5-azaC Treatment Research Articles

Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells

Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-β isoforms, TGF-β1 and TGF-β2, and their combination. TGF-β1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCR-based array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCR-based array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, −200b, −200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-β1-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.

The effects of the DNA Demethylating reagent, 5-azacytidine on SMCHD1 genomic localization

BackgroundDNA methylation is an epigenetic modification that mainly repress expression of genes essential during embryogenesis and development. There are key ATPase-dependent enzymes that read or write DNA methylation to remodel chromatin and regulate gene expression. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is an architectural protein that regulates expression of numerous genes, some of which are imprinted, that are sensitive to DNA methylation. In addition, SMCHD1 germline mutations lead to developmental diseases; facioscapulohumoral muscular dystrophy (FSHD), bosma arhinia and micropthalmia (BAMS). Current evidence suggests that SMCHD1 functions through maintenance or de novo DNA methylation required for chromatin compaction. However, it is unclear if DNA methylation is also essential for genomic recruitment of SMCHD1 and its role as an architectural protein. We previously isolated SMCHD1 using a methylated DNA region from mouse pituitary growth hormone (Gh1) promoter, suggesting that methylation is required for SMCHD1 DNA binding. The goal of this study was to further understand DNA methylation directed role of SMCHD1 in regulating gene expression. Therefore, we profiled SMCHD1 genome wide occupancy in human neuroblastoma SH-SY5Y cells and evaluated if DNA methylation is required for SMCHD1 genomic binding by treating cells with the DNA demethylating reagent, 5-azacytidine (5-azaC).ResultsOur data suggest that the majority of SMCHD1 binding occurs in intron and intergenic regions. Gene ontology analysis of genes associated with SMCHD1 genomic occupancy that is sensitive to 5-azaC treatment suggests SMCHD1 involvement in central nervous system development. The potassium voltage-gated channel subfamily Q member1 (KCNQ1) gene that associates with central nervous system is a known SMCHD1 target. We showed SMCHD1 binding to an intronic region of KCNQ1 that is lost following 5-azaC treatment suggesting DNA methylation facilitated binding of SMCHD1. Indeed, deletion of SMCHD1 by CRISPR- Cas9 increases KCNQ1 gene expression confirming its role in regulating KCNQ1 gene expression.ConclusionThese findings provide novel insights on DNA methylation directed function of SMCHD1 in regulating expression of genes associated with central nervous system development that impact future drug development strategies.

Application of 5-azacytidine induces DNA hypomethylation and accelerates dormancy release in buds of tree peony

Release of bud dormancy is a prerequisite for the growth resumption and production in perennial plants such as tree peony. DNA methylation plays a pivotal role in regulating gene expression. In this study, combination of morphologic observation and DNA methylation analysis indicated that 5-azacytidine (5-azaC) application for 7 d declined 5 mC quantities and promoted dormancy release. After 5-azaC treatment, total 174,341 unigenes and 1818 differentially expression genes (DEGs) were obtained by RNA-seq, of which there were 1194 DEGs after 1 d 5-azaC treatment (AD1 vs CD1), and 624 DEGs after 7 d (AD7 vs CD7), respectively. The KEGG pathway analysis identified that totally 10 DEGs annotated in DNA replication pathway were enriched when AD7 compared with CD7. Furthermore, the expression patterns of several DEGs by real-time quantitative RT-PCR were consistent with that of RNA-seq data. 5-azaC application significantly decreased the expression levels of DNA methyltransferase genes, PsCMT3, PsMET1 and PsDRM2, and increased the transcript of demethylase gene PsROS1. Simultaneously, total methyltransferases activity decreased, and demethylase activity was induced by 5-azaC. In summary, application of 5-azaC inhibited the expression of the genes related to growth and development in short-term, indicating a possible toxic effect to plant, and its long-term effect was to induce hypomethylation by increasing demethylase genes transcripts and decreasing the expressions of methyltransferase genes, and then activate cell cycle, DNA replication and glycol-metabolism processes, which subsequently accelerated dormancy release. All these would provide a new strategy to further understand the molecular mechanism of dormancy release in tree peony.

