cotransfection with siEZH2 and siTSSC3 could partially reverse the ability of siEZH2 alone. We have demonstrated that EZH2 plays a crucial role in tumor growth and distant metastasis in osteosarcoma; its oncogenic role is related to its regulation of the expression of TSSC3. PMID:26265454

  15. DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia

    PubMed Central

    Chen, C.W.; Koche, R.P.; Sinha, A.U.; Deshpande, A.J.; Zhu, N.; Eng, R.; Doench, J.G.; Xu, H.; Chu, S.H.; Qi, J.; Wang, X.; Delaney, C.; Bernt, K.M.; Root, D.E.; Hahn, W.C.; Bradner, J.E.; Armstrong, S.A.

    2015-01-01

    MLL -rearrangements generate MLL-fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL-fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL-fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling activation and repression of gene expression could provide novel opportunities for combination therapy. PMID:25822366

  16. Silencing of CD59 enhanced the sensitivity of HT29 cells to 5-Fluorouracil and Oxaliplatin.

    PubMed

    Yin, Haipeng; Li, Cuiling; Wang, Shaoyu; Guo, Qiang; Ren, Xia; Jiang, Guosheng

    2015-01-01

    Complement regulatory proteins (CD55 and CD59) were known to be expressed in many tumors and tumor cell lines including colorectal carcinoma, and were proposed as immunotherapy targets, however whether knocking down of CD55 and CD59 will affect the sensitivity of HT-29 cells to chemotherapy drugs for example, 5-Fluorouracil and Oxaliplatin and their possible mechanisms haven't been studied. To address this question, SiRNAs targeting CD55 and CD59 were chemically synthesized and transfected into HT-29 cells by lipofectamine. HT-29 growth curves of CD55 and CD59 knockdown cells were detected by MTT assay, HT29 inhibition curves to chemotherapy drugs (5-Fu and Oxaliplatin) were also assayed, in addition, chemotherapy sensitivity changes of HT29 affected by CD55 and CD59 knockdown were equally detected. Complement mediated lysis was examined by calcein-AM. We found that silencing CD59 in HT-29 cells could significantly enhance their sensitivity to 5-FU (P < 0.05) and Oxaliplatin (P < 0.05), and significantly reduced their IC50 concentration. On the contrary, knocking down of CD55 could inhibit HT-29 growth (P < 0.05). Mechanisms included increasing apoptosis rate of HT-29 by CD59 knocking down and G1/G0 blocking by silencing CD55. Our results thus shed light on the novel mechanism of chemotherapy resistance and provide an alternative strategy to overcome the resistance problem. PMID:25444672

  17. Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair

    PubMed Central

    Zhu, Kai; Lai, Hao; Guo, Changfa; Li, Jun; Wang, Yulin; Wang, Lingyan; Wang, Chunsheng

    2014-01-01

    Mesenchymal stem cell (MSC) transplantation has attracted much attention in myocardial infarction therapy. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. Small interfering RNA-mediated prolyl hydroxylase domain protein 2 (PHD2) silencing in MSCs holds tremendous potential to enhance their survival and paracrine effect after transplantation. However, an efficient and biocompatible PHD2 silencing system for clinical application is lacking. Herein, we developed a novel PHD2 silencing system based on arginine-terminated generation 4 poly(amidoamine) (Arg-G4) nanoparticles. The system exhibited effective and biocompatible small interfering RNA delivery and PHD2 silencing in MSCs in vitro. After genetically modified MSC transplantation in myocardial infarction models, MSC survival and paracrine function of IGF-1 were enhanced significantly in vivo. As a result, we observed decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angiogenesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This work demonstrated that an Arg-G4 nanovector-based PHD2 silencing system could enhance the efficiency of MSC transplantation for infarcted myocardium repair. PMID:25429216

  18. Pre-neoplastic epigenetic disruption of transcriptional enhancers in chronic inflammation

    PubMed Central

    Kron, Ken J.; Bailey, Swneke D.; Roulois, David; Lupien, Mathieu; Line, Sérgio R. Peres; de Souza, Ana Paula; De Carvalho, Daniel D.

    2016-01-01

    Chronic periodontitis (CP) is a chronic inflammatory disease independently associated with higher incidence of oral cavity squamous cell carcinoma (OSCC). However, the molecular mechanism responsible for this increased incidence is unknown. Here we profiled the DNA methylome of CP patients and healthy controls and compared to a large set of OSCC samples from TCGA. We observed a significant overlap between the altered DNA methylation patterns in CP and in OSCC, suggesting an emergence of a pre-neoplastic epigenome in CP. Remarkably, the hypermethylated CpGs in CP were significantly enriched for enhancer elements. This aberrant enhancer methylation is functional and able to disrupt enhancer activity by preventing the binding of chromatin looping factors. This study provides new insights on the molecular mechanisms linking chronic inflammation and tumor predisposition, highlighting the role of epigenetic disruption of transcriptional enhancers. PMID:26908456

  19. In vivo epigenetic reprogramming of primary human colon cancer cells enhances metastases

    PubMed Central

    Singovski, Grigori; Bernal, Carolina; Kuciak, Monika; Siegl-Cachedenier, Irene; Conod, Arwen; Ruiz i Altaba, Ariel

    2016-01-01

    How metastases develop is not well understood and no genetic mutations have been reported as specific metastatic drivers. Here we have addressed the idea that epigenetic reprogramming by GLI-regulated pluripotent stemness factors promotes metastases. Using primary human colon cancer cells engrafted in mice, we find that transient expression of OCT4, SOX2, KLF4 +/− cMYC establishes an enhanced pro-metastatic state in the primary tumor that is stable through sequential engraftments and is transmitted through clonogenic cancer stem cells. Metastatic reprogramming alters NANOG methylation and stably boosts NANOG and NANOGP8 expression. Metastases and reprogrammed EMT-like phenotypes require endogenous NANOG, but enhanced NANOG is not sufficient to induce these phenotypes. Finally, reprogrammed tumors enhance GLI2, and we show that GLI2high and AXIN2low, which are markers of the metastatic transition of colon cancers, are prognostic of poor disease outcome in patients. We propose that metastases arise through epigenetic reprogramming of cancer stem cells within primary tumors. PMID:26031752

  20. In vivo epigenetic reprogramming of primary human colon cancer cells enhances metastases.

    PubMed

    Singovski, Grigori; Bernal, Carolina; Kuciak, Monika; Siegl-Cachedenier, Irene; Conod, Arwen; Ruiz I Altaba, Ariel

    2016-04-01

    How metastases develop is not well understood and no genetic mutations have been reported as specific metastatic drivers. Here we have addressed the idea that epigenetic reprogramming by GLI-regulated pluripotent stemness factors promotes metastases. Using primary human colon cancer cells engrafted in mice, we find that transient expression of OCT4, SOX2, KLF4 +/- cMYC establishes an enhanced pro-metastatic state in the primary tumor that is stable through sequential engraftments and is transmitted through clonogenic cancer stem cells. Metastatic reprogramming alters NANOG methylation and stably boosts NANOG and NANOGP8 expression. Metastases and reprogrammed EMT-like phenotypes require endogenous NANOG, but enhanced NANOG is not sufficient to induce these phenotypes. Finally, reprogrammed tumors enhance GLI2, and we show that GLI2(high) and AXIN2(low), which are markers of the metastatic transition of colon cancers, are prognostic of poor disease outcome in patients. We propose that metastases arise through epigenetic reprogramming of cancer stem cells within primary tumors. PMID:26031752

  1. Genome Wide Mapping of NR4A Binding Reveals Cooperativity with ETS Factors to Promote Epigenetic Activation of Distal Enhancers in Acute Myeloid Leukemia Cells

    PubMed Central

    Duren, Ryan P.; Boudreaux, Seth P.; Conneely, Orla M.

    2016-01-01

    Members of the NR4A subfamily of orphan nuclear receptors regulate cell fate decisions via both genomic and non-genomic mechanisms in a cell and tissue selective manner. NR4As play a key role in maintenance of hematopoietic stem cell homeostasis and are critical tumor suppressors of acute myeloid leukemia (AML). Expression of NR4As is broadly silenced in leukemia initiating cell enriched populations from human patients relative to normal hematopoietic stem/progenitor cells. Rescue of NR4A expression in human AML cells inhibits proliferation and reprograms AML gene signatures via transcriptional mechanisms that remain to be elucidated. By intersecting an acutely regulated NR4A1 dependent transcriptional profile with genome wide NR4A binding distribution, we now identify an NR4A targetome of 685 genes that are directly regulated by NR4A1. We show that NR4As regulate gene transcription primarily through interaction with distal enhancers that are co-enriched for NR4A1 and ETS transcription factor motifs. Using a subset of NR4A activated genes, we demonstrate that the ETS factors ERG and FLI-1 are required for activation of NR4A bound enhancers and NR4A target gene induction. NR4A1 dependent recruitment of ERG and FLI-1 promotes binding of p300 histone acetyltransferase to epigenetically activate NR4A bound enhancers via acetylation at histone H3K27. These findings disclose novel epigenetic mechanisms by which NR4As and ETS factors cooperate to drive NR4A dependent gene transcription in human AML cells. PMID:26938745

  2. Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

    PubMed

    Tsang, Daisy P F; Wu, William K K; Kang, Wei; Lee, Ying-Ying; Wu, Feng; Yu, Zhuo; Xiong, Lei; Chan, Anthony W; Tong, Joanna H; Yang, Weiqin; Li, May S M; Lau, Suki S; Li, Xiangchun; Lee, Sau-Dan; Yang, Yihua; Lai, Paul B S; Yu, Dae-Yeul; Xu, Gang; Lo, Kwok-Wai; Chan, Matthew T V; Wang, Huating; Lee, Tin L; Yu, Jun; Wong, Nathalie; Yip, Kevin Y; To, Ka-Fai; Cheng, Alfred S L

    2016-04-01

    Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis. PMID:26800240

  3. Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum).

    PubMed

    Fragkostefanakis, Sotirios; Sedeek, Khalid E M; Raad, Maya; Zaki, Marwa Samir; Kalaitzis, Panagiotis

    2014-07-01

    Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves. PMID:24803411

  4. Gene silencing of TACE enhances plaque stability and improves vascular remodeling in a rabbit model of atherosclerosis

    PubMed Central

    Zhao, Xueqiang; Kong, Jing; Zhao, Yuxia; Wang, Xuping; Bu, Peili; Zhang, Cheng; Zhang, Yun

    2015-01-01

    We aimed to test the hypothesis that gene silencing of tumor necrosis factor alpha converting enzyme (TACE) may attenuate lesion inflammation and positive vascular remodeling and enhance plaque stability in a rabbit model of atherosclerosis. Lentivirus-mediated TACE shRNA was injected into the abdominal aortic plaques of rabbits which effectively down-regulated TACE expression and activities from week 8 to week 16. TACE gene silencing reduced remodeling index and plaque burden, and diminished the content of macrophages and lipids while increased that of smooth muscle cells and collagen in the aortic plaques. In addition, TACE gene silencing attenuated the local expression of P65, iNOS, ICAM-1, VEGF and Flt-1 and activities of MMP9 and MMP2 while increased the local expression of TGF-β1 together with reduced number of neovessels in the aorta. TACE shRNA treatment resulted in down-regulated expression of TACE in macrophages and blunted ERK-P38 phosphorylation and tube formation of co-cultured mouse vascular smooth muscle cells or human umbilical vein endothelial cells. In conclusion, gene silencing of TACE enhanced plaque stability and improved vascular positive remodeling. The mechanisms may involve attenuated local inflammation, neovascularization and MMP activation, as well as enhanced collagen production probably via down-regulated ERK-NF-κB and up-regulated TGF-β1 signaling pathways. PMID:26655882

  5. Conserved enhancer and silencer elements responsible for differential Adh transcription in Drosophila cell lines.

    PubMed Central

    Ayer, S; Benyajati, C

    1990-01-01

    The distal promoter of Adh is differentially expressed in Drosophila tissue culture cell lines. After transfection with an exogenous Adh gene, there was a specific increase in distal alcohol dehydrogenase (ADH) transcripts in ADH-expressing (ADH+) cells above the levels observed in transfected ADH-nonexpressing (ADH-) cells. We used deletion mutations and a comparative transient-expression assay to identify the cis-acting elements responsible for enhanced Adh distal transcription in ADH+ cells. DNA sequences controlling high levels of distal transcription were localized to a 15-base-pair (bp) region nearly 500 bp upstream of the distal RNA start site. In addition, a 61-bp negative cis-acting element was found upstream from and adjacent to the enhancer. When this silencer element was deleted, distal transcription increased only in the ADH+ cell line. These distant upstream elements must interact with the promoter elements, the Adf-1-binding site and the TATA box, as they only influenced transcription when at least one of these two positive distal promoter elements was present. Internal deletions targeted to the Adf-1-binding site or the TATA box reduced transcription in both cell types but did not affect the transcription initiation site. Distal transcription in transfected ADH- cells appears to be controlled primarily through these promoter elements and does not involve the upstream regulatory elements. Evolutionary conservation in distantly related Drosophila species suggests the importance of these upstream elements in correct developmental and tissue-specific expression of ADH. Images PMID:1694013

  6. Silencing of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

    SciTech Connect

    Kim, Sung Youl; Yoo, Young Hyun; Park, Jeen-Woo

    2013-04-05

    Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report an autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.

  7. Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression

    PubMed Central

    Watari, Hidemichi; Hanley, Sharon J.B.; Yamada, Takahiro; Hosaka, Masayoshi; Kudo, Masataka; Yue, Junming; Sakuragi, Noriaki

    2016-01-01

    Overexpression of IQGAP1 and microRNA (miRNA) dysregulation are frequent in human tumors, but little is known about the role of IQGAP1 and its relationship to miRNA in endometrial carcinogenesis. We demonstrate that IQGAP1 activates the epithelial–mesenchymal transition (EMT) program and that miR-124 directly represses IQGAP1 expression in endometrial cancer (EC) cells. The overexpression of IQGAP1 stimulates EMT features and enhances migration, invasion and proliferation of EC cells, whereas knocking down IQGAP1 expression reverses EMT and inhibits these malignant properties. Using miRNA microarray profiling, we identified 29 miRNAs (let-7b, let-7f, miR-10b, miR-15b, miR-23a, miR-24, miR-25, miR-27a, miR-29b, miR-30a-5p, miR-34a, miR-124, miR-127, miR-130b, miR-148a, miR-155, miR-191*, miR-194, miR-224, miR-362, miR-409-3p, miR-422b, miR-424, miR-453, miR-497, miR-518d, miR-518f*, miR-526a and miR-656) that are significantly down-regulated in an in vitro-selected highly invasive derivative cell line (HEC-50-HI) relative to the parental HEC-50 cells. We further identified miR-124 as a direct regulator of IQGAP1 in EC cells. Enforced expression of miR-124 suppresses EC cell invasion and proliferation. The expression of IQGAP1 mRNA was significantly elevated in EC tissues, while the expression of miR-124 was decreased. The downregulation of miR-124 correlates with a poor survival outcome for patients with EC. Treating EC cells with the demethylating agent 5-aza-2′-deoxycytidine increased miR-124 expression and down-regulated IQGAP1 levels. Our data suggest that IQGAP1 promotes EMT, migration and invasion of EC cells. MiR-124, a novel tumor suppressor miRNA that is epigenetically silenced in EC, can reverse EMT and the invasive properties, by attenuating the expression of the IQGAP1 oncogene. PMID:26934121

  8. Long-lasting pro-inflammatory suppression of microglia by LPS-preconditioning is mediated by RelB-dependent epigenetic silencing.

    PubMed

    Schaafsma, W; Zhang, X; van Zomeren, K C; Jacobs, S; Georgieva, P B; Wolf, S A; Kettenmann, H; Janova, H; Saiepour, N; Hanisch, U-K; Meerlo, P; van den Elsen, P J; Brouwer, N; Boddeke, H W G M; Eggen, B J L

    2015-08-01

    Microglia, the innate immune cells of the central nervous system (CNS), react to endotoxins like bacterial lipopolysaccharides (LPS) with a pronounced inflammatory response. To avoid excess damage to the CNS, the microglia inflammatory response needs to be tightly regulated. Here we report that a single LPS challenge results in a prolonged blunted pro-inflammatory response to a subsequent LPS stimulation, both in primary microglia cultures (100 ng/ml) and in vivo after intraperitoneal (0.25 and 1mg/kg) or intracerebroventricular (5 μg) LPS administration. Chromatin immunoprecipitation (ChIP) experiments with primary microglia and microglia acutely isolated from mice showed that LPS preconditioning was accompanied by a reduction in active histone modifications AcH3 and H3K4me3 in the promoters of the IL-1β and TNF-α genes. Furthermore, LPS preconditioning resulted in an increase in the amount of repressive histone modification H3K9me2 in the IL-1β promoter. ChIP and knock-down experiments showed that NF-κB subunit RelB was bound to the IL-1β promoter in preconditioned microglia and that RelB is required for the attenuated LPS response. In addition to a suppressed pro-inflammatory response, preconditioned primary microglia displayed enhanced phagocytic activity, increased outward potassium currents and nitric oxide production in response to a second LPS challenge. In vivo, a single i.p. LPS injection resulted in reduced performance in a spatial learning task 4 weeks later, indicating that a single inflammatory episode affected memory formation in these mice. Summarizing, we show that LPS-preconditioned microglia acquire an epigenetically regulated, immune-suppressed phenotype, possibly to prevent excessive damage to the central nervous system in case of recurrent (peripheral) inflammation. PMID:25843371

  9. A CTCF-binding silencer regulates the imprinted genes AWT1 and WT1-AS and exhibits sequential epigenetic defects during Wilms' tumourigenesis.

    PubMed

    Hancock, Anne L; Brown, Keith W; Moorwood, Kim; Moon, Hanlim; Holmgren, Claes; Mardikar, Sudhanshu H; Dallosso, Anthony R; Klenova, Elena; Loukinov, Dmitri; Ohlsson, Rolf; Lobanenkov, Victor V; Malik, Karim

    2007-02-01

    We have shown previously that AWT1 and WT1-AS are functionally imprinted in human kidney. In the adult kidney, expression of both transcripts is restricted to the paternal allele, with the silent maternal allele retaining methylation at the WT1 antisense regulatory region (WT1 ARR). Here, we report characterization of the WT1 ARR differentially methylated region and show that it contains a transcriptional silencer element acting on both the AWT1 and WT1-AS promoters. DNA methylation of the silencer results in increased transcriptional repression, and the silencer is also shown to be an in vitro and in vivo target site for the imprinting regulator protein CTCF. Binding of CTCF is methylation-sensitive and limited to the unmethylated silencer. Potentiation of the silencer activity is demonstrated after CTCF protein is knocked down, suggesting a novel silencer-blocking activity for CTCF. We also report assessment of WT1 ARR methylation in developmental and tumour tissues, including the first analysis of Wilms' tumour precursor lesions, nephrogenic rests. Nephrogenic rests show increases in methylation levels relative to foetal kidney and reductions relative to the adult kidney, together with biallelic expression of AWT1 and WT1-AS. Notably, the methylation status of CpG residues within the CTCF target site appears to distinguish monoallelic and biallelic expression states. Our data suggest that failure of methylation spreading at the WT1 ARR early in renal development, followed by imprint erasure, occurs during Wilms' tumourigenesis. We propose a model wherein imprinting defects at chromosome 11p13 may contribute to Wilms' tumourigenesis. PMID:17210670

  10. Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

    PubMed Central

    Gaziova, Zuzana; Baumann, Volker; Winkler, Anna-Maria; Winkler, Johannes

    2014-01-01

    The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles. PMID:24613624

  11. RNAi revised - target mRNA-dependent enhancement of gene silencing

    PubMed Central

    Dornseifer, Simon; Willkomm, Sarah; Far, Rosel Kretschmer-Kazemi; Liebschwager, Janine; Beltsiou, Foteini; Frank, Kirsten; Laufer, Sandra D.; Martinetz, Thomas; Sczakiel, Georg; Claussen, Jens Christian; Restle, Tobias

    2015-01-01

    The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery. PMID:26578554

  12. RNAi revised--target mRNA-dependent enhancement of gene silencing.

    PubMed

    Dornseifer, Simon; Willkomm, Sarah; Far, Rosel Kretschmer-Kazemi; Liebschwager, Janine; Beltsiou, Foteini; Frank, Kirsten; Laufer, Sandra D; Martinetz, Thomas; Sczakiel, Georg; Claussen, Jens Christian; Restle, Tobias

    2015-12-15

    The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery. PMID:26578554

  13. Phylogenetic and Epigenetic Footprinting of the Putative Enhancers of the Peg3 Domain

    PubMed Central

    Kim, Joomyeong; Ye, An

    2016-01-01

    The Peg3 (Paternally Expressed Gene 3) imprinted domain is predicted to be regulated through a large number of evolutionarily conserved regions (ECRs) that are localized within its middle 200-kb region. In the current study, we characterized these potential cis-regulatory regions using phylogenetic and epigenetic approaches. According to the results, the majority of these ECRs are potential enhancers for the transcription of the Peg3 domain. Also, these potential enhancers can be divided into two groups based on their histone modification and DNA methylation patterns: ubiquitous and tissue-specific enhancers. Phylogenetic and bioinformatic analyses further revealed that several cis-regulatory motifs are frequently associated with the ECRs, such as the E box, PITX2, NF-κB and RFX1 motifs. A series of subsequent ChIP experiments demonstrated that the trans factor MYOD indeed binds to the E box of several ECRs, further suggesting that MYOD may play significant roles in the transcriptional control of the Peg3 domain. Overall, the current study identifies, for the first time, a set of cis-regulatory motifs and corresponding trans factors that may be critical for the transcriptional regulation of the Peg3 domain. PMID:27104590

  14. Review-Epigenetic therapy for cancer.

    PubMed

    Saleem, Mohammad; Abbas, Khizar; Manan, Maria; Ijaz, Hira; Ahmed, Bilal; Ali, Muhammad; Hanif, Muhammad; Farooqi, Ammad Ahmad; Qadir, Muhammad Imran

    2015-05-01

    Epigenetics means the study of alterations in the genetic material that affect the phenotype but does not affect the genotype. Epigenetics cause alterations in cell properties, which are inherited; but it does not cause alterations in DNA sequence. Epigenetic mediated silencing of gene is of four types, which are DNA methylation, histone deacetylation, RNA associated silencing and Genomic imprinting. Other factors (environmental and xenobiotics) can also cause gene silencing but DNA methytlation and changes in histones of chromatin are two important changes, which are responsible for malignant diseases. Two groups of drugs are under development, which corrects the epigenetic alterations. These are histone deacetylation (HDAC) inhibitors and DNA methytransferase (DNMT) inhibitors. These drugs may be used in cancer because in cancer, hypermethylation of cancer suppressor gene causes gene silencing. Epigenetic therapy scope is likely to increase in future. PMID:26004710

  15. LincHOTAIR epigenetically silences miR34a by binding to PRC2 to promote the epithelial-to-mesenchymal transition in human gastric cancer

    PubMed Central

    Liu, Y-w; Sun, M; Xia, R; Zhang, E-b; Liu, X-h; Zhang, Z-h; Xu, T-p; De, W; Liu, B-r; Wang, Z-x

    2015-01-01

    lncRNAs play important roles in the epigenetic regulation of carcinogenesis and progression. Previous studies suggest that HOTAIR contributes to gastric cancer (GC) development, and the overexpression of HOTAIR predicts a poor prognosis. In this study, we found that HOTAIR was more highly expressed in diffuse-type GC than in intestinal type (P=0.048). In the diffuse type, there is significant relationship between HOTAIR expression and DFS (P<0.001). CDH1 was downregulated in diffuse-type GC tissues (P=0.0007) and showed a negative relationship with HOTAIR (r2=0.154, P=0.0354). In addition, HOTAIR knockdown significantly repressed migration, invasion and metastasis both in vitro and vivo and reversed the epithelial-to-mesenchymal transition in GC cells. We also showed that HOTAIR recruiting and binding to PRC2 epigenetically represses miR34a, which controls the targets C-Met (HGF/C-Met/Snail pathway) and Snail, thus contributing to GC cell-EMT process and accelerating tumor metastasis. Moreover, it is demonstrated that HOTAIR crosstalk with microRNAs during epigenetic regulation. Our results suggest that HOTAIR acts as an EMT regulator and may be a candidate prognostic biomarker and a target for new therapies in GC patients. PMID:26136075

  16. Tex10 Coordinates Epigenetic Control of Super-Enhancer Activity in Pluripotency and Reprogramming.

    PubMed

    Ding, Junjun; Huang, Xin; Shao, Ningyi; Zhou, Hongwei; Lee, Dung-Fang; Faiola, Francesco; Fidalgo, Miguel; Guallar, Diana; Saunders, Arven; Shliaha, Pavel V; Wang, Hailong; Waghray, Avinash; Papatsenko, Dmitri; Sánchez-Priego, Carlos; Li, Dan; Yuan, Ye; Lemischka, Ihor R; Shen, Li; Kelley, Kevin; Deng, Haiteng; Shen, Xiaohua; Wang, Jianlong

    2015-06-01

    Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination. PMID:25936917

  17. E2 Regulates Epigenetic Signature on Neuroglobin Enhancer-Promoter in Neuronal Cells

    PubMed Central

    Guglielmotto, Michela; Reineri, Stefania; Iannello, Andrea; Ferrero, Giulio; Vanzan, Ludovica; Miano, Valentina; Ricci, Laura; Tamagno, Elena; De Bortoli, Michele; Cutrupi, Santina

    2016-01-01

    Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERβ) that bind to specific regulatory genomic regions. We focused our attention on 17β-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases. PMID:27313512

  18. Tex10 Coordinates Epigenetic Control of Super-Enhancer Activity in Pluripotency and Reprogramming

    PubMed Central

    Ding, Junjun; Huang, Xin; Shao, Ningyi; Zhou, Hongwei; Lee, Dung-Fang; Faiola, Francesco; Fidalgo, Miguel; Guallar, Diana; Saunders, Arven; Shliaha, Pavel V.; Wang, Hailong; Waghray, Avinash; Papatsenko, Dmitri; Sánchez-Priego, Carlos; Li, Dan; Yuan, Ye; Lemischka, Ihor R.; Shen, Li; Kelley, Kevin; Deng, Haiteng; Shen, Xiaohua; Wang, Jianlong

    2015-01-01

    SUMMARY Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination. PMID:25936917

  19. Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming.

    PubMed

    Chen, Jun; Chen, Xiaolong; Li, Min; Liu, Xiaoyu; Gao, Yawei; Kou, Xiaochen; Zhao, Yanhong; Zheng, Weisheng; Zhang, Xiaobai; Huo, Yi; Chen, Chuan; Wu, You; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

    2016-02-16

    The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies. PMID:26832419

  20. Enhanced B Cell Alloantigen Presentation and Its Epigenetic Dysregulation in Liver Transplant Rejection.

    PubMed

    Ningappa, M; Ashokkumar, C; Higgs, B W; Sun, Q; Jaffe, R; Mazariegos, G; Li, D; Weeks, D E; Subramaniam, S; Ferrell, R; Hakonarson, H; Sindhi, R

    2016-02-01

    T cell suppression prevents acute cellular rejection but causes life-threatening infections and malignancies. Previously, liver transplant (LTx) rejection in children was associated with the single-nucleotide polymorphism (SNP) rs9296068 upstream of the HLA-DOA gene. HLA-DOA inhibits B cell presentation of antigen, a potentially novel antirejection drug target. Using archived samples from 122 white pediatric LTx patients (including 77 described previously), we confirmed the association between rs9296068 and LTx rejection (p = 0.001, odds ratio [OR] 2.55). Next-generation sequencing revealed that the putative transcription factor (CCCTC binding factor [CTCF]) binding SNP locus rs2395304, in linkage disequilibrium with rs9296068 (D' 0.578, r(2) = 0.4), is also associated with LTx rejection (p = 0.008, OR 2.34). Furthermore, LTx rejection is associated with enhanced B cell presentation of donor antigen relative to HLA-nonidentical antigen in a novel cell-based assay and with a downregulated HLA-DOA gene in a subset of these children. In lymphoblastoid B (Raji) cells, rs2395304 coimmunoprecipitates with CTCF, and CTCF knockdown with morpholino antisense oligonucleotides enhances alloantigen presentation and downregulates the HLA-DOA gene, reproducing observations made with HLA-DOA knockdown and clinical rejection. Alloantigen presentation is suppressed by inhibitors of methylation and histone deacetylation, reproducing observations made during resolution of rejection. Enhanced donor antigen presentation by B cells and its epigenetic dysregulation via the HLA-DOA gene represent novel opportunities for surveillance and treatment of transplant rejection. PMID:26663361

  1. Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway.

    PubMed

    Naim, Fatima; Shrestha, Pushkar; Singh, Surinder P; Waterhouse, Peter M; Wood, Craig C

    2016-06-01

    Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application. PMID:26628000

  2. Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of HC-Pro has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses post-transcriptional gene silencing (PTGS). PT...

  3. Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

    PubMed

    Guzman-Villanueva, Diana; El-Sherbiny, Ibrahim M; Vlassov, Alexander V; Herrera-Ruiz, Dea; Smyth, Hugh D C

    2014-10-01

    The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA. PMID:25063077

  4. Increased expression of long noncoding RNA TUG1 predicts a poor prognosis of gastric cancer and regulates cell proliferation by epigenetically silencing of p57

    PubMed Central

    Zhang, E; He, X; Yin, D; Han, L; Qiu, M; Xu, T; Xia, R; Xu, L; Yin, R; De, W

    2016-01-01

    Recent evidence highlights long noncoding RNAs (lncRNAs) as crucial regulators of cancer biology that contribute to tumorigenesis. LncRNA TUG1 was initially detected in a genomic screen for genes upregulated in response to taurine treatment in developing mouse retinal cells. Our previous study showed that TUG1 could affect cell proliferation through epigenetically regulating HOXB7 in human non-small cell lung cancer. However, the clinical significance and potential role of TUG1 in GC remains unclear. In this study, we found that TUG1 is significantly increased and is correlated with outcomes in gastric cancer (GC). Further experiments revealed that knockdown of TUG1 repressed GC proliferation both in vitro and in vivo. Mechanistic investigations showed that TUG1 has a key role in G0/G1 arrest. We further demonstrated that TUG1 was associated with PRC2 and that this association was required for epigenetic repression of cyclin-dependent protein kinase inhibitors, including p15, p16, p21, p27 and p57, thus contributing to the regulation of GC cell cycle and proliferation. Together, our results suggest that TUG1, as a regulator of proliferation, may serve as a candidate prognostic biomarker and target for new therapies in human GC. PMID:26913601

  5. Silencing HO-1 sensitizes SKM-1 cells to apoptosis induced by low concentration 5-azacytidine through enhancing p16 demethylation.

    PubMed

    Wang, Ping; Ma, Dan; Wang, Jishi; Fang, Qin; Gao, Rui; Wu, Weibing; Lu, Tangsheng; Cao, Lu

    2015-03-01

    Heme oxygenase-1 was reported previously as a resistance target on acute myelocytic leukemia (AML). We found that HO-1 was resistant to 5-azacytidine (AZA) treatment of myelodysplastic syndrome (MDS), and explored further the relative mechanisms. Patient bone marrow mononuclear cells (n=48) diagnosed as different levels of MDS were collected. Cell growth was evaluated by MTT assay; cell cycle and apoptosis were detected by flow cytometry; mRNA expression was assessed by real-time PCR, protein expression was analyzed through western blotting. Methylation was assessed by MSP. The survival time, and weight of mice were recorded. HO-1 overexpression was observed in SKM-1 cells after AZA treatment comparing to other cell lines. The HO-1 expression in MDS patients with high-risk was higher than in low-risk patients. After HO-1 was silenced by lentivirus-mediated siRNA, the proliferation of SKM-1 cells was effectively inhibited by low concentration AZA, and the cell cycle was arrested in the G0/G1 phase. Upregulation of p16 and changing of p16-relative cell cycle protein was observed after silencing HO-1 in AZA treated SKM-1 cells. In addition, DNMT1 was downregulated following the decrease of HO-1 expression. In vivo, silencing HO-1 inhibited SKM-1 cell growth induced by AZA in a NOD/SCID mouse model. Silencing HO-1 sensitized SKM-1 cells toward AZA, which may be attributed to the influence of HO-1 on AZA-induced p16 demethylation. HO-1 may be one of the targets that enhance the therapeutic effects of AZA on MDS malignant transformation inspiring new treatment methods for high-risk and very high-risk MDS patients in clinical practice. PMID:25585641

  6. A hypoxia-inducible factor (HIF)-3α splicing variant, HIF-3α4 impairs angiogenesis in hypervascular malignant meningiomas with epigenetically silenced HIF-3α4

    SciTech Connect

    Ando, Hitoshi; Natsume, Atsushi; Iwami, Kenichiro; Ohka, Fumiharu; Kuchimaru, Takahiro; Kizaka-Kondoh, Shinae; Ito, Kengo; Saito, Kiyoshi; Sugita, Sachi; Hoshino, Tsuneyoshi; Wakabayashi, Toshihiko

    2013-03-29

    Highlights: ► HIF-3α4 is silenced by DNA methylation in meningiomas. ► Induction of HIF-3α4 impaired angiogenesis in meningiomas. ► Induction of HIF-3α4 impaired proliferation and oxygen-dependent metabolism. -- Abstract: Hypoxia inducible factor is a dominant regulator of adaptive cellular responses to hypoxia and controls the expression of a large number of genes regulating angiogenesis as well as metabolism, cell survival, apoptosis, and other cellular functions in an oxygen level-dependent manner. When a neoplasm is able to induce angiogenesis, tumor progression occurs more rapidly because of the nutrients provided by the neovasculature. Meningioma is one of the most hypervascular brain tumors, making anti-angiogenic therapy an attractive novel therapy for these tumors. HIF-3α has been conventionally regarded as a dominant-negative regulator of HIF-1α, and although alternative HIF-3α splicing variants are extensively reported, their specific functions have not yet been determined. In this study, we found that the transcription of HIF-3α4 was silenced by the promoter DNA methylation in meningiomas, and inducible HIF-3α4 impaired angiogenesis, proliferation, and metabolism/oxidation in hypervascular meningiomas. Thus, HIF-3α4 could be a potential molecular target in meningiomas.