Influence of demethylation on regulatory T and Th17 cells in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) represents a heterogeneous hematopoietic disorder in which mature blood cells are derived from an abnormal multipotent progenitor cell. The current therapy for MDS involves repeated cycles of DNA methyltransferase (DNMT) inhibitors, particularly the demethylation drug 5-azacytidine (5-azaC) which has been shown to increase the survival of patients with high-risk MDS. The mechanisms behind the therapeutic effects of 5-azaC are not yet clear. In this study the effect of 5-azaC on the development of regulatory T cells (Tregs) and T-helper 17 (Th17) cells was investigated. The numbers of CD4+ T-cell subsets in 30 patients with intermediate-2/high-risk MDS were serially assessed at diagnosis and following 5-azaC treatment. The number of FoxP3+ Tregs was significantly higher after 3 months of therapy. However, there was no statistical difference in the number of Th17 cells following treatment. In vitro, 5-azaC enhanced the overall proportion of Tregs, but not Th17, in CD4+ T cells from patients with MDS. Addition of 5-azaC reduced the proliferative capacity of Tregs, suggesting that the increase in Tregs was due to conversion of conventional CD25− cells, rather than proliferation of CD25+FoxP3+ cells. The FoxP3 expression in 5-azaC-treated T effectors was also increased. Interestingly, while Tbet and RORγT mRNA transcription had no obvious changes, due to the demethylation of the FoxP3 promoter, these findings are important in associating the induction of DNA hypomethylation and the clinical response to 5-azaC.

Transcriptome profiling of \u2018Kyoho\u2019 grape at different stages of berry development following 5-azaC treatment

Background5-Azacytidine (5-azaC) promotes the development of ‘Kyoho’ grape berry but the associated changes in gene expression have not been reported. In this study, we performed transcriptome analysis of grape berry at five developmental stages after 5-azaC treatment to elucidate the gene expression networks controlling berry ripening.ResultsThe expression patterns of most genes across the time series were similar between the 5-azaC treatment and control groups. The number of differentially expressed genes (DEGs) at a given developmental stage ranged from 9 (A3_C3) to 690 (A5_C5). The results indicated that 5-azaC treatment had not very great influences on the expressions of most genes. Functional annotation of the DEGs revealed that they were mainly related to fruit softening, photosynthesis, protein phosphorylation, and heat stress. Eight modules showed high correlation with specific developmental stages and hub genes such as PEROXIDASE 4, CAFFEIC ACID 3-O-METHYLTRANSFERASE 1, and HISTONE-LYSINE N-METHYLTRANSFERASE EZA1 were identified by weighted gene correlation network analysis.Conclusions5-AzaC treatment alters the transcriptional profile of grape berry at different stages of development, which may involve changes in DNA methylation.

Transgenerational activation of an autonomous DNA transposon, Dart1-24, by 5-azaC treatment in rice.

Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.

EPIGENETIC DYSREGULATION OF CEBPE ENHANCER FUNCTION IN MLL-REARRANGED LEUKEMIA

Mixed lineage leukemia (MLL)-rearranged leukemia is characterized by the presence of MLL fusion proteins that mediate differentiation blockage through aberrant epigenetic regulation. CCAAT enhancer binding proteins (CEBPs) are key transcription factors involved in myeloid lineage commitment and maturation, among which CEBPE is crucial in terminal differentiation of granulocytes/macrophages. Here we report that CEBPE expression is significantly lower in human MLL acute myeloid leukemia (AML) cell lines (HL60, NB4, U937 and Kasumi-1), when compared to the non-MLL AML cell lines (ML-2, MV4:11, Monomac-6 and THP-1). Importantly, TCGA dataset shows that low CEBPE expression is significantly associated with poor survival of AML patients. We further identified the putative CEBPE enhancer and validated its interaction with promoter by chromatin conformation capture (3C) assay. Strikingly, DNA hypermethylation is observed specially at the CEBPE enhancer in MLL AML cells. We further demonstrated an induction of CEBPE and myeloid marker CD11b in MLL AML cells treated with DNA methylation inhibitor 5’-aza-cytidine (5’-aza-C), suggesting the crucial epigenetic regulatory role of CEBPE enhancer. While all-trans retinoid acid failed to trigger cell differentiation in MLL AML cells, co-treatment with 5’-aza-C resulted in strong induction of CEBPE with terminal differentiated cell morphology, compared to 5’-aza-C treatment per se. It therefore suggests that de-repression of CEBPE by removal of enhancer DNA methylation may prone MLL AML cells to undergo differentiation upon chemical/pharmaceutical induction. Collectively, our work showed that aberrant DNA methylation mediated by MLL fusion proteins represses CEBPE expression through altering its enhancer activity, which subsequently leads to cell differentiation blockage in MLL AML cells.

Morphological variation of Chrysanthemum lavandulifolium induced by 5-azaC treatment

To explore the possible role of DNA methylation in plant development. Different concentrations of the DNA methyltransferase inhibitor 5-azacytidine were used on different components of Chrysanthemum lavandulifolium (stem and hypocotyl). The effects of 5-azaC on plantlet growth and flower development were investigated. Results showed that low concentrations of 5-azaC (20 μmol L−1–100 μmol L−1) inhibited plantlet growth (from the stem segments), and high concentrations of 5-azaC (150 μmol L−1–200 μmol L−1) promoted plantlet growth. This inhibition is similar in the regeneration of hypocotyls, except that the concentration range of 5-azaC is different. Low concentration of 5-azaC (10 μmol L−1–20 μmol L−1) inhibit generation or growth of callus-generated plantlets, high concentration of 5-azaC (150 μmol L−1–200 μmol L−1) promoted the growth of hypocotyl regeneration seedlings. In thus, the inhibitory effect of plantlet was similar in hypocotyl regeneration. Therefore, different explants have different sensitivities to methyltransferase inhibitor (5-azaC) treatment. Progeny plants treated with stems of C. lavandulifolium under the short days condition, the control plants (about 15 leaves) did not bloom, plants undergoing high concentration treatments flowered in some strains (flowering rate 80% in treatment 6), and the pitch of all unflowering plants was dwarfed. It seems that 5-azaC can break the C. lavandulifolium adolescent period limit, potentially by inducing the photoperiodic in advance or through another mechanism, after 5-azaC processing may alters key gene methylation, resulting in flowering. In the hypocotyl-treated progeny after multiple passages, all treatments had flowering and non-flowering lines under short-day conditions, and the flowering rate was between 39.29% and 78.43%. We also obtained a continuous flowering line was obtained (2-8-2), and this trait exhibited stable by asexual reproduction. In addition, abundant variation in leaf morphology, flower shape and stem color variation were observed in hypocotyl regeneration plants. All the evidence suggests that the methyltransferase inhibitor (5-azaC) may affect the DNA methylation level of many key genes regulating plant growth and development. This study provides phenotypic data and materials for further exploring the role of DNA methylation in the growth and flower induction of C. lavandulifolium.

5-Azacytidine promotes shoot regeneration during Agrobacterium-mediated soybean transformation

Agrobacterium-mediated soybean transformation has been greatly improved in recent years, however the transformation efficiency is still low and highly genotype-dependent when compared to other species. Here, we characterized seventeen soybean genotypes based on their genetic transformation efficiencies, i.e., high and low, during Agrobacterium-mediated transformation. To reveal the molecular basis of this transformation difference, we constructed a highly efficient transient transgene expression system using soybean cotyledon protoplasts and then assess the methylation levels of promoter and coding regions of an EYFP (enhanced yellow fluorescent protein) gene introduced into the protoplast cultures of various soybean genotypes using BSP (bisulfite sequencing PCR). Increased methylation was found to be associated with the considerably decreased transfection efficiency (as percentage of EYFP fluorescent protoplasts) in low-efficacy genotypes as compared with those in high-efficacy on three DAT (day after transfection). 5-Azacytidine (5-Azac), a demethylating reagent commonly applied in epigenetic researches, significantly improved the transient transfection efficiency and transgene expression level in low-efficiency genotypes. Furthermore, the shoot regeneration efficiency in low-efficiency genotypes was substantially increased by 5-Azac treatment in an Agrobacterium-mediated soybean transformation system. Taken together, we concluded that lower methylation level in transgene contributed to enhanced shoot regeneration in Agrobacterium-mediated soybean transformation.