  7. Silencing c-Kit expression in human DCs suppresses Th2, Th17 response but enhances Th1 response

    PubMed Central

    Yang, Bin; Yang, Qin; Huang, Qianchuan; Yan, Hongbo; Sun, Ting; Tong, Hui

    2015-01-01

    Dendritic cells (DCs) are integral to the differentiation of T helper cells into T helper type 1 TH1, TH2 and TH17 subsets. RNA interference (RNAi), which causes the degradation of any RNA in a sequence specific manner, is a posttranscriptional gene silencing mechanism. Targeting the c-Kit in DCs has been used as an approach to enhance antitumor immunity. Here, we shwed that transfection of DCs with siRNA specific for c-Kit gene can significantly knock down c-Kit. When exposed to TNF-α, immature DCs transfected with c-Kit siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production. The c-Kit siRNA-treated DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay. Furthermore, c-Kit siRNA-transfected DCs enhanced TH1 responses by increasing IFN-γ and decreasing IL-4 production, and much stronger cytotoxic activity was observed when DCs were co-transfected with c-Kit siRNA and an endogenous tumor antigen in vitro. Our findings indicate that silencing the c-Kit gene in DCs with siRNA may offer a potential approach to enhance antitumor immunotherapy. PMID:26550451

  8. Enhancement of the Seed-Target Recognition Step in RNA Silencing by a PIWI/MID Domain Protein

    PubMed Central

    Parker, James S.; Parizotto, Eneida A.; Wang, Muhan; Roe, S. Mark; Barford, David

    2009-01-01

    Summary Target recognition in RNA silencing is governed by the “seed sequence” of a guide RNA strand associated with the PIWI/MID domain of an Argonaute protein in RISC. Using a reconstituted in vitro target recognition system, we show that a model PIWI/MID domain protein confers position-dependent tightening and loosening of guide-strand-target interactions. Over the seed sequence, the interaction affinity is enhanced up to ˜300-fold. Enhancement is achieved through a reduced entropy penalty for the interaction. In contrast, interactions 3′ of the seed are inhibited. We quantified mismatched target recognition inside and outside the seed, revealing amplified discrimination at the third position in the seed mediated by the PIWI/MID domain. Thus, association of the guide strand with the PIWI/MID domain generates an enhanced affinity anchor site over the seed that can promote fidelity in target recognition and stabilize and guide the assembly of the active silencing complex. PMID:19187762

  9. STAT3-silenced human dendritic cells have an enhanced ability to prime IFNγ production by both αβ and γδ T lymphocytes.

    PubMed

    Sanseverino, Isabella; Purificato, Cristina; Varano, Barbara; Conti, Lucia; Gessani, Sandra; Gauzzi, M Cristina

    2014-07-01

    Dendritic cells (DC) are an attractive target for therapeutic manipulation of the immune system to enhance insufficient immune responses, such those occurring in cancer, or to dampen dangerous responses in allergic and autoimmune diseases. Main goal of this study was to manipulate human monocyte-derived DC (MDDC) function by silencing STAT3, since this transcription factor plays a key role as a negative regulator of immune surveillance, and is strongly involved in inflammation. STAT3 silencing did not affect the immunophenotype of both immature and toll-like receptor (TLR) ligand-matured DC. However, an altered cytokine secretion profile, characterized by lower IL10 and higher IL12 and TNFα levels, was observed in silenced DC with respect to control cells upon TLR triggering. Accordingly, STAT3 silenced MDDC promoted a higher IFNγ production by CD4(+) naïve T cells. Furthermore, STAT3 silencing in MDDC favored the activation of γδ T lymphocytes, an immune cell population with important antitumor effector activities. This effect was at least in part mediated by the increased IL12 production by silenced cells. STAT3 silencing also increased the levels of CCL4, a CCR5-binding chemokine known to be involved in T helper 1 (Th1) cell recruitment. Altogether these results strengthen the role of STAT3 as a critical check point of the suppression of Th1 responses, unraveling its potential to dampen DC capability to both induce and recruit different IFNγ producing T lymphocyte subsets. PMID:24674241

  10. EMT and stem cell-like properties associated with miR-205 and miR-200 epigenetic silencing are early manifestations during carcinogen-induced transformation of human lung epithelial cells

    PubMed Central

    Tellez, Carmen S.; Juri, Daniel E.; Do, Kieu; Bernauer, Amanda M.; Thomas, Cindy L.; Damiani, Leah A.; Tessema, Mathewos; Leng, Shuguang; Belinsky, Steven A.

    2011-01-01

    Epithelial mesenchymal transition (EMT) is strongly associated with cancer progression, but its potential role during premalignant development has not been studied. Here we show that a four-week exposure of immortalized human bronchial epithelial cells (HBECs) to tobacco carcinogens can induce a persistent, irreversible, and multifaceted dedifferentiation program marked by EMT and the emergence of stem cell-like properties. EMT induction was epigenetically driven, initially by chromatin remodeling through H3K27me3 enrichment and later by ensuing DNA methylation to sustain silencing of tumor suppressive microRNAs miR-200b, miR-200c, and miR-205, which were implicated in the dedifferentiation program in HBECs and also in primary lung tumors. Carcinogen-treated HBECs acquired stem-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44high/CD24low, CD133, and ALDH1 stem cell-like markers. miRNA overexpression studies indicated that regulation of the EMT, stem-like, and transformed phenotypes in HBECs were distinct events. Our findings extend present concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can participate in cancer initiation to promote the clonal expansion of premalignant lung epithelial cells. PMID:21363915

  11. Epigenetic silencing of miR-218 by the lncRNA CCAT1, acting via BMI1, promotes an altered cell cycle transition in the malignant transformation of HBE cells induced by cigarette smoke extract.

    PubMed

    Lu, Lu; Xu, Hui; Luo, Fei; Liu, Xinlu; Lu, Xiaolin; Yang, Qianlei; Xue, Junchao; Chen, Chao; Shi, Le; Liu, Qizhan

    2016-08-01

    Cigarette smoking is the strongest risk factor for the development of lung cancer, the leading cause of cancer-related deaths. However, the molecular mechanisms leading to lung cancer are largely unknown. A long-noncoding RNA (lncRNA), CCAT1, regarded as cancer-associated, has been investigated extensively. Moreover, the molecular mechanisms of lncRNAs in regulation of microRNAs (miRNAs) induced by cigarette smoke remain unclear. In the present investigation, cigarette smoke extract (CSE) caused an altered cell cycle and increased CCAT1 levels and decreased miR-218 levels in human bronchial epithelial (HBE) cells. Depletion of CCAT1 attenuated the CSE-induced decreases of miR-218 levels, suggesting that miR-218 is negatively regulated by CCAT1 in HBE cells exposed to CSE. The CSE-induced increases of BMI1 levels and blocked by CCAT1 siRNA were attenuated by an miR-218 inhibitor. Moreover, in CSE-transformed HBE cells, the CSE-induced cell cycle changes and elevated neoplastic capacity were reversed by CCAT1 siRNA or BMI1 siRNA. This epigenetic silencing of miR-218 by CCAT1 induces an altered cell cycle transition through BMI1 and provides a new mechanism for CSE-induced lung carcinogenesis. PMID:27212446

  12. RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae1

    PubMed Central

    Subramanian, Senthil; Graham, Madge Y.; Yu, Oliver; Graham, Terrence L.

    2005-01-01

    Isoflavones are thought to play diverse roles in plant-microbe interactions and are also potentially important to human nutrition and medicine. Isoflavone synthase (IFS) is a key enzyme for the formation of the isoflavones. Here, we examined the consequences of RNAi silencing of genes for this enzyme in soybean (Glycine max). Soybean cotyledon tissues were transformed with Agrobacterium rhizogenes carrying an RNAi silencing construct designed to silence expression of both copies of IFS genes. Approximately 50% of emerging roots were transformed with the RNAi construct, and most transformed roots exhibited >95% silencing of isoflavone accumulation. Silencing of IFS was also demonstrated throughout the entire cotyledon (in tissues distal to the transformation site) both by high-performance liquid chromatography analysis of isoflavones and by real-time reverse transcription-PCR. This distal silencing led to a nearly complete suppression of mRNA accumulation for both the IFS1 and IFS2 genes and of isoflavone accumulations induced by wounding or treatment with the cell wall glucan elicitor from Phytophthora sojae. Preformed isoflavone conjugates were not reduced in distal tissues, suggesting little turnover of these stored isoflavone pools. Distal silencing was established within just 5 d of transformation and was highly efficient for a 3- to 4-d period, after which it was no longer apparent in most experiments. Silencing of IFS was effective in at least two genotypes and led to enhanced susceptibility to P. sojae, disrupting both R gene-mediated resistance in roots and nonrace-specific resistance in cotyledon tissues. The soybean cotyledon system, already a model system for defense signal-response and cell-to-cell signaling, may provide a convenient and effective system for functional analysis of plant genes through gene silencing. PMID:15778457

  13. Enhanced Generation of Myeloid Lineages in Hematopoietic Differentiation from Embryonic Stem Cells by Silencing Transcriptional Repressor Twist-2

    PubMed Central

    Sharabi, Andrew B.; Lee, Sung-Hyung; Goodell, Margaret A.; Huang, Xue F.

    2009-01-01

    Abstract The self-renewal and multilineage differentiation of embryonic stem cells (ESC) is largely governed by transcription factors or repressors. Extensive efforts have focused on elucidating critical factors that control the differentiation of specific cell lineages, for instance, myeloid lineages in hematopoietic development. In this study, we found that Twist-2, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in inhibiting the differentiation of ESC. Murine ES cells, in which Twist-2 expression is silenced by lentivirally delivered shRNA, exhibit an enhanced formation of primary embryoid bodies (EB) and enhanced differentiation into mesodermally derived hematopoietic colonies. Furthermore, Twist-2 silenced (LV-siTwist-2) ESC display significantly increased generation of myeloid lineages (Gr-1+ and F4/80+ cells) during in vitro hematopoietic differentiation. Treatment with the Toll-like receptor (TLR) 4 ligand synergistically stimulates the generation of primary EB formation as well as of hematopoietic progenitors differentiated from LV-siTwist-2 ES cells. Thus, this study reveals the critical role of the transcriptional repressor Twist-2 in regulating the development of myeloid lineage in hematopoietic differentiation from ESC. This study also suggests a potential strategy for directional differentiation of ESC by inhibiting a transcriptional repressor. PMID:20025523

  14. Adaption by Rewiring Epigenetic Landscapes

    PubMed Central

    Liu, Yifei; Xiao, Andrew

    2016-01-01

    Embryonic stem cells (ESCs) generally rely on repressive histone modifications to silence retrotransposons, rather than DNA methylation as in differentiated cells. In this issue of Cell Stem Cell, He et al. (2015) show that Daxx/Atrx repress transposons in ESCs devoid of 5mC, demonstrating dynamic reorganization of epigenetic networks and crosstalk between distinct repressive mechanisms to maintain transposon silencing. PMID:26340521

  15. Long noncoding RNA HOXA-AS2 promotes gastric cancer proliferation by epigenetically silencing P21/PLK3/DDIT3 expression

    PubMed Central

    Xia, Rui; Liu, Yan-wen; Ding, Jie; Ma, Hong-wei; He, Xue-zhi; Zhang, Zhi-hong; Liu, Zhi-jun; Liu, Xiang-hua; De, Wei

    2015-01-01

    Current evidence suggests that long noncoding RNAs (lncRNAs) may be an important class of functional regulators involved in human cancers development, including gastric cancer (GC). Here, we reported that HOXA cluster antisense RNA2 (HOXA-AS2), a 1048bp RNA, was upregulated in GC. Increased HOXA-AS2 expression in GC was associated with larger tumor size and higher clinical stage; patients with higher levels of HOXA-AS2 expression had a relatively poor prognosis. Further experiments revealed that HOXA-AS2 knockdown significantly inhibited GC cells proliferation by causing G1 arrest and promoting apoptosis, whereas HOXA-AS2 overexpression promoted cell growth. Furthermore, HOXA-AS2 could epigenetically repress the expression of P21, PLK3, and DDIT3 via binding with EZH2 (enhaner of zeste homolog 2), a key component of PRC2; ChIP assays demonstrated that EZH2 could directly bind to the promoter of P21, PLK3 and DDIT3, inducing H3K27 trimethylated. In conclusion, these data suggest that HOXA-AS2 could be an oncogene for GC partly through suppressing P21, PLK3, and DDIT3 expression; HOXA-AS2 may be served as a candidate prognostic biomarker and target for new therapies in human GC. PMID:26384350

  16. Epigenetic engineering shows that a human centromere resists silencing mediated by H3K27me3/K9me3

    PubMed Central

    Martins, Nuno M. C.; Bergmann, Jan H.; Shono, Nobuaki; Kimura, Hiroshi; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C.

    2016-01-01

    Centromeres are characterized by the centromere-specific H3 variant CENP-A, which is embedded in chromatin with a pattern characteristic of active transcription that is required for centromere identity. It is unclear how centromeres remain transcriptionally active despite being flanked by repressive pericentric heterochromatin. To further understand centrochromatin’s response to repressive signals, we nucleated a Polycomb-like chromatin state within the centromere of a human artificial chromosome (HAC) by tethering the methyltransferase EZH2. This led to deposition of the H3K27me3 mark and PRC1 repressor binding. Surprisingly, this state did not abolish HAC centromere function or transcription, and this apparent resistance was not observed on a noncentromeric locus, where transcription was silenced. Directly tethering the reader/repressor PRC1 bypassed this resistance, inactivating the centromere. We observed analogous responses when tethering the heterochromatin Editor Suv39h1-methyltransferase domain (centromere resistance) or reader HP1α (centromere inactivation), respectively. Our results reveal that the HAC centromere can resist repressive pathways driven by H3K9me3/H3K27me3 and may help to explain how centromeres are able to resist inactivation by flanking heterochromatin. PMID:26564795

  17. Silencing Fibronectin Extra Domain A Enhances Radiosensitivity in Nasopharyngeal Carcinomas Involving an FAK/Akt/JNK Pathway

    SciTech Connect

    Ou Juanjuan; Pan Feng; Geng Peiliang; Wei Xing; Xie Ganfeng; Deng Jia; Pang Xueli; Liang Houjie

    2012-03-15

    Purpose: Fibronectin extra domain A (EDA) is known to play important roles in angiogenesis, lymphangiogenesis, and metastasis in malignant tumors. The present study examined the effect of EDA on the radioresistance potential of nasopharyngeal carcinoma (NPC). Methods and Materials: EDA expression levels in blood samples and tumor tissues of NPC patients were tested by enzyme-linked immunosorbent assay and immunohistochemistry. Radiosensitivity was tested by colony survival assay. Apoptosis was determined by flow cytometry. The expressions of EDA, cleaved caspase 9, cleaved caspase 3, cleaved PARP, Bcl-2, and the levels of phosphorylated FAK, Akt, and JNK were measured by Western blot. Xenografts were used to confirm the effect of EDA on radiosensitivity in vivo. Results: EDA levels in blood samples of advanced NPC patients were much higher than those in early-stage patients. In tumor tissues, the positive expressions of EDA in NPC tumor tissues were shown to be correlated with the differentiation degrees of cancer cells and lymph node metastases. Additionally, the expression of EDA is positively correlated with the expression of antiapoptotic gene (Bcl2), but negatively correlated with the expressions of apoptotic genes (cleaved caspase-3, cleaved caspase-9, cleaved PARP). In vitro, EDA-silenced NPC cells CNE-2 shows substantially enhanced radiosensitivity with lower colony survival and more apoptosis in response to radiation. In vivo, EDA-silenced xenografts were more sensitive to radiation. At the molecular level, FAK/Akt/JNK signaling was demonstrated to be inactivated in EDA-silenced CNE-2 cells. Conclusions: EDA strongly affected the radiosensitivity of NPC cells. FAK/Akt/JNK signaling was found to be a potential signaling mediating EDA function.