Triterpenoid content and expression of triterpenoid biosynthetic genes in birch (Betula platyphylla Suk) treated with 5-azacytidine

DNA methylation is widespread in plants and associated with plant development and defense mechanisms. However, the relationship between DNA methylation and plant secondary metabolism has rarely been reported. Here, when birch suspension cells were treated with 5-azacytidine (5-azaC), which blocks DNA methylation, triterpenoid accumulation was significantly promoted and antioxidant and defense enzymatic activity changed. For studying triterpenoid accumulation, 0.1 mM azaC was optimal. A qRT-PCR assay revealed increased expression of genes encoding key triterpenoid biosynthetic enzymes. Evaluation of methylation polymorphisms at CCGG sites showed that the methylation level was lower in cells treated with 5-azaC. These results demonstrated that 5-azaC treatment led to an increase in the production of triterpenoids in cell cultures through a mechanism that involved in DNA methylation, which resulted in the induction of genes encoding the key enzymes. The study provides evidence of a relationship between DNA methylation and regulation of secondary metabolism.

The Effects of DNA Methylation Inhibition on Flower Development in the Dioecious Plant Salix Viminalis

DNA methylation, an important epigenetic modification, regulates the expression of genes and is therefore involved in the transitions between floral developmental stages in flowering plants. To explore whether DNA methylation plays different roles in the floral development of individual male and female dioecious plants, we injected 5-azacytidine (5-azaC), a DNA methylation inhibitor, into the trunks of female and male basket willow (Salix viminalis L.) trees before flower bud initiation. As expected, 5-azaC decreased the level of DNA methylation in the leaves of both male and female trees during floral development; however, it increased DNA methylation in the leaves of male trees at the flower transition stage. Furthermore, 5-azaC increased the number, length and diameter of flower buds in the female trees but decreased these parameters in the male trees. The 5-azaC treatment also decreased the contents of soluble sugars, starch and reducing sugars in the leaves of the female plants, while increasing them in the male plants at the flower transition stage; however, this situation was largely reversed at the flower development stage. In addition, 5-azaC treatment decreased the contents of auxin indoleacetic acid (IAA) in both male and female trees at the flower transition stage. These results indicate that hypomethylation in leaves at the flower transition stage promotes the initiation of flowering and subsequent floral growth in Salix viminalis, suggesting that DNA methylation plays a similar role in vegetative–reproductive transition and early floral development. Furthermore, methylation changes during the vegetative–reproductive transition and floral development were closely associated with the biosynthesis, metabolism and transportation of carbohydrates and IAA. These results provide insight into the epigenetic regulation of carbohydrate accumulation.

The variation of berry development and DNA methylation after treatment with 5-azaC on ‘Kyoho’ grape

DNA methylation has been proved to function in fruit ripening. The different concentrations of azacitidine (5-azaC), an inhibitor of methyltransferase, were sprayed on ‘Kyoho’ grape berries to explore the changes of DNA methylation, the effect of 5-azaC on berry ripening, and the relationship between DNA methylation and reactive oxygen species (ROS) metabolism during the grape berry development. The results showed that the treatment of 100 μM 5-azaC could promote the berry ripening of ‘Kyoho’ 20 days earlier than the control. The assessment of methylation by methylation sensitive amplification polymorphism (MSAP) markers after the treatment of 100 μM 5-azaC, demonstrated that the methylation rate of the treatment was all lower than the control at any berry development periods. 5-azaC treatments influenced the ROS metabolic activities and related physiological indexes of berries. The sucrose and soluble solid content of 5-azaC-treated berries were significantly higher than control at 75 dpa. The change trend of H2O2 content of the control was similar with the 100 μM 5-azaC treatment. All treatments enhanced rates of superoxide anion (O2−) production. Superoxide dismutase (SOD) activity in treated berries were higher than the control and had significant difference with the control at 35 dpa. The results demonstrated that 5-azaC could accelerate grape berries ripening, and there was a connection between ROS metabolism and DNA methylation during the grape berry development.

5-Azacytidine treatment and TaPBF-D over-expression increases glutenin accumulation within the wheat grain by hypomethylating the Glu-1 promoters.