  18. Silencing of ataxia-telangiectasia mutated by siRNA enhances the in vitro and in vivo radiosensitivity of glioma.

    PubMed

    Li, Yan; Li, Luchun; Li, Bo; Wu, Zhijuan; Wu, Yongzhong; Wang, Ying; Jin, Fu; Li, Dairong; Ma, Huiwen; Wang, Donglin

    2016-06-01

    It is reported that high expression of the ataxia-telangiectasia mutated (ATM) gene is linked with radioresistance in glioma. We hypothesized that the radiosensitivity of this brain tumor is enhanced by silencing of the ATM gene. We transfected the glioma cell line U251 with the siRNA-ATMpuro (group A) lentivirus or the siRNA-HKpuro (group N, negative control) lentivirus before irradiation. RT-qPCR and western blotting were performed to verify the efficiency of siRNA‑mediated ATM silencing. Expression levels of the ATM gene and protein were obviously downregulated after transfection. Moreover, the expression of the p53, PCNA and survivin genes, which are related to radiosensitivity, was also decreased. CCK-8 and colony formation assays showed lower cell proliferation and survival in group A than in groups N and C (control group that was not transfected with any siRNA). The level of double-stranded DNA breaks was also greater in group A, as determined by the comet tail assay. Flow cytometry showed a higher rate of cell apoptosis and a higher number of cells in the G2 phase in group A. Furthermore, caspase-3, caspase-8 and caspase-9 activity was also higher in group A. In vivo analysis in mouse models created by implantation of the transfected cell lines showed that the amount of necrosis and hemorrhage was higher in group A than that in the control groups. In conclusion, silencing of ATM via the siRNA technique could improve the in vitro and in vivo radiosensitivity of glioma cells. PMID:27108486

  19. Enhanced Gene Silencing through Human Serum Albumin-Mediated Delivery of Polyethylenimine-siRNA Polyplexes

    PubMed Central

    Nicolì, Elena; Syga, Marie Isabel; Bosetti, Michela; Shastri, V. Prasad

    2015-01-01

    Small interfering RNA (siRNA) targeted therapeutics (STT) offers a compelling alternative to tradition medications for treatment of genetic diseases by providing a means to silence the expression of specific aberrant proteins, through interference at the expression level. The perceived advantage of siRNA therapy is its ability to target, through synthetic antisense oligonucleotides, any part of the genome. Although STT provides a high level of specificity, it is also hindered by poor intracellular uptake, limited blood stability, high degradability and non-specific immune stimulation. Since serum proteins has been considered as useful vehicles for targeting tumors, in this study we investigated the effect of incorporation of human serum albumin (HSA) in branched polyethylenimine (bPEI)-siRNA polyplexes in their internalization in epithelial and endothelial cells. We observed that introduction of HSA preserves the capacity of bPEI to complex with siRNA and protect it against extracellular endonucleases, while affording significantly improved internalization and silencing efficiency, compared to bPEI-siRNA polyplexes in endothelial and metastatic breast cancer epithelial cells. Furthermore, the uptake of the HSA-bPEI-siRNA ternary polyplexes occurred primarily through a caveolae-mediated endocytosis, thus providing evidence for a clear role for HSA in polyplex internalization. These results provide further impetus to explore the role of serum proteins in delivery of siRNA. PMID:25856158

  20. ZNF300P1 Encodes a lincRNA that regulates cell polarity and is epigenetically silenced in type II epithelial ovarian cancer

    PubMed Central

    2014-01-01

    Background We previously identified that the CpG island-associated promoter of the novel lincRNA ZNF300P1 (also known as LOC134466) is frequently hypermethylated and silenced in ovarian cancer tissues. However, the function of ZNF300P1 was unknown. In this report we demonstrate that ZNF300P1 is involved in the regulation of key cell cycle and cell motility networks in human ovarian surface epithelial cells, and may play a role in promoting metastasis in ovarian cancer cells. Methods We applied methylated DNA immunoprecipitation on whole genome promoter tiling arrays and Sequenom assays to examine methylation status of ZNF300P1 in multiple ovarian cancer cell lines, as well as in normal ovarian and ovarian tumor tissues. Transcript profiling was used to investigate the effects of ZNF300P1 suppression in ovarian cancer cells. We utilized siRNA knockdown in normal ovarian surface epithelial cells and performed cellular proliferation, migration and adhesion assays to validate and explore the profiling results. Results We demonstrate that ZNF300P1 is methylated in multiple ovarian cancer cell lines. Loss of ZNF300P1 results in decreased cell proliferation and colony formation. In addition, knockdown of the ZNF300P1 transcript results in aberrant and less persistent migration in wound healing assays due to a loss of cellular polarity. Using an ex vivo peritoneal adhesion assay, we also reveal a role for ZNF300P1 in the attachment of ovarian cancer cells to peritoneal membranes, indicating a potential function of ZNF300P1 expression in metastasis of ovarian cancer cells to sites within the peritoneal cavity. Conclusion Our findings further support ZNF300P1 as frequently methylated in ovarian cancer and reveal a novel function for ZNF300P1 lincRNA expression in regulating cell polarity, motility, and adhesion and loss of expression may contribute to the metastatic potential of ovarian cancer cells. PMID:24393131

  1. Epigenetic regulation of spinal cord gene expression contributes to enhanced postoperative pain and analgesic tolerance subsequent to continuous opioid exposure

    PubMed Central

    Liang, De-Yong; Shi, Xiao-You; Sun, Yuan; Clark, J David

    2016-01-01

    Background Opioids have become the mainstay for treatment of moderate to severe pain and are commonly used to treat surgical pain. While opioid administration has been shown to cause opioid-induced hyperalgesia and tolerance, interactions between opioid administration and surgery with respect to these problematic adaptations have scarcely been addressed. Accumulating evidence suggests opioids and nociceptive signaling may converge on epigenetic mechanisms in spinal cord to enhance or prolong neuroplastic changes. Epigenetic regulation of Bdnf (brain-derived neurotrophic factor) and Pdyn (prodynorphin) genes may be involved. Results Four days of ascending doses of morphine treatment caused opioid-induced hyperalgesia and reduced opioid analgesic efficacy in mice. Both opioid-induced hyperalgesia and the reduced opioid analgesic efficacy were enhanced in mice that received hindpaw incisions. The expression of Bdnf and Pdyn (qPCR) was increased after morphine treatment and incision. Chromatin immunoprecipitation assays demonstrated that the Pdyn and Bdnf promoters were more strongly associated with acetylated H3K9 after morphine plus incision than in the morphine or incision alone groups. Selective tropomyosin-related kinase B (ANA-12) and κ-opioid receptor (nor-binaltorphimine) antagonists were administered intrathecally, both reduced hyperalgesia one or three days after surgery. Administration of ANA-12 or nor-binaltorphimine attenuated the decreased morphine analgesic efficacy on day 1, but only nor-binaltorphimine was effective on day 3 after incision in opioid-exposed group. Coadministration of histone acetyltransferase inhibitor anacardic acid daily with morphine blocked the development of opioid-induced hyperalgesia and attenuated incision-enhanced hyperalgesia in morphine-treated mice. Anacardic acid had similar effects on analgesic tolerance, showing the involvement of histone acetylation in the interactions detected. Conclusions Spinal epigenetic changes

  2. A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation

    PubMed Central

    Chakraborty, Tirtha; Perlot, Thomas; Subrahmanyam, Ramesh; Jani, Anant; Goff, Peter H.; Zhang, Yu; Ivanova, Irina; Alt, Frederick W.

    2009-01-01

    A tissue-specific transcriptional enhancer, Eμ, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Eμ results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Eμ-independent and Eμ-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression. PMID:19414554

  3. Multitarget Drugs: an Epigenetic Epiphany.

    PubMed

    Ganesan, A

    2016-06-20

    Epigenetics refers to changes in a biological phenotype that are not due to an underlying change in genotype. In eukaryotes, epigenetics involves a set of chemical modifications of the DNA and the histone proteins in nucleosomes. These dynamic changes are carried out by enzymes and modulate protein-protein and protein-nucleic acid interactions to determine whether specific genes are expressed or silenced. Both the epigenetic enzymes and recognition domains are currently important drug discovery targets, particularly for the treatment of cancer. This review summarizes the progress of epigenetic targets that have reached a clinical stage: DNA methyltransferases, histone deacetylases, lysine methyltransferases, lysine demethylases, and bromodomains; this is followed by a comprehensive survey of multitarget drugs that have included an epigenetic target as one of their mechanisms of action. PMID:26891251

  4. Epigenetic reprogramming in plant sexual reproduction.

    PubMed

    Kawashima, Tomokazu; Berger, Frédéric

    2014-09-01

    Epigenetic reprogramming consists of global changes in DNA methylation and histone modifications. In mammals, epigenetic reprogramming is primarily associated with sexual reproduction and occurs during both gametogenesis and early embryonic development. Such reprogramming is crucial not only to maintain genomic integrity through silencing transposable elements but also to reset the silenced status of imprinted genes. In plants, observations of stable transgenerational inheritance of epialleles have argued against reprogramming. However, emerging evidence supports that epigenetic reprogramming indeed occurs during sexual reproduction in plants and that it has a major role in maintaining genome integrity and a potential contribution to epiallelic variation. PMID:25048170

  5. Gli2 silencing enhances TRAIL-induced apoptosis and reduces tumor growth in human hepatoma cells in vivo

    PubMed Central

    Zhang, Da-wei; Li, Hai-yan; Lau, Wan-yee; Cao, Liang-qi; Li, Yue; Jiang, Xiao-feng; Yang, Xue-wei; Xue, Ping

    2014-01-01

    Our previous studies have showed that Gli2 played a predominant role in proliferation and apoptosis resistance to TRAIL in hepatoma cells. The purpose of this study was to explore whether Gli2 silencing enhances efficiency of TRAIL for hepatoma in vivo. SMMC-7721-shRNA cells were implanted subcutaneously into nude mices and TRAIL was injected into the peritoneal space. TUNEL assay was used to detect apoptosis of tumor cells. The expression of Gli2, c-FLIPL, c-FLIPS, and Bcl-2 protein was determined by immunohistochemistry, respectively. Apoptosis and the level of caspases proteins in SMMC-7721 and HepG2 cells were detected by Flow cytometry and Western blot. Transcriptional activity of c-FLIP induced by Gli2 was measured by luciferase reporter gene assay. The results showed that lower volumes and weights of tumor were found in mice xenografted with SMMC-7721-shRNA cells as compared with control cells in the presence of TRAIL (P < 0.05). TUNEL assay showed significantly higher apoptosis index (AI) in the SMMC-7721-shRNA group than in the control groups (P < 0.05). There were remarkable positive correlations between Gli2 and c-FLIPL, c-FLIPS, Bcl-2 protein expression. Over-expression of c-FLIP or Bcl-2 in HepG2 cells attenuated TRAIL-induced apoptosis via suppression of caspase-8 or caspase-9 activity, respectively. Luciferase reporter gene assay found a regulatory sequence by which Gli2 activated transcription between -1007 to -244 in the c-FLIP promoter region. This study demonstrates that Gli2 showed regulatory activity on transcription of c-FLIP gene, and Gli2 silencing enhances TRAIL-induced apoptosis via down-regulation of c-FLIP and Bcl-2 in human hepatoma cells in vivo. PMID:25535898

  6. Paternal care in a fish: epigenetics and fitness enhancing effects on offspring anxiety

    PubMed Central

    McGhee, Katie E.; Bell, Alison M.

    2014-01-01

    In many animals, including humans, interactions with caring parents can have long-lasting effects on offspring sensitivity to stressors. However, whether these parental effects impact offspring fitness in nature is often unclear. In addition, despite evidence that maternal care can influence offspring behaviour via epigenetic alterations to the genome, it remains unclear whether paternal care has similar effects. Here, we show in three-spined sticklebacks, a fish in which fathers are the sole provider of offspring care, that the direct care provided by fathers affects offspring anxiety and the potential for epigenetic alterations to the offspring genome. We find that families are differentially vulnerable to early stress and fathers can compensate for this differential sensitivity with the quality of their care. This variation in paternal care is also linked to the expression in offspring brains of a DNA methyltransferase (Dnmt3a) responsible for de novo methylation. We show that these paternal effects are potentially adaptive and anxious offspring are unlikely to survive an encounter with a predator. By supplying offspring care, fathers reduce offspring anxiety thereby increasing the survival of their offspring—not in the traditional sense through resource provisioning but through an epigenetic effect on offspring behavioural development. PMID:25232132

  7. Enhanced extravasation, stability and in vivo cardiac gene silencing via in situ siRNA-albumin conjugation.

    PubMed

    Lau, Shannen; Graham, Bim; Cao, Nga; Boyd, Ben J; Pouton, Colin W; White, Paul J

    2012-01-01

    A potential barrier to progression of siRNA therapeutics to the clinic is the ability of these agents to cross the vascular endothelium to reach target cells. This study aimed to bypass the endothelial barrier by harnessing the extravasation capability of the serum protein albumin to allow siRNA to reach cardiomyocytes. A strategy for conjugating siRNA to albumin in vivo was developed that involved activating 3'-amine, 2'-O-methyl, phosphorothioate modified siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to yield maleimide-functionalized siRNA ("activated siRNA"); this thiol-reactive species can then irreversibly link to the single surface-exposed cysteine residue of endogenous albumin following intravenous administration. An IGF-I-receptor (IGF-IR) siRNA sequence which was effective in vitro was used to test the ability of the siRNA-albumin conjugate to bypass the endothelial barrier in Balb/C mice and produce silencing. In situ conjugation of maleimide-functionalized siRNA to albumin in mouse serum occurred within minutes of addition, and the resulting conjugate was found to be nuclease stable by SDS-PAGE analysis. In Sprague-Dawley rats, activated siRNA showed a significantly enhanced elimination half-life (75.9 ± 18.2 min) compared to unactivated siRNA (5.1 ± 0.2 min). Intravenous injection of this activated siRNA (1 mg/kg daily for four days) significantly reduced left ventricle IGF-IR mRNA to 64.1 ± 4.1% of that in vehicle-treated animals (mean ± SEM), while the control siRNA (unconjugated) had no effect (n = 4, P > 0.05). Imaging of microvessels from mice treated with fluorescein-labeled activated siRNA showed clear evidence of extravasation and cellular uptake in capillary endothelial cells, cardiomyocytes and vascular smooth muscle cells for mice treated with the activated siRNA but not mice treated with the unactivated siRNA. siRNA-albumin constructs are therefore capable of extravasation to the myocardium resulting in

  8. Silent assassin: oncogenic ras directs epigenetic inactivation of target genes.

    PubMed

    Cheng, Xiaodong

    2008-01-01

    Oncogenic transformation is associated with genetic changes and epigenetic alterations. A study now shows that oncogenic Ras uses a complex and elaborate epigenetic silencing program to specifically repress the expression of multiple unrelated cancer-suppressing genes through a common pathway. These results suggest that cancer-related epigenetic modifications may arise through a specific and instructive mechanism and that genetic changes and epigenetic alterations are intimately connected and contribute to tumorigenesis cooperatively. PMID:18385037

  9. Expression of TET2 enzyme indicates enhanced epigenetic modification of cells in periodontitis.

    PubMed

    Larsson, Lena; Thorbert-Mros, Sara; Lopez-Lago, Aaron; Kalm, Josephine; Shikhan, Asal; Berglundh, Tord

    2016-08-01

    DNA methylation is an important epigenetic mechanism involved in the regulation of gene expression, and a reduction in DNA methylation influences cell-cycle progression and cell differentiation in inflammatory cells. The aim of the present study was to analyze the DNA-methylation pattern at local and global/systemic levels in patients with periodontitis and gingivitis. Twenty-one subjects with generalized, severe periodontitis and 17 subjects with gingival inflammation but no attachment loss were recruited. Gingival biopsies and peripheral blood samples were collected and prepared for immunohistochemical analysis of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), ten-eleven translocation 2 (TET2), and DNA methyltransferase 1 (DNMT1). Whilst a similar pattern for 5mC and 5hmC DNA methylation was found in both types of lesions, a significantly larger proportion of TET2-positive cells was found in periodontitis lesions than in gingivitis lesions. Quantitative real-time PCR analysis showed no differences between gingivitis and periodontitis lesions regarding expression of TET2 and isocitrate dehydrogenase (IDH) genes, while the global level of 5hmC was significantly higher in blood than in tissue in patients with periodontitis. It is suggested that epigenetic changes are more common in periodontitis lesions than in gingivitis lesions and that such changes are tissue specific. PMID:27297088

  10. Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR.