5-azaC treatment and TaPBF - D over-expression decrease C-methylation status of three Glu - 1 gene promoters, and aid in enhancing the expression of the Glu - 1 genes. The wheat glutenins exert a strong influence over dough elasticity, but the regulation of their encoding genes has not been firmly established. Following treatment with 5-azacytidine (5-azaC), both the weight and glutenin content of the developing and mature grains were significantly increased. The abundance of transcript produced by the Glu-1 genes (encoding high-molecular-weight glutenin subunits), as well as those encoding demethylases and transcriptional factors associated with prolamin synthesis was higher than in grain of non-treated plants. These grains also contained an enhanced content of the prolamin box binding factor (PBF) protein. Bisulfite sequencing indicated that the Glu-1 promoters were less strongly C-methylated in the developing grain than in the flag leaf, while in the developing grain of 5-azaC treated plants, the C-methylation level was lower than in equivalent grains of non-treated plants. Both Glu-1 transcript abundance and glutenin content were higher in the grain set by three independent over-expressors of the D genome homoeolog of TaPBF than in the grain set by wild type plants. When assessed 10days after flowering, the Glu-1 promoters' methylation level was lower in the developing grains set by the TaPBF-D over-expressor than in the wild type control. An electrophoretic mobility shift assay showed that PBF-D was able to bind in vitro to the P-box of Glu-1By8 and -1Dx2, while a ChIP-qPCR analysis revealed that a lower level of C-methylation in the Glu-1By8 and -1Dx2 promoters improved the TaPBF binding. We suggest that promoter DNA C-methylation is a key determinant of Glu-1 transcription.

Tissue-Specific Response to Experimental Demethylation at Seed Germination in the Non-Model Herb Erodium cicutarium

Experimental alteration of DNA methylation is a suitable tool to infer the relationship between phenotypic and epigenetic variation in plants. A detailed analysis of the genome-wide effect of demethylating agents, such as 5-azacytidine (5azaC), and zebularine is only available for the model species Arabidopsis thaliana, which suggests that 5azaC may have a slightly larger effect. In this study, global methylation estimates obtained by high-performance liquid chromatography (HPLC) analyses were conducted to investigate the impact of 5azaC treatment on leaf and root tissue in Erodium cicutarium (Geraniaceae), which is an annual herb native to Mediterranean Europe that is currently naturalized in all continents, sometimes becoming invasive. We used seeds collected from two natural populations in SE Spain. Root tissue of the second generation (F2) greenhouse-grown seedlings had a significantly lower global cytosine methylation content than leaf tissue (13.0 vs. 17.7% of all cytosines). Leaf tissue consistently decreased methylation after treatment, but the response of root tissue varied according to seed provenance, suggesting that genetic background can mediate the response to experimental demethylation. We also found that both leaf number and leaf length were reduced in treated seedlings supporting a consistent phenotypic effect of the treatment regardless of seedling provenance. These findings suggest that, although the consequences of experimental demethylation may be tissue- and background-specific, this method is effective in altering early seedling development, and can thus be useful in ecological epigenetic studies that are aiming to investigate the links between epigenetic and phenotypic variation in non-model plant species.

Dynamic changes in methylome and transcriptome patterns in response to methyltransferase inhibitor 5-azacytidine treatment in citrus.

DNA methylation is known to play an important role in various developmental processes in plants. However, there is a general lack of understanding about the possible functions of DNA methylation in fruit trees. Using callus as a model, methylome, transcriptome and metabolite changes were assessed after treatment with the DNA methyltransferase inhibitor 5-azacytidine (5azaC). Genome-wide methylome analysis revealed the demethylation of a diverse of genes, including many genes encoding transcription factors (TFs), genes involved in biological processes, and the up-regulation of a wide range of transposable elements (TEs). Combined with the RNA-seq data, we observed no obvious genome-wide correlation between the changes in methylation status and expression levels. Furthermore, 5azaC treatment induced carotenoid degradation along with strong activation of carotenoid cleavage dioxygenases 1 (CpCCD1). Functional complementation analysis in bacterial system showed that CpCCD1 exhibited strong catalytic activities toward zeaxanthin, β-carotene and lycopene. In summary, 5azaC treatments induced carotenoid degradation by CpCCD1 activation and led to a genome-wide demethylation effect.