    PubMed

    Vizoso, Miguel; Ferreira, Humberto J; Lopez-Serra, Paula; Carmona, F Javier; Martínez-Cardús, Anna; Girotti, Maria Romina; Villanueva, Alberto; Guil, Sonia; Moutinho, Catia; Liz, Julia; Portela, Anna; Heyn, Holger; Moran, Sebastian; Vidal, August; Martinez-Iniesta, Maria; Manzano, Jose L; Fernandez-Figueras, Maria Teresa; Elez, Elena; Muñoz-Couselo, Eva; Botella-Estrada, Rafael; Berrocal, Alfonso; Pontén, Fredrik; Oord, Joost van den; Gallagher, William M; Frederick, Dennie T; Flaherty, Keith T; McDermott, Ultan; Lorigan, Paul; Marais, Richard; Esteller, Manel

    2015-07-01

    Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors. PMID:26030178

  11. Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR

    PubMed Central

    Vizoso, Miguel; Ferreira, Humberto J; Lopez-Serra, Paula; Javier Carmona, F; Martínez-Cardús, Anna; Girotti, Maria Romina; Villanueva, Alberto; Guil, Sonia; Moutinho, Catia; Liz, Julia; Portela, Anna; Heyn, Holger; Moran, Sebastian; Vidal, August; Martinez-Iniesta, Maria; Manzano, Jose L; Fernandez-Figueras, Maria Teresa; Elez, Elena; Muñoz-Couselo, Eva; Botella-Estrada, Rafael; Berrocal, Alfonso; Pontén, Fredrik; van den Oord, Joost; Gallagher, William M; Frederick, Dennie T; Flaherty, Keith T; McDermott, Ultan; Lorigan, Paul; Marais, Richard; Esteller, Manel

    2016-01-01

    Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors. PMID:26030178

  12. Transcriptional gene silencing in humans.

    PubMed

    Weinberg, Marc S; Morris, Kevin V

    2016-08-19

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

  13. Mapping epigenetic mutations in fission yeast using whole-genome next-generation sequencing

    PubMed Central

    Irvine, Danielle V.; Goto, Derek B.; Vaughn, Matthew W.; Nakaseko, Yukinobu; McCombie, W. Richard; Yanagida, Mitsuhiro; Martienssen, Rob

    2009-01-01

    Fission yeast is an important model for epigenetic studies due to the ease with which genetic mutants can be isolated. However, it can be difficult to complement epigenetic phenotypes with genomic libraries in order to identify the genes responsible. This is because epigenetic phenotypes are typically unstable, and can prohibit complementation if silencing cannot be reestablished. Here we have resequenced the fission yeast genome following mutagenesis to readily identify a novel mutant involved in heterochromatic silencing. Candidate genes were identified as functional single base changes linked to the mutation, which were then reconstituted in a wild-type strain to recapitulate the mutant phenotype. By this procedure we identified a weak allele of ubc4, which encodes an essential E2 ubiquitin ligase, as responsible for the swi*603 mutant phenotype. In combination with a large collection of mutants and suppressor plasmids, next-generation genomic resequencing promises to dramatically enhance the power of yeast genetics, permitting the isolation of subtle alleles of essential genes, alleles with quantitative effects, and enhancers and suppressors of heterochromatic silencing. PMID:19423874

  14. Nucleosome Positioning and Epigenetics

    NASA Astrophysics Data System (ADS)

    Schwab, David; Bruinsma, Robijn

    2008-03-01

    The role of chromatin structure in gene regulation has recently taken center stage in the field of epigenetics, phenomena that change the phenotype without changing the DNA sequence. Recent work has also shown that nucleosomes, a complex of DNA wrapped around a histone octamer, experience a sequence dependent energy landscape due to the variation in DNA bend stiffness with sequence composition. In this talk, we consider the role nucleosome positioning might play in the formation of heterochromatin, a compact form of DNA generically responsible for gene silencing. In particular, we discuss how different patterns of nucleosome positions, periodic or random, could either facilitate or suppress heterochromatin stability and formation.

  15. Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing

    PubMed Central

    Ahlenstiel, Chantelle; Mendez, Catalina; Lim, Steven T H; Marks, Katherine; Turville, Stuart; Cooper, David A; Kelleher, Anthony D; Suzuki, Kazuo

    2015-01-01

    Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-κB motifs within the viral 5′LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5′LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation. PMID:26506039

  16. Epigenetics and Nutritional Environmental Signals

    PubMed Central

    Mazzio, Elizabeth A.; Soliman, Karam F. A.

    2014-01-01

    All terrestrial life is influenced by multi-directional flows of information about its environment, enabling malleable phenotypic change through signals, chemical processes, or various forms of energy that facilitate acclimatization. Billions of biological co-inhabitants of the earth, including all plants and animals, collectively make up a genetic/epigenetic ecosystem by which adaptation/survival (inputs and outputs) are highly interdependent on one another. As an ecosystem, the solar system, rotation of the planets, changes in sunlight, and gravitational pull influence cyclic epigenetic transitions and chromatin remodeling that constitute biological circadian rhythms controlling senescence. In humans, adverse environmental conditions such as poverty, stress, alcohol, malnutrition, exposure to pollutants generated from industrialization, man-made chemicals, and use of synthetic drugs can lead to maladaptive epigenetic-related illnesses with disease-specific genes being atypically activated or silenced. Nutrition and dietary practices are one of the largest facets in epigenetic-related metabolism, where specific “epi-nutrients” can stabilize the genome, given established roles in DNA methylation, histone modification, and chromatin remodeling. Moreover, food-based “epi-bioactive” constituents may reverse maladaptive epigenetic patterns, not only prior to conception and during fetal/early postnatal development but also through adulthood. In summary, in contrast to a static genomic DNA structure, epigenetic changes are potentially reversible, raising the hope for therapeutic and/or dietary interventions that can reverse deleterious epigenetic programing as a means to prevent or treat major illnesses. PMID:24861811

  17. Epigenetics and nutritional environmental signals.

    PubMed

    Mazzio, Elizabeth A; Soliman, Karam F A

    2014-07-01

    All terrestrial life is influenced by multi-directional flows of information about its environment, enabling malleable phenotypic change through signals, chemical processes, or various forms of energy that facilitate acclimatization. Billions of biological co-inhabitants of the earth, including all plants and animals, collectively make up a genetic/epigenetic ecosystem by which adaptation/survival (inputs and outputs) are highly interdependent on one another. As an ecosystem, the solar system, rotation of the planets, changes in sunlight, and gravitational pull influence cyclic epigenetic transitions and chromatin remodeling that constitute biological circadian rhythms controlling senescence. In humans, adverse environmental conditions such as poverty, stress, alcohol, malnutrition, exposure to pollutants generated from industrialization, man-made chemicals, and use of synthetic drugs can lead to maladaptive epigenetic-related illnesses with disease-specific genes being atypically activated or silenced. Nutrition and dietary practices are one of the largest facets in epigenetic-related metabolism, where specific "epi-nutrients" can stabilize the genome, given established roles in DNA methylation, histone modification, and chromatin remodeling. Moreover, food-based "epi-bioactive" constituents may reverse maladaptive epigenetic patterns, not only prior to conception and during fetal/early postnatal development but also through adulthood. In summary, in contrast to a static genomic DNA structure, epigenetic changes are potentially reversible, raising the hope for therapeutic and/or dietary interventions that can reverse deleterious epigenetic programing as a means to prevent or treat major illnesses. PMID:24861811

  18. Virus-viroid interactions: Citrus Tristeza Virus enhances the accumulation of Citrus Dwarfing Viroid in Mexican lime via virus-encoded silencing suppressors.

    PubMed

    Serra, Pedro; Bani Hashemian, Seyed M; Fagoaga, Carmen; Romero, Juan; Ruiz-Ruiz, Susana; Gorris, Maria T; Bertolini, Edson; Duran-Vila, Núria

    2014-01-01

    An assay to identify interactions between Citrus Dwarfing Viroid (CDVd) and Citrus Tristeza Virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd. PMID:24227850

  19. Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors.

    PubMed

    Guo, Xiaoli; Chronis, Demosthenis; De La Torre, Carola M; Smeda, John; Wang, Xiaohong; Mitchum, Melissa G

    2015-08-01

    CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant-parasitic cyst nematodes secrete CLE-like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode-induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock-down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants. PMID:25581705

  20. Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery

    PubMed Central

    Shen, Haifa; Rodriguez-Aguayo, Cristian; Xu, Rong; Gonzalez-Villasana, Vianey; Mai, Junhua; Huang, Yi; Zhang, Guodong; Guo, Xiaojing; Bai, Litao; Qin, Guoting; Deng, Xiaoyong; Li, Qingpo; Erm, Donald R.; Liu, Xuewu; Sakamoto, Jason; Chavez-Reyes, Arturo; Han, Hee-Dong; Sood, Anil K.; Ferrari, Mauro; Lopez-Berestein, Gabriel

    2013-01-01

    Purpose RNA interference has the potential to specifically knock down the expression of target genes, and thereby transform cancer therapy. However, lack of effective delivery of small inhibitory RNA (siRNA) has dramatically limited its in vivo applications. We have developed a multistage vector (MSV) system, composed of discoidal porous silicon particles loaded with nanotherapeutics, that directs effective delivery and sustained release of siRNA in tumor tissues. In this study, we evaluated therapeutic efficacy of MSV-loaded EphA2 siRNA (MSV/EphA2) with murine orthotopic models of metastatic ovarian cancers as a first step towards development of a new class of nanotherapeutics for the treatment of ovarian cancer. Experimental design Tumor accumulation of MSV/EphA2 and sustained release of siRNA from MSV were analyzed after i.v. administration of MSV/siRNA. Nude mice with metastatic SKOV3ip2 tumors were treated with MSV/EphA2 and paclitaxel, and therapeutic efficacy was assessed. Mice with chemotherapy-resistant HeyA8 ovarian tumors were treated with a combination of MSV/EphA2 and docetaxel, and enhanced therapeutic efficacy was evaluated. Results Treatment of SKOV3ip2 tumor mice with MSV/EphA2 biweekly for 6 weeks resulted in dose-dependent (5, 10 and 15 μg/mice) reduction of tumor weight (36%, 64%, and 83%) and number of tumor nodules compared with the control groups. In addition, tumor growth was completely inhibited when mice were treated with MSV/EphA2 in combination with paclitaxel. Furthermore, combination treatment with MSV/EphA2 and docetaxel inhibited growth of HeyA8-MDR tumors, which were otherwise resistant to docetaxel treatment. Conclusion These findings indicate that MSV/EphA2 merits further development as a novel therapeutic agent for ovarian cancer. PMID:23386691

  1. Acrolein enhances epigenetic modifications, FasL expression and hepatocyte toxicity induced by anti-HIV drug Zidovudine.

    PubMed

    Ghare, Smita S; Donde, Hridgandh; Chen, Wei-Yang; Barker, David F; Gobejishvilli, Leila; McClain, Craig J; Barve, Shirish S; Joshi-Barve, Swati

    2016-09-01

    Zidovudine (AZT) remains the mainstay of antiretroviral therapy against HIV in resource-poor countries; however, its use is frequently associated with hepatotoxicity. Not all HIV patients on AZT develop hepatotoxicity, and the determining factors are unclear. Alcohol consumption and cigarette smoking are known risk factors for HIV hepatotoxicity, and both are significant sources of acrolein, a highly reactive and toxic aldehyde. This study examines the potential hepatotoxic interactions between acrolein and AZT. Our data demonstrate that acrolein markedly enhanced AZT-induced transcriptionally permissive histone modifications (H3K9Ac and H3K9Me3) allowing the recruitment of transcription factor NF-kB and RNA polymerase II at the FasL gene promoter, resulting in FasL upregulation and apoptosis in hepatocytes. Notably, the acrolein scavenger, hydralazine prevented these promoter-associated epigenetic changes and inhibited FasL upregulation and apoptosis induced by the combination of AZT and acrolein, as well as AZT alone. Our data strongly suggest that acrolein enhancement of promoter histone modifications and FasL upregulation are major pathogenic mechanisms driving AZT-induced hepatotoxicity. Moreover, these data also indicate the therapeutic potential of hydralazine in mitigating AZT hepatotoxicity. PMID:27238871

  2. Bypassing the Requirements for Epigenetic Modifications in Gene Transcription by Increasing Enhancer Strength▿

    PubMed Central

    Koutroubas, George; Merika, Menie; Thanos, Dimitris

    2008-01-01

    Our current concept postulates that histone acetylation is required for the recruitment of bromodomain-containing transcription complexes, such as the chromatin-remodeling machine SWI/SNF and the basal transcription factor TFIID. We generated simple NF-κB-dependent enhancers of increasing transcriptional strengths and found that the histone acetylation requirements for activation of transcription depended on the strengths of these enhancers. All enhancers function by recruiting SWI/SNF and TFIID to induce nucleosome sliding, a prerequisite for transcriptional activation. However, histone acetylation, although it occurs, is dispensable for TFIID and SWI/SNF recruitment by the strong enhancers, indicating that strong activators can overcome the chromatin barrier by directly recruiting the necessary transcriptional complexes. Weak enhancers depend on histone acetylation for recruitment, and this requirement is independent of a histone acetylation code. Thus, the need for nucleosome modifications is imposed on genes and translated according to the quality and strengths of the activators. PMID:18025106

  3. The use of pH-sensitive functional selenium nanoparticles shows enhanced in vivo VEGF-siRNA silencing and fluorescence imaging.

    PubMed

    Yu, Qianqian; Liu, Yanan; Cao, Chengwen; Le, Fangling; Qin, Xiuying; Sun, Dongdong; Liu, Jie

    2014-08-01

    The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH-Cit/SeNPs@siRNA led to significantly higher accumulation of siRNA within the tumor itself, VEGF gene silencing, and reduced angiogenesis in the tumor. Furthermore, the G2/PAH-Cit/SeNP delivery system not only enhanced anti-tumor effects on tumor-bearing nude mice as compared to SeNPs@siRNA, but also resulted in weak occurrence of lesions in major target organs. In sum, this study provides a new class of siRNA delivery system, thereby providing an alternative therapeutic route for cancer treatment. PMID:24986368

  4. The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

    PubMed

    de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

    2015-11-01

    In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects. PMID:26323262

  5. Nutritional epigenetics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter is intended to provide a timely overview of the current state of research at the intersection of nutrition and epigenetics. I begin by describing epigenetics and molecular mechanisms of eigenetic regulation, then highlight four classes of nutritional exposures currently being investiga...

  6. The analysis of novel distal Cebpa enhancers and silencers using a transcriptional model reveals the complex regulatory logic of hematopoietic lineage specification.

    PubMed

    Bertolino, Eric; Reinitz, John; Manu

    2016-05-01

    C/EBPα plays an instructive role in the macrophage-neutrophil cell-fate decision and its expression is necessary for neutrophil development. How Cebpa itself is regulated in the myeloid lineage is not known. We decoded the cis-regulatory logic of Cebpa, and two other myeloid transcription factors, Egr1 and Egr2, using a combined experimental-computational approach. With a reporter design capable of detecting both distal enhancers and silencers, we analyzed 46 putative cis-regulatory modules (CRMs) in cells representing myeloid progenitors, and derived early macrophages or neutrophils. In addition to novel enhancers, this analysis revealed a surprisingly large number of silencers. We determined the regulatory roles of 15 potential transcriptional regulators by testing 32,768 alternative sequence-based transcriptional models against CRM activity data. This comprehensive analysis allowed us to infer the cis-regulatory logic for most of the CRMs. Silencer-mediated repression of Cebpa was found to be effected mainly by TFs expressed in non-myeloid lineages, highlighting a previously unappreciated contribution of long-distance silencing to hematopoietic lineage resolution. The repression of Cebpa by multiple factors expressed in alternative lineages suggests that hematopoietic genes are organized into densely interconnected repressive networks instead of hierarchies of mutually repressive pairs of pivotal TFs. More generally, our results demonstrate that de novo cis-regulatory dissection is feasible on a large scale with the aid of transcriptional modeling. Current address: Department of Biology, University of North Dakota, 10 Cornell Street, Stop 9019, Grand Forks, ND 58202-9019, USA. PMID:26945717

  7. Alzheimer's disease and epigenetic diet.

    PubMed

    Sezgin, Zeynep; Dincer, Yildiz

    2014-12-01

    Alzheimer's disease (AD) is the most common neurodegenerative disease. Many efforts have been directed to prevent AD due to its rising prevalence and the lack of an effective curative treatment. Various epigenetic mechanisms are linked to pathogenesis of AD. Epigenetic alterations may occur through external factors and are known for their reversibility. Dietary factors can influence epigenetic mechanisms. Several neuroprotective nutrients have been shown to enhance cognition, memory and other impaired functions seen in AD. Within recent years neuroprotective nutrients have gained more attention in the field of epigenetic. A growing body of evidence suggest that epigenetic changes triggered by dietary nutrients have an important role in health and in prevention of some diseases, especially neurodegenerative disorders. Several studies have shown that folic acid, vitamin B12, choline, zinc, selenium, dietary polyphenols are capable of interacting with epigenetic mechanisms and ultimately gene expression. Epigenetic mechanisms resulting in neuronal dysfunction may be modified by diet. Therefore manipulation of epigenetic mechanisms via dietary nutrients may affect influence the vulnerability of neurons to degeneration which is seen in AD. The aim of this article is to provide a brief overview about the recent findings related to epigenetic alterations that are linked to AD pathogenesis, and to discuss the bioactive nutrients which can affect these epigenetic mechanisms. PMID:25290336

  8. Epigenetic Regulation in Plant Responses to the Environment

    PubMed Central

    Baulcombe, David C.; Dean, Caroline

    2014-01-01

    In this article, we review environmentally mediated epigenetic regulation in plants using two case histories. One of these, vernalization, mediates adaptation of plants to different environments and it exemplifies processes that are reset in each generation. The other, virus-induced silencing, involves transgenerationally inherited epigenetic modifications. Heritable epigenetic marks may result in heritable phenotypic variation, influencing fitness, and so be subject to natural selection. However, unlike genetic inheritance, the epigenetic modifications show instability and are influenced by the environment. These two case histories are then compared with other phenomena in plant biology that are likely to represent epigenetic regulation in response to the environment. PMID:25183832

  9. Epigenetics across the human lifespan

    PubMed Central

    Kanherkar, Riya R.; Bhatia-Dey, Naina; Csoka, Antonei B.

    2014-01-01

    Epigenetics has the potential to explain various biological phenomena that have heretofore defied complete explication. This review describes the various types of endogenous human developmental milestones such as birth, puberty, and menopause, as well as the diverse exogenous environmental factors that influence human health, in a chronological epigenetic context. We describe the entire course of human life from periconception to death and chronologically note all of the potential internal timepoints and external factors that influence the human epigenome. Ultimately, the environment presents these various factors to the individual that influence the epigenome, and the unique epigenetic and genetic profile of each individual also modulates the specific response to these factors. During the course of human life, we are exposed to an environment that abounds with a potent and dynamic milieu capable of triggering chemical changes that activate or silence genes. There is constant interaction between the external and internal environments that is required for normal development and health maintenance as well as for influencing disease load and resistance. For example, exposure to pharmaceutical and toxic chemicals, diet, stress, exercise, and other environmental factors are capable of eliciting positive or negative epigenetic modifications with lasting effects on development, metabolism and health. These can impact the body so profoundly as to permanently alter the epigenetic profile of an individual. We also present a comprehensive new hypothesis of how these diverse environmental factors cause both direct and indirect epigenetic changes and how this knowledge can ultimately be used to improve personalized medicine. PMID:25364756

  10. Implications of Genetic and Epigenetic Alterations of CDKN2A (p16(INK4a)) in Cancer.

    PubMed

    Zhao, Ran; Choi, Bu Young; Lee, Mee-Hyun; Bode, Ann M; Dong, Zigang

    2016-06-01

    Aberrant gene silencing is highly associated with altered cell cycle regulation during carcinogenesis. In particular, silencing of the CDKN2A tumor suppressor gene, which encodes the p16(INK4a) protein, has a causal link with several different types of cancers. The p16(INK4a) protein plays an executional role in cell cycle and senescence through the regulation of the cyclin-dependent kinase (CDK) 4/6 and cyclin D complexes. Several genetic and epigenetic aberrations of CDKN2A lead to enhanced tumorigenesis and metastasis with recurrence of cancer and poor prognosis. In these cases, the restoration of genetic and epigenetic reactivation of CDKN2A is a practical approach for the prevention and therapy of cancer. This review highlights the genetic status of CDKN2A as a prognostic and predictive biomarker in various cancers. PMID:27428416

  11. Use of Epigenetic Drugs in Disease: An Overview

    PubMed Central

    Heerboth, Sarah; Lapinska, Karolina; Snyder, Nicole; Leary, Meghan; Rollinson, Sarah; Sarkar, Sibaji

    2014-01-01

    Epigenetic changes such as DNA methylation and histone methylation and acetylation alter gene expression at the level of transcription by upregulating, downregulating, or silencing genes completely. Dysregulation of epigenetic events can be pathological, leading to cardiovascular disease, neurological disorders, metabolic disorders, and cancer development. Therefore, identifying drugs that inhibit these epigenetic changes are of great clinical interest. In this review, we summarize the epigenetic events associated with different disorders and diseases including cardiovascular, neurological, and metabolic disorders, and cancer. Knowledge of the specific epigenetic changes associated with these types of diseases facilitates the development of specific inhibitors, which can be used as epigenetic drugs. In this review, we discuss the major classes of epigenetic drugs currently in use, such as DNA methylation inhibiting drugs, bromodomain inhibitors, histone acetyl transferase inhibitors, histone deacetylase inhibitors, protein methyltransferase inhibitors, and histone methylation inhibitors and their role in reversing epigenetic changes and treating disease. PMID:25512710

  12. Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells.

    PubMed

    Li, Ju; Han, Suhyoun; Cousin, Wendy; Conboy, Irina M

    2015-03-01

    The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here, we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of cyclin/cyclin-dependent kinase (CDK) inhibitors (CDKIs) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells, FGF2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15(INK4B) and p27(KIP1) , become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration. PMID:25447026

  13. Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells

    PubMed Central

    Li, Ju; Han, Suhyoun; Cousin, Wendy; Conboy, Irina M.

    2014-01-01

    The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of CDK inhibitors (CDKI) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF-2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells FGF-2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15INK4B and p27KIP1, become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration. PMID:25447026

  14. Silencing MRP1-4 genes by RNA interference enhances sensitivity of human hepatoma cells to chemotherapy

    PubMed Central

    Su, Zheng; Liu, Gaojie; Fang, Tingfeng; Wang, Yang; Zhang, Huayao; Yang, Shanglin; Wei, Jinxing; Lv, Zejian; Tan, Langping; Liu, Jianping

    2016-01-01

    Aim: Besides surgical treatment, systematic chemotherapy plays a crucial role in HCC treatment, especially for patients with advanced HCC. However, none of the single-drug-treatment strategies have shown significant survival benefit due to a high incidence rate of chemoresistance. This study was designed to observe the effect of small interfering of RNA (SiRNA) targeting multidrug resistance-related protein 1-4 (MRP1, MRP2, MRP3, and MRP4) in modulating drug resistance of HepG2/ADM and SMMC7721/ADM cells. Methods: HepG2/Adriamycin (ADM) and SMMC7721/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity and half inhibitory concentration (IC50) of drugs was calculated. Flow cytometry was employed to analyze cell cycle distribution. MRP1-4 mRNA expression levels were measured by quantitative real-time PCR (QRT-PCR). Expression of proteins was analyzed by Western blot. The growth curve was draw and the cell apoptosis was also observed. Animal experiment was used to compare the cell growth. Results: MTT assay showed that the values of IC50 and RI of HepG2/ADM and SMMC7721/ADM decreased after siRNA treatment in HepG2/ADM cells and SMMC7721/ADM cells. QRT-PCR analysis demonstrated the MRP1-4 mRNA expression decreased significantly in HepG2/ADM cells and SMMC7721/ADM cells after siRNA transfection. In addition, compared with parental cells, MRP1-4 protein expressions apparently decreased in SMMC7721/ADM and HepG2/ADM cells. Flow cytometry showed significantly elevated apoptosis rate following MRP1-4 siRNA transfection. Animal experiment suggested that silencing MRP1-4 gene in vivo inhibited tumor growth. Conclusion: Inhibition of MRP1-4 by small interfering RNA enhanced and selectively restored sensitivity of hepatoma cells to drugs. MRP1-4 siRNA might represent a new therapeutic option for HCC. PMID:27398162

  15. The presence of tomato leaf curl Kerala virus AC3 protein enhances viral DNA replication and modulates virus induced gene-silencing mechanism in tomato plants

    PubMed Central

    2011-01-01

    Background Geminiviruses encode few viral proteins. Most of the geminiviral proteins are multifunctional and influence various host cellular processes for the successful viral infection. Though few viral proteins like AC1 and AC2 are well characterized for their multiple functions, role of AC3 in the successful viral infection has not been investigated in detail. Results We performed phage display analysis with the purified recombinant AC3 protein with Maltose Binding Protein as fusion tag (MBP-AC3). Putative AC3 interacting peptides identified through phage display were observed to be homologous to peptides of proteins from various metabolisms. We grouped these putative AC3 interacting peptides according to the known metabolic function of the homologous peptide containing proteins. In order to check if AC3 influences any of these particular metabolic pathways, we designed vectors for assaying DNA replication and virus induced gene-silencing of host gene PCNA. Investigation with these vectors indicated that AC3 enhances viral replication in the host plant tomato. In the PCNA gene-silencing experiment, we observed that the presence of functional AC3 ORF strongly manifested the stunted phenotype associated with the virus induced gene-silencing of PCNA in tomato plants. Conclusions Through the phage display analysis proteins from various metabolic pathways were identified as putative AC3 interacting proteins. By utilizing the vectors developed, we could analyze the role of AC3 in viral DNA replication and host gene-silencing. Our studies indicate that AC3 is also a multifunctional protein. PMID:21496351

  16. Transcriptional and Epigenetic Mechanisms Underlying Enhanced in Vitro Adipocyte Differentiation by the Brominated Flame Retardant BDE-47

    PubMed Central

    2015-01-01

    Recent studies suggest that exposure to endocrine-disrupting compounds (EDCs) may play a role in the development of obesity. EDCs such as the flame retardant 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47) have been shown to enhance adipocyte differentiation in the murine 3T3-L1 model. The mechanisms by which EDCs direct preadipocytes to form adipocytes are poorly understood. Here, we examined transcriptional and epigenetic mechanisms underlying the induction of in vitro adipocyte differentiation by BDE-47. Quantitative high content microscopy revealed concentration-dependent enhanced adipocyte differentiation following exposure to BDE-47 or the antidiabetic drug troglitazone (TROG). BDE-47 modestly activated the key adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) in COS7 cells, transiently transfected with a GAL4 reporter construct. Increased gene expression was observed for Pparγ2, leptin (Lep), and glucose-6-phophatase catalytic subunit (G6pc) in differentiated 3T3-L1 cells after BDE-47 exposure compared to TROG. Methylation-sensitive high resolution melting (MS-HRM) revealed significant demethylation of three CpG sites in the Pparγ2 promoter after exposure to both BDE-47 and TROG in differentiated 3T3-L1 cells. This study shows the potential of BDE-47 to induce adipocyte differentiation through various mechanisms that include Pparγ2 gene induction and promoter demethylation accompanied by activation of PPARγ, and possible disruption of glucose homeostasis and IGF1 signaling. PMID:24559133

  17. Benzyl butyl phthalate induces epigenetic stress to enhance adipogenesis in mesenchymal stem cells.

    PubMed

    Sonkar, Ravi; Powell, Catherine A; Choudhury, Mahua

    2016-08-15

    Endocrine disruptors, phthalates, may have contributed to recent global obesity health crisis. Our study investigated the potential of benzyl butyl phthalate (BBP) to regulate the mesenchymal stem cell epigenome to drive adipogenesis. BBP exposure enhanced lipid accumulation and adipogenesis in a dose-dependent manner compared to control (P < 0.001). Adipogenesis markers, PPARγ (P < 0.001), C/EBPα (P < 0.01), and aP2 (P < 0.001) were significantly upregulated by increasing concentrations of BBP when compared to DMSO. BBP enhanced H3K9 acetylation while decreasing H3K9 dimethylation. Fifty μM BBP increased histone acetyltransferases, p300 (P < 0.05) and GCN5 (P < 0.01) gene expression. Furthermore, histone deacetylases (HDACs), HDAC3 (P < 0.01) and HDAC10 (P < 0.01, 10 μM BBP; P < 0.001, 50 μM BBP) and histone methyltransferases, SETDB1 (P < 0.01) and G9a (P < 0.01), were significantly downregulated by BBP exposure. BBP acts, in part, through PPARγ, as PPARγ knockdown led to decreased H3K9ac and rescued H3K9me2 during BBP exposure. In conclusion, BBP regulated MSCs towards adipogenesis by tipping the epigenomic balance. PMID:27164441

  18. Epigenetics and Chromatin Remodeling Play a Role in Lung Disease

    PubMed Central

    Mortaz, Esmaeil; Masjedi, Mohammad Reza; Barnes, Peter J

    2011-01-01

    Epigenetics is defined as heritable changes that affect gene expression without altering the DNA sequence. Epigenetic regulation of gene expression is facilitated through different mechanisms such as DNA methylation, histone modifications and RNA-associated silencing by small non-coding RNAs. All these mechanisms are crucial for normal development, differentiation and tissue-specific gene expression. These three systems interact and stabilize one another and can initiate and sustain epigenetic silencing, thus determining heritable changes in gene expression. Histone acetylation regulates diverse cellular functions including inflammatory gene expression, DNA repair and cell proliferation. Transcriptional coactivators possess intrinsic histone acetyltransferase activity and this activity drives inflammatory gene expression. Eleven classical histone deacetylases (HDACs) act to regulate the expression of distinct subsets of inflammatory/immune genes. Thus, loss of HDAC activity or the presence of HDAC inhibitors can further enhance inflammatory gene expression by producing a gene-specific change in HAT activity. For example, HDAC2 expression and activity are reduced in lung macrophages, biopsy specimens, and blood cells from patients with severe asthma and smoking asthmatics, as well as in patients with chronic obstructive pulmonary disease (COPD). This may account, at least in part, for the enhanced inflammation and reduced steroid responsiveness seen in these patients. Other proteins, particularly transcription factors, are also acetylated and are targets for deacetylation by HDACs and sirtuins, a related family of 7 predominantly protein deacetylases. Thus the acetylation/deacetylation status of NF-κB and the glucocorticoid receptor can also affect the overall expression pattern of inflammatory genes and regulate the inflammatory response. Understanding and targeting specific enzymes involved in this process might lead to new therapeutic agents, particularly in

  19. The use of pH-sensitive functional selenium nanoparticles shows enhanced in vivo VEGF-siRNA silencing and fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Yu, Qianqian; Liu, Yanan; Cao, Chengwen; Le, Fangling; Qin, Xiuying; Sun, Dongdong; Liu, Jie

    2014-07-01

    The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH-Cit/SeNPs@siRNA led to significantly higher accumulation of siRNA within the tumor itself, VEGF gene silencing, and reduced angiogenesis in the tumor. Furthermore, the G2/PAH-Cit/SeNP delivery system not only enhanced anti-tumor effects on tumor-bearing nude mice as compared to SeNPs@siRNA, but also resulted in weak occurrence of lesions in major target organs. In sum, this study provides a new class of siRNA delivery system, thereby providing an alternative therapeutic route for cancer treatment.The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH

  20. Melatonin relieves neuropathic allodynia through spinal MT2-enhanced PP2Ac and downstream HDAC4 shuttling-dependent epigenetic modification of hmgb1 transcription.