The Biomphalaria glabrata DNA methylation machinery displays spatial tissue expression, is differentially active in distinct snail populations and is modulated by interactions with Schistosoma mansoni.

BackgroundThe debilitating human disease schistosomiasis is caused by infection with schistosome parasites that maintain a complex lifecycle alternating between definitive (human) and intermediate (snail) hosts. While much is known about how the definitive host responds to schistosome infection, there is comparably less information available describing the snail’s response to infection.Methodology/Principle findingsHere, using information recently revealed by sequencing of the Biomphalaria glabrata intermediate host genome, we provide evidence that the predicted core snail DNA methylation machinery components are associated with both intra-species reproduction processes and inter-species interactions. Firstly, methyl-CpG binding domain protein (Bgmbd2/3) and DNA methyltransferase 1 (Bgdnmt1) genes are transcriptionally enriched in gonadal compared to somatic tissues with 5-azacytidine (5-AzaC) treatment significantly inhibiting oviposition. Secondly, elevated levels of 5-methyl cytosine (5mC), DNA methyltransferase activity and 5mC binding in pigmented hybrid- compared to inbred (NMRI)- B. glabrata populations indicate a role for the snail’s DNA methylation machinery in maintaining hybrid vigour or heterosis. Thirdly, locus-specific detection of 5mC by bisulfite (BS)-PCR revealed 5mC within an exonic region of a housekeeping protein-coding gene (Bg14-3-3), supporting previous in silico predictions and whole genome BS-Seq analysis of this species’ genome. Finally, we provide preliminary evidence for parasite-mediated host epigenetic reprogramming in the schistosome/snail system, as demonstrated by the increase in Bgdnmt1 and Bgmbd2/3 transcript abundance following Bge (B. glabrata embryonic cell line) exposure to parasite larval transformation products (LTP).Conclusions/SignificanceThe presence of a functional DNA methylation machinery in B. glabrata as well as the modulation of these gene products in response to schistosome products, suggests a vital role for DNA methylation during snail development/oviposition and parasite interactions. Further deciphering the role of this epigenetic process during Biomphalaria/Schistosoma co-evolutionary biology may reveal key factors associated with disease transmission and, moreover, enable the discovery of novel lifecycle intervention strategies.

Demethylation of miR-495 inhibits cell proliferation, migration and promotes apoptosis by targeting STAT-3 in breast cancer.

In breast cancer (BC), silencing of miRNA genes due to miRNA gene promoter methylation are the important mechanisms directly contributing to tumorigenesis and tumor progression. miRNA-495 (miR-495) has been reported to be a tumor suppressor gene in various cancers, but its role and regulation in BC remains unclear. In the present study, the level of miR-495 was inversely correlated with the expression of STAT-3 in BC tissues and cell lines. miR-495 can directly target 3'-UTR of STAT-3 mRNA and thereby decrease the expression of STAT-3 in MCF-7 and HCC1973 cells by Targetscan and Dual-luciferase assay. We further analyzed miR-495 promoter methylation by sodium bisulfite sequencing method (BSP), and found DNA methyltransferase inhibitor, 5-AzaC concomitantly upregulated expression of miR-495 and downregulated its target gene STAT-3 and its downstream target VEGF. Furthermore, we further observed that 5-AzaC treatment, miR-495 mimics and STAT-3 knockdown significantly inhibited cell function in breast cancer by Transwell assay, EdU flow cytometry, AnnexinV-FITC/PI combined with flow cytometry and Hoechst staining. Taken together, our data are first to demonstrate that the miR-495 is silenced due to promoter methylation in breast cancer. DNA methyltransferase inhibitor 5-AzaC could reverse miR‑495 (suppressor gene) and STAT-3 (oncogene). The anticancer properties of 5-AzaC were preliminarily confirmed in breast cancer.

Inhibin-α gene mutations and mRNA levels in human lymphoid and myeloid leukemia cells.