    PubMed

    Lin, Tzer-Bin; Hsieh, Ming-Chun; Lai, Cheng-Yuan; Cheng, Jen-Kun; Wang, Hsueh-Hsiao; Chau, Yat-Pang; Chen, Gin-Den; Peng, Hsien-Yu

    2016-04-01

    Melatonin (MLT; N-acetyl-5-methoxytryptamine) exhibits analgesic properties in chronic pain conditions. While researches linking MLT to epigenetic mechanisms have grown exponentially over recent years, very few studies have investigated the contribution of MLT-associated epigenetic modification to pain states. Here, we report that together with behavioral allodynia, spinal nerve ligation (SNL) induced a decrease in the expression of catalytic subunit of phosphatase 2A (PP2Ac) and enhanced histone deacetylase 4 (HDAC4) phosphorylation and cytoplasmic accumulation, which epigenetically alleviated HDAC4-suppressed hmgb1 gene transcription, resulting in increased high-mobility group protein B1 (HMGB1) expression selectively in the ipsilateral dorsal horn of rats. Focal knock-down of spinal PP2Ac expression also resulted in behavioral allodynia in association with similar protein expression as observed with SNL. Notably, intrathecal administration with MLT increased PP2Ac expression, HDAC4 dephosphorylation and nuclear accumulation, restored HDAC4-mediated hmgb1 suppression and relieved SNL-sensitized behavioral pain; these effects were all inhibited by spinal injection of 4P-PDOT (a MT2 receptor antagonist, 30 minutes before MLT) and okadaic acid (OA, a PP2A inhibitor, 3 hr after MLT). Our findings demonstrate a novel mechanism by which MLT ameliorates neuropathic allodynia via epigenetic modification. This MLT-exhibited anti-allodynia is mediated by MT2-enhanced PP2Ac expression that couples PP2Ac with HDAC4 to induce HDAC4 dephosphorylation and nuclear import, herein increases HDAC4 binding to the promoter of hmgb1 gene and upregulates HMGB1 expression in dorsal horn neurons. PMID:26732138

  1. MutS HOMOLOG1-derived epigenetic breeding potential in tomato.

    PubMed

    Yang, Xiaodong; Kundariya, Hardik; Xu, Ying-Zhi; Sandhu, Ajay; Yu, Jiantao; Hutton, Samuel F; Zhang, Mingfang; Mackenzie, Sally A

    2015-05-01

    Evidence is compelling in support of a naturally occurring epigenetic influence on phenotype expression in land plants, although discerning the epigenetic contribution is difficult. Agriculturally important attributes like heterosis, inbreeding depression, phenotypic plasticity, and environmental stress response are thought to have significant epigenetic components, but unequivocal demonstration of this is often infeasible. Here, we investigate gene silencing of a single nuclear gene, MutS HOMOLOG1 (MSH1), in the tomato (Solanum lycopersicum) 'Rutgers' to effect developmental reprogramming of the plant. The condition is heritable in subsequent generations independent of the MSH1-RNA interference transgene. Crossing these transgene-null, developmentally altered plants to the isogenic cv Rutgers wild type results in progeny lines that show enhanced, heritable growth vigor under both greenhouse and field conditions. This boosted vigor appears to be graft transmissible and is partially reversed by treatment with the methylation inhibitor 5-azacytidine, implying the influence of mobile, epigenetic factors and DNA methylation changes. These data provide compelling evidence for the feasibility of epigenetic breeding in a crop plant. PMID:25736208

  2. MutS HOMOLOG1-Derived Epigenetic Breeding Potential in Tomato1[OPEN

    PubMed Central

    Kundariya, Hardik; Xu, Ying-Zhi; Sandhu, Ajay; Yu, Jiantao; Zhang, Mingfang

    2015-01-01

    Evidence is compelling in support of a naturally occurring epigenetic influence on phenotype expression in land plants, although discerning the epigenetic contribution is difficult. Agriculturally important attributes like heterosis, inbreeding depression, phenotypic plasticity, and environmental stress response are thought to have significant epigenetic components, but unequivocal demonstration of this is often infeasible. Here, we investigate gene silencing of a single nuclear gene, MutS HOMOLOG1 (MSH1), in the tomato (Solanum lycopersicum) ‘Rutgers’ to effect developmental reprogramming of the plant. The condition is heritable in subsequent generations independent of the MSH1-RNA interference transgene. Crossing these transgene-null, developmentally altered plants to the isogenic cv Rutgers wild type results in progeny lines that show enhanced, heritable growth vigor under both greenhouse and field conditions. This boosted vigor appears to be graft transmissible and is partially reversed by treatment with the methylation inhibitor 5-azacytidine, implying the influence of mobile, epigenetic factors and DNA methylation changes. These data provide compelling evidence for the feasibility of epigenetic breeding in a crop plant. PMID:25736208

  3. Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements

    PubMed Central

    Thakore, Pratiksha I.; D’Ippolito, Anthony M; Song, Lingyun; Safi, Alexias; Shivakumar, Nishkala K.; Kabadi, Ami M.; Reddy, Timothy E.; Crawford, Gregory E.; Gersbach, Charles A.

    2015-01-01

    Epigenome editing with the CRISPR/Cas9 platform is a promising technology to modulate gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of the nuclease-inactive dCas9 to the KRAB repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB is not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates expression of multiple globin genes. Genome-wide analyses demonstrated that localization of dCas9-KRAB to HS2 specifically induced H3K9 tri-methylation (H3K9me3) at the enhancer and reduced the chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome. PMID:26501517

  4. MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs

    PubMed Central

    Wood, C David; Veenstra, Hildegonda; Khasnis, Sarika; Gunnell, Andrea; Webb, Helen M; Shannon-Lowe, Claire; Andrews, Simon; Osborne, Cameron S; West, Michelle J

    2016-01-01

    Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma. DOI: http://dx.doi.org/10.7554/eLife.18270.001 PMID:27490482

  5. MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs.

    PubMed

    Wood, C David; Veenstra, Hildegonda; Khasnis, Sarika; Gunnell, Andrea; Webb, Helen M; Shannon-Lowe, Claire; Andrews, Simon; Osborne, Cameron S; West, Michelle J

    2016-01-01

    Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma. PMID:27490482

  6. Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects

    PubMed Central

    Shang, Renfu; Zhang, Fengjuan; Xu, Beiying; Xi, Hairui; Zhang, Xue; Wang, Weihua; Wu, Ligang

    2015-01-01

    Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16–18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 3′ end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 3′ overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications. PMID:26455506

  7. Epigenetics in acute kidney injury

    PubMed Central

    Tang, Jinhua; Zhuang, Shougang

    2015-01-01

    Purpose of review Recent advances in epigenetics indicate the involvement of several epigenetic modifications in the pathogenesis of acute kidney injury (AKI). The purpose of this review is to summarize our understanding of recent advances in epigenetic regulation of AKI and provide mechanistic insight into the role of acetylation, methylation, and microRNA expression in the pathological processes of AKI. Recent findings Enhancement of protein acetylation by pharmacological inhibition of histone deacetylases (HDACs) leads to more severe tubular injury and impairment of renal structural and functional recovery. The changes in promoter DNA methylation occur in the kidney with ischemia/reperfusion. microRNA expression is associated with regulation of both renal injury and regeneration after AKI. Summary Recent studies on epigenetic regulation indicate that acetylation, methylation, and microRNA expression are critically implicated in the pathogenesis of AKI. Strategies targeting epigenetic processes may hold a therapeutic potential for patients with AKI. PMID:26050122

  8. Epigenetic Virtues of Chromodomains

    PubMed Central

    Blus, Bartlomiej J; Wiggins, Kimberly; Khorasanizadeh, Sepideh

    2011-01-01

    The chromatin organization modifier domain (chromodomain) was first identified as a motif associated with chromatin silencing in Drosophila. There is growing evidence that chromodomains are evolutionary conserved across different eukaryotic species to control diverse aspects of epigenetic regulation. Although originally reported as histone H3 methyllysine readers, the chromodomain functions have now expanded to recognition of other histone and non-histone partners as well as interaction with nucleic acids. Chromodomain binding to a diverse group of targets is mediated by a conserved substructure called the chromobox homology region. This motif can be used to predict methyllysine binding and distinguish chromodomains from related Tudor “Royal” family members. In this review, we discuss and classify various chromodomains according to their context, structure and the mechanism of target recognition. PMID:22023491

  9. Position-Effect Variegation, Heterochromatin Formation, and Gene Silencing in Drosophila

    PubMed Central

    Elgin, Sarah C.R.; Reuter, Gunter

    2013-01-01

    Position-effect variegation (PEV) results when a gene normally in euchromatin is juxtaposed with heterochromatin by rearrangement or transposition. When heterochromatin packaging spreads across the heterochromatin/euchromatin border, it causes transcriptional silencing in a stochastic pattern. PEV is intensely studied in Drosophila using the white gene. Screens for dominant mutations that suppress or enhance white variegation have identified many conserved epigenetic factors, including the histone H3 lysine 9 methyltransferase SU(VAR)3-9. Heterochromatin protein HP1a binds H3K9me2/3 and interacts with SU(VAR)3-9, creating a core memory system. Genetic, molecular, and biochemical analysis of PEV in Drosophila has contributed many key findings concerning establishment and maintenance of heterochromatin with concomitant gene silencing. PMID:23906716

  10. Silencing of Tuberin Enhances Photoreceptor Survival and Function in a Preclinical Model of Retinitis Pigmentosa (An American Ophthalmological Society Thesis)

    PubMed Central

    Tsang, Stephen H.; Chan, Lawrence; Tsai, Yi-Ting; Wu, Wen-Hsuan; Hsu, Chun-Wei; Yang, Jin; Tosi, Joaquin; Wert, Katherine J.; Davis, Richard J.; Mahajan, Vinit B.

    2014-01-01

    Purpose: To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis. Methods: In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6bH620Q/Pde6bH620Q at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6bH620Q/Pde6bH620Q rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin’s role in regulating survival. Results: At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration. Conclusions: Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP. PMID:25646031

  11. Surface coating of siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers: enhanced gene silencing and reduced adverse effects in vitro

    NASA Astrophysics Data System (ADS)

    Zeng, Xianghui; de Groot, Anne Marit; Sijts, Alice J. A. M.; Broere, Femke; Oude Blenke, Erik; Colombo, Stefano; van Eden, Willem; Franzyk, Henrik; Nielsen, Hanne Mørck; Foged, Camilla

    2015-11-01

    Cationic vectors have demonstrated the potential to facilitate intracellular delivery of therapeutic oligonucleotides. However, enhanced transfection efficiency is usually associated with adverse effects, which also proves to be a challenge for vectors based on cationic peptides. In this study a series of proteolytically stable palmitoylated α-peptide/β-peptoid peptidomimetics with a systematically varied number of repeating lysine and homoarginine residues was shown to self-assemble with small interfering RNA (siRNA). The resulting well-defined nanocomplexes were coated with anionic lipids giving rise to net anionic liposomes. These complexes and the corresponding liposomes were optimized towards efficient gene silencing and low adverse effects. The optimal anionic liposomes mediated a high silencing effect, which was comparable to that of the control (cationic Lipofectamine 2000), and did not display any noticeable cytotoxicity and immunogenicity in vitro. In contrast, the corresponding nanocomplexes mediated a reduced silencing effect with a more narrow safety window. The surface coating with anionic lipid bilayers led to partial decomplexation of the siRNA-peptidomimetic nanocomplex core of the liposomes, which facilitated siRNA release. Additionally, the optimal anionic liposomes showed efficient intracellular uptake and endosomal escape. Therefore, these findings suggest that a more efficacious and safe formulation can be achieved by surface coating of the siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers.Cationic vectors have demonstrated the potential to facilitate intracellular delivery of therapeutic oligonucleotides. However, enhanced transfection efficiency is usually associated with adverse effects, which also proves to be a challenge for vectors based on cationic peptides. In this study a series of proteolytically stable palmitoylated α-peptide/β-peptoid peptidomimetics with a systematically varied number of repeating lysine

  12. Epigenetics in Breast and Prostate Cancer

    PubMed Central

    Wu, Yanyuan; Sarkissyan, Marianna; Vadgama, Jaydutt V.

    2015-01-01

    SUMMARY Most recent investigations into cancer etiology have identified a key role played by epigenetics. Specifically, aberrant DNA and histone modifications which silence tumor suppressor genes or promote oncogenes have been demonstrated in multiple cancer models. While the role of epigenetics in several solid tumor cancers such as colorectal cancer are well established, there is emerging evidence that epigenetics also plays a critical role in breast and prostate cancer. In breast cancer, DNA methylation profiles have been linked to hormone receptor status and tumor progression. Similarly in prostate cancer, epigenetic patterns have been associated with androgen receptor status and response to therapy. The regulation of key receptor pathways and activities which affect clinical therapy treatment options by epigenetics renders this field high priority for elucidating mechanisms and potential targets. A new set of methylation arrays are now available to screen epigenetic changes and provide the cuttingedge tools needed to perform such investigations. The role of nutritional interventions affecting epigenetic changes particularly holds promise. Ultimately, determining the causes and outcomes from epigenetic changes will inform translational applications for utilization as biomarkers for risk and prognosis as well as candidates for therapy. PMID:25421674

  13. The Use of Mouse Models to Study Epigenetics

    PubMed Central

    Blewitt, Marnie; Whitelaw, Emma

    2013-01-01

    Much of what we know about the role of epigenetics in the determination of phenotype has come from studies of inbred mice. Some unusual expression patterns arising from endogenous and transgenic murine alleles, such as the Agouti coat color alleles, have allowed the study of variegation, variable expressivity, transgenerational epigenetic inheritance, parent-of-origin effects, and position effects. These phenomena have taught us much about gene silencing and the probabilistic nature of epigenetic processes. Based on some of these alleles, large-scale mutagenesis screens have broadened our knowledge of epigenetic control by identifying and characterizing novel genes involved in these processes. PMID:24186070

  14. TLR agonist–Stat3 siRNA conjugates: cell-specific gene silencing and enhanced antitumor immune responses

    PubMed Central

    Kortylewski, Marcin; Swiderski, Piotr; Herrmann, Andreas; Wang, Lin; Kowolik, Claudia; Kujawski, Maciej; Lee, Heehyoung; Scuto, Anna; Liu, Yong; Yang, Chunmei; Deng, Jiehui; Soifer, Harris S.; Raubitschek, Andrew; Forman, Stephen; Rossi, John J.; Pardoll, Drew M.; Jove, Richard; Yu, Hua

    2010-01-01

    Efficient delivery of siRNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We describe a novel siRNA-based approach – synthetically linking siRNA to an oligonucleotide TLR9 agonist – that targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. Because Stat3 in tumor-associated immune cells suppresses antitumor immune responses and hinders TLR9-induced immune stimulation, we tested CpG-Stat3siRNA conjugates for anti-tumor effects. When injected locally at the tumor site or systemically through an intravenous route, the CpG-Stat3siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of tumor-associated immune cells, and ultimately potent anti-tumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment. PMID:19749770

  15. Epigenetics and Autism

    PubMed Central

    Millis, Richard M.

    2013-01-01

    This review identifies mechanisms for altering DNA-histone interactions of cell chromatin to upregulate or downregulate gene expression that could serve as epigenetic targets for therapeutic interventions in autism. DNA methyltransferases (DNMTs) can phosphorylate histone H3 at T6. Aided by protein kinase Cβ1, the DNMT lysine-specific demethylase-1 prevents demethylation of H3 at K4. During androgen-receptor-(AR-) dependent gene activation, this sequence may produce AR-dependent gene overactivation which may partly explain the male predominance of autism. AR-dependent gene overactivation in conjunction with a DNMT mechanism for methylating oxytocin receptors could produce high arousal inputs to the amygdala resulting in aberrant socialization, a prime characteristic of autism. Dysregulation of histone methyltransferases and histone deacetylases (HDACs) associated with low activity of methyl CpG binding protein-2 at cytosine-guanine sites in genes may reduce the capacity for condensing chromatin and silencing genes in frontal cortex, a site characterized by decreased cortical interconnectivity in autistic subjects. HDAC1 inhibition can overactivate mRNA transcription, a putative mechanism for the increased number of cerebral cortical columns and local frontal cortex hyperactivity in autistic individuals. These epigenetic mechanisms underlying male predominance, aberrant social interaction, and low functioning frontal cortex may be novel targets for autism prevention and treatment strategies. PMID:24151554

  16. Silencing NFBD1/MDC1 enhances the radiosensitivity of human nasopharyngeal cancer CNE1 cells and results in tumor growth inhibition

    PubMed Central

    Wang, Z; Zeng, Q; Chen, T; Liao, K; Bu, Y; Hong, S; Hu, G

    2015-01-01

    NFBD1 functions in cell cycle checkpoint activation and DNA repair following ionizing radiation (IR). In this study, we defined the NFBD1 as a tractable molecular target to radiosensitize nasopharyngeal carcinoma (NPC) cells. Silencing NFBD1 using lentivirus-mediated shRNA-sensitized NPC cells to radiation in a dose-dependent manner, increasing apoptotic cell death, decreasing clonogenic survival and delaying DNA damage repair. Furthermore, downregulation of NFBD1 inhibited the amplification of the IR-induced DNA damage signal, and failed to accumulate and retain DNA damage-response proteins at the DNA damage sites, which leaded to defective checkpoint activation following DNA damage. We also implicated the involvement of NFBD1 in IR-induced Rad51 and DNA-dependent protein kinase catalytic subunit foci formation. Xenografts models in nude mice showed that silencing NFBD1 significantly enhanced the antitumor activity of IR, leading to tumor growth inhibition of the combination therapy. Our studies suggested that a combination of gene therapy and radiation therapy may be an effective strategy for human NPC treatment. PMID:26247734

  17. Silencing NFBD1/MDC1 enhances the radiosensitivity of human nasopharyngeal cancer CNE1 cells and results in tumor growth inhibition.

    PubMed

    Wang, Z; Zeng, Q; Chen, T; Liao, K; Bu, Y; Hong, S; Hu, G

    2015-01-01

    NFBD1 functions in cell cycle checkpoint activation and DNA repair following ionizing radiation (IR). In this study, we defined the NFBD1 as a tractable molecular target to radiosensitize nasopharyngeal carcinoma (NPC) cells. Silencing NFBD1 using lentivirus-mediated shRNA-sensitized NPC cells to radiation in a dose-dependent manner, increasing apoptotic cell death, decreasing clonogenic survival and delaying DNA damage repair. Furthermore, downregulation of NFBD1 inhibited the amplification of the IR-induced DNA damage signal, and failed to accumulate and retain DNA damage-response proteins at the DNA damage sites, which leaded to defective checkpoint activation following DNA damage. We also implicated the involvement of NFBD1 in IR-induced Rad51 and DNA-dependent protein kinase catalytic subunit foci formation. Xenografts models in nude mice showed that silencing NFBD1 significantly enhanced the antitumor activity of IR, leading to tumor growth inhibition of the combination therapy. Our studies suggested that a combination of gene therapy and radiation therapy may be an effective strategy for human NPC treatment. PMID:26247734

  18. Arabidopsis transcriptional repressor VAL1 triggers Polycomb silencing at FLC during vernalization.

    PubMed

    Qüesta, Julia I; Song, Jie; Geraldo, Nuno; An, Hailong; Dean, Caroline

    2016-07-29

    The determinants that specify the genomic targets of Polycomb silencing complexes are still unclear. Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC) accelerates flowering and involves a cold-dependent epigenetic switch. Here we identify a single point mutation at an intragenic nucleation site within FLC that prevents this epigenetic switch from taking place. The mutation blocks nucleation of plant homeodomain-Polycomb repressive complex 2 (PHD-PRC2) and indicates a role for the transcriptional repressor VAL1 in the silencing mechanism. VAL1 localizes to the nucleation region in vivo, promoting histone deacetylation and FLC transcriptional silencing, and interacts with components of the conserved apoptosis- and splicing-associated protein (ASAP) complex. Sequence-specific targeting of transcriptional repressors thus recruits the machinery for PHD-PRC2 nucleation and epigenetic silencing. PMID:27471304

  19. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    PubMed

    Zhang, Yu-Qin; Xiao, Chuan-Xing; Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy. PMID:23874836

  20. Silencing of Pokemon Enhances Caspase-Dependent Apoptosis via Fas- and Mitochondria-Mediated Pathways in Hepatocellular Carcinoma Cells

    PubMed Central

    Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy. PMID:23874836

  1. Practising Silence in Teaching

    ERIC Educational Resources Information Center

    Forrest, Michelle

    2013-01-01

    The concept "silence" has diametrically opposed meanings; it connotes peace and contemplation as well as death and oblivion. Silence can also be considered a practice. There is keeping the rule of silence to still the mind and find inner truth, as well as forcibly silencing in the sense of subjugating another to one's own purposes.…

  2. Long noncoding RNA ANRIL indicates a poor prognosis of gastric cancer and promotes tumor growth by epigenetically silencing of miR-99a/miR-449a.

    PubMed

    Zhang, Er-bao; Kong, Rong; Yin, Dan-dan; You, Liang-hui; Sun, Ming; Han, Liang; Xu, Tong-peng; Xia, Rui; Yang, Jin-song; De, Wei; Chen, Jin fei

    2014-04-30

    Long noncoding RNAs are involved in diseases including cancer. Here, we reported that ANRIL (CDKN2B-AS1), a 3.8-kb long noncoding RNA, recruiting and binding to PRC2, was generally upregulated in human gastric cancer (GC) tissues. In a cohort of 120 GC patients, the higher expression of ANRIL was significantly correlated with a higher TNM stage (P=0.041) and tumor size (P=0.001). Multivariate analyses revealed that ANRIL expression served as an independent predictor for overall survival (P=0.036). Further experiments revealed that ANRIL knockdown significantly repressed the proliferation both in vitro and in vivo. We also showed that E2F1 could induce ANRIL and ANRIL-mediated growth promotion is in part due to epigenetic repression of miR-99a/miR-449a in Trans (controlling the targets--mTOR and CDK6/E2F1 pathway) by binding to PRC2, thus forming a positive feedback loop, continuing to promote GC cell proliferation. To our knowledge, this is the first report showed that the role of ANRIL in the progression of GC and ANRIL could crosstalk with microRNAs in epigenetic level. Our results suggest that ANRIL, as a growth regulator, may serve as a candidate prognostic biomarker and target for new therapies in human gastric cancer. PMID:24810364

  3. Long noncoding RNA ANRIL indicates a poor prognosis of gastric cancer and promotes tumor growth by epigenetically silencing of miR-99a/miR-449a

    PubMed Central

    You, Liang-hui; Sun, Ming; Han, Liang; Xu, Tong-peng; Xia, Rui; Yang, Jin-song; Chen, Jin fei

    2014-01-01

    Long noncoding RNAs are involved in diseases including cancer. Here, we reported that ANRIL (CDKN2B-AS1), a 3.8-kb long noncoding RNA, recruiting and binding to PRC2, was generally upregulated in human gastric cancer (GC) tissues. In a cohort of 120 GC patients, the higher expression of ANRIL was significantly correlated with a higher TNM stage (P=0.041) and tumor size (P=0.001). Multivariate analyses revealed that ANRIL expression served as an independent predictor for overall survival (P=0.036). Further experiments revealed that ANRIL knockdown significantly repressed the proliferation both in vitro and in vivo. We also showed that E2F1 could induce ANRIL and ANRIL-mediated growth promotion is in part due to epigenetic repression of miR-99a/miR-449a in Trans (controlling the targets—mTOR and CDK6/E2F1 pathway) by binding to PRC2, thus forming a positive feedback loop, continuing to promote GC cell proliferation. To our knowledge, this is the first report showed that the role of ANRIL in the progression of GC and ANRIL could crosstalk with microRNAs in epigenetic level. Our results suggest that ANRIL, as a growth regulator, may serve as a candidate prognostic biomarker and target for new therapies in human gastric cancer. PMID:24810364

  4. MAGE-C2-Specific TCRs Combined with Epigenetic Drug-Enhanced Antigenicity Yield Robust and Tumor-Selective T Cell Responses.

    PubMed

    Kunert, Andre; van Brakel, Mandy; van Steenbergen-Langeveld, Sabine; da Silva, Marvin; Coulie, Pierre G; Lamers, Cor; Sleijfer, Stefan; Debets, Reno

    2016-09-15

    Adoptive T cell therapy has shown significant clinical success for patients with advanced melanoma and other tumors. Further development of T cell therapy requires improved strategies to select effective, yet nonself-reactive, TCRs. In this study, we isolated 10 TCR sequences against four MAGE-C2 (MC2) epitopes from melanoma patients who showed clinical responses following vaccination that were accompanied by significant frequencies of anti-MC2 CD8 T cells in blood and tumor without apparent side effects. We introduced these TCRs into T cells, pretreated tumor cells of different histological origins with the epigenetic drugs azacytidine and valproate, and tested tumor and self-reactivities of these TCRs. Pretreatment of tumor cells upregulated MC2 gene expression and enhanced recognition by T cells. In contrast, a panel of normal cell types did not express MC2 mRNA, and similar pretreatment did not result in recognition by MC2-directed T cells. Interestingly, the expression levels of MC2, but not those of CD80, CD86, or programmed death-ligand 1 or 2, correlated with T cell responsiveness. One of the tested TCRs consistently recognized pretreated MC2(+) cell lines from melanoma, head and neck, bladder, and triple-negative breast cancers but showed no response to MHC-eluted peptides or peptides highly similar to MC2. We conclude that targeting MC2 Ag, combined with epigenetic drug-enhanced antigenicity, allows for significant and tumor-selective T cell responses. PMID:27489285

  5. Epigenetics and inheritance of phenotype variation in livestock.

    PubMed

    Triantaphyllopoulos, Kostas A; Ikonomopoulos, Ioannis; Bannister, Andrew J

    2016-01-01

    Epigenetic inheritance plays a crucial role in many biological processes, such as gene expression in early embryo development, imprinting and the silencing of transposons. It has recently been established that epigenetic effects can be inherited from one generation to the next. Here, we review examples of epigenetic mechanisms governing animal phenotype and behaviour, and we discuss the importance of these findings in respect to animal studies, and livestock in general. Epigenetic parameters orchestrating transgenerational effects, as well as heritable disorders, and the often-overlooked areas of livestock immunity and stress, are also discussed. We highlight the importance of nutrition and how it is linked to epigenetic alteration. Finally, we describe how our understanding of epigenetics is underpinning the latest cancer research and how this can be translated into directed efforts to improve animal health and welfare. PMID:27446239

  6. Enhancing glioblastoma cell sensitivity to chemotherapeutics: A strategy involving survivin gene silencing mediated by gemini surfactant-based complexes.

    PubMed

    Cruz, Rita Q; Morais, Catarina M; Cardoso, Ana M; Silva, Sandra G; Vale, Maria L; Marques, Eduardo F; Pedroso de Lima, Maria C; Jurado, Amália S

    2016-07-01

    Glioblastoma (GBM), the highest grade astrocytoma, is one of the most aggressive and challenging cancers to treat. The standard treatment is usually limited due to the intrinsic resistance of GBM to chemotherapy and drug non-specific effects. Therefore, new therapeutic strategies need to be developed to target tumor cells, sparing healthy tissues. In this context, the inhibitor-of-apoptosis protein (IAP) survivin emerges as an ideal target for a gene silencing approach, since it is sharply differentially expressed in cancer tissues. In this work, two different families of cationic gemini surfactants (bis-quat conventional and serine-derived) were tested regarding their efficiency to deliver small interfering RNAs (siRNAs) in a human GBM cell line (U87), in order to select an effective siRNA anti-survivin carrier. Importantly, survivin downregulation combined with administration of the chemotherapeutic agents temozolomide or etoposide resulted in a synergistic cytotoxic effect, thus revealing to be a promising strategy to reduce the chemotherapeutic doses for GBM treatment. PMID:27106606

  7. Arsenic silences hepatic PDK4 expression through activation of histone H3K9 methylatransferase G9a.

    PubMed

    Zhang, Xi; Wu, Jianguo; Choiniere, Jonathan; Yang, Zhihong; Huang, Yi; Bennett, Jason; Wang, Li

    2016-08-01

    It is well established that increased liver cancer incidence is strongly associated with epigenetic silencing of tumor suppressor genes; the latter is contributed by the environmental exposure to arsenic. Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates the TCA cycle. However, the epigenetic mechanisms mediated by arsenic to control PDK4 expression remain elusive. In the present study, we showed that histone methyltransferase G9a- and Suv39H-mediated histone H3 lysine 9 (H3K9) methylations contributed to PDK4 silencing in hepatic cells. The PDK4 expression was induced by G9a inhibitor BRD4770 (BRD) and Suv39H inhibitor Chaetocin (CHA). In contrast, arsenic exposure decreased PDK4 expression by inducing G9a and increasing H3K9 di- and tri-methylations levels (H3K9me2/3). In addition, arsenic exposure antagonizes the effect of BRD by enhancing the enrichment of H3K9me2/3 in the PKD4 promoter. Moreover, knockdown of G9a using siRNA induced PDK4 expression in HCC cells. Furthermore, arsenic decreased hepatic PDK4 expression as well as diminished the induction of PDK4 by BRD in mouse liver and hepatocytes. Overall, the results suggest that arsenic causes aberrant repressive histone modification to silence PDK4 in both HCC cells and in mouse liver. PMID:27217333

  8. Epigenetic memory: the Lamarckian brain

    PubMed Central

    Fischer, Andre

    2014-01-01

    Recent data support the view that epigenetic processes play a role in memory consolidation and help to transmit acquired memories even across generations in a Lamarckian manner. Drugs that target the epigenetic machinery were found to enhance memory function in rodents and ameliorate disease phenotypes in models for brain diseases such as Alzheimer's disease, Chorea Huntington, Depression or Schizophrenia. In this review, I will give an overview on the current knowledge of epigenetic processes in memory function and brain disease with a focus on Morbus Alzheimer as the most common neurodegenerative disease. I will address the question whether an epigenetic therapy could indeed be a suitable therapeutic avenue to treat brain diseases and discuss the necessary steps that should help to take neuroepigenetic research to the next level. PMID:24719207

  9. Dynamic Contrast Enhanced MRI Assessing the Antiangiogenic Effect of Silencing HIF-1α with Targeted Multifunctional ECO/siRNA Nanoparticles.

    PubMed

    Malamas, Anthony S; Jin, Erlei; Gujrati, Maneesh; Lu, Zheng-Rong

    2016-07-01

    Stabilization of hypoxia inducible factor 1α (HIF-1α), a biomarker of hypoxia, in hypoxic tumors mediates a variety of downstream genes promoting tumor angiogenesis and cancer cell survival as well as invasion, and compromising therapeutic outcome. In this study, dynamic contrast enhanced MRI (DCE-MRI) with a biodegradable macromolecular MRI contrast agent was used to noninvasively assess the antiangiogenic effect of RGD-targeted multifunctional lipid ECO/siHIF-1α nanoparticles in a mouse HT29 colon cancer model. The RGD-targeted ECO/siHIF-1α nanoparticles resulted in over 50% reduction in tumor size after intravenous injection at a dose of 2.0 mg of siRNA/kg every 3 days for 3 weeks compared to a saline control. DCE-MRI revealed significant decline in vascularity and over a 70% reduction in the tumor blood flow, permeability-surface area product, and plasma volume fraction vascular parameters in the tumor treated with the targeted ECO/siHIF-1α nanoparticles. The treatment with targeted ECO/siRNA nanoparticles resulted in significant silencing of HIF-1α expression at the protein level, which also significantly suppressed the expression of VEGF, Glut-1, HKII, PDK-1, LDHA, and CAIX, which are all important players in tumor angiogenesis, glycolytic metabolism, and pH regulation. By possessing the ability to elicit a multifaceted effect on tumor biology, silencing HIF-1α with RGD-targeted ECO/siHIF-1α nanoparticles has great promise as a single therapy or in combination with traditional chemotherapy or radiation strategies to improve cancer treatment. PMID:27264671

  10. Complete Genome Sequence of Bacillus megaterium Siphophage Silence

    PubMed Central

    Solis, Jonathan A.; Farmer, Nicholas G.; Cahill, Jesse L.; Rasche, Eric S.

    2015-01-01

    Silence is a newly isolated siphophage that infects Bacillus megaterium, a soil bacterium that is used readily in research and commercial applications. A study of B. megaterium phage Silence will enhance our knowledge of the diversity of Bacillus phages. Here, we describe the complete genome sequence and annotated features of Silence. PMID:26450722