The inhibin-α gene was identified as a tumor suppressor gene in the gonads and adrenal glands by functional studies using knockout mice. Methylation of CpG sites within the regulatory regions of tumor suppressor gene is frequently associated with their transcriptional silencing. We investigated epigenetic modifications, changes in loss of heterozygosity (LOH), and mutation of the inhibin-α gene, and regulation of transcriptional expression in response to inhibitors of DNA methylation (5-aza-2'-deoxycytidine, 5-AzaC) in human lymphoid (Jurkat, Molt-4, Raji, and IM-9) and myeloid (HL-60, Kasumi-1, and K562) leukemia cells. The inhibin-α promoter was hypermethylated in lymphoid (Molt-4 and Raji) and myeloid (HL-60 and Kasumi-1) leukemia cells. Inhibin-α gene mutations differed significantly between lymphoid (heterozygote) and myeloid (homozygote) leukemia cells. LOH in the inhibin-α gene was detected in lymphoid and myeloid leukemia cells, with the exception of Jurkat cells. Treatment with 5-AzaC, a demethylating agent, resulted in increased inhibin-α mRNA and protein levels in most of the cell lines. Also, 5-AzaC treatment inhibited cell proliferation and induced apoptosis. Taken together, our results reveal that the inhibin-α gene is transcriptionally silenced in human leukemia cells and that reactivation is suppressed by a demethylating agent. In addition, mutations in, and expression levels of, the inhibin-α gene differed between human lymphoid and myeloid leukemia cells.

Abstract A55: Human endogenous retroviruses differentially regulate immune checkpoints and modulation of immune responses in serous ovarian carcinoma.

Abstract Purpose: The first aim is to investigate the activation of 7 immune checkpoints at different time points following transfection of three diverse ERV envelope (env) genes in serous ovarian carcinoma cells. The second aim is to determine if the ERV-W1 Syncytin-1 env and ERV-3 protein are expressed in primary serous OvCa tumors and ascites fluids to explore a possible role in the tumor microenvironment. Introduction: The overall survival of serous OvCa has not greatly improved. We and others have demonstrated up-regulation of interferon (IFN) type I signaling pathway viral defense genes including IFN stimulated genes (ISGs), in a variety of human tumors (ovary, breast, lung) but also in ovarian and colon carcinoma cell lines treated with the DNA methyltransferase inhibitor Aza-C. Aza-C treatment of serous OvCa cell lines leads to an induction of PD-L1 expression as mediated downstream signaling via IFNg. We recently demonstrated that activated ERVs function at the RNA level to ignite the IFN response. Exogenous retroviruses like HIV have evolved to suppress the immune response by up-regulation of PD-L1 on target cells. Tumors mimic regulatory mechanisms involving immunosuppression and inflammation, which we propose involves ERVs. Experimental Procedures: ERV-3, Syncytin-1 and ERV-W2 env CMV overexpression vectors and a control EGFP-vector were transfected into OvCa serous cell lines (TykNu, A2780 and Hey) and RNA expression of 7 immune checkpoint molecules (LGALS9, PDL-2, CD80, CD276, PD-L1, VTCN1 and PD1) were monitored at days 3 and 7 post transfection using real time PCR. Immunohistochemistry (IHC) and Western analyses using antibodies for Syncytin-1 and ERV-3 were performed with primary serous OvCa tumors. Western and ELISA analyses of serous OvCa ascites were executed to detect Syncytin-1 protein levels. Syncytin-1 protein concentrations were quantified in ascites with two different antibodies using ELISA and correlated to a standard curve based on purified Syncytin-1 levels. Results: Three serous OvCa cell lines following ERV env transfection showed differences of immune checkpoint activation. TykNu demonstrated the highest fold-levels of induction for LGALS9 (73.1x), PD-L1 (15.2x), PD-L2 (9.8x) and PD1 (6.1x) at day 3 for all ERV expression vectors, which decreased by day 7 except for PD1. A2780 showed increased levels for LGALS9 (7.2x) and PD1 (5.2x) with all ERV expression vectors at day 3, which decreased by day 7. In contrast, Hey cells showed no significant increase of the analyzed immune checkpoint genes. Syncytin-1 and ERV-3 protein was highly expressed in primary OvCa epithelial cells using IHC, compared to patient matched control ovarian tissues. Western analyses of ascites from OvCa patients showed high levels of Syncytin-1 in the supernatant, supporting protein secretion. Absolute quantification using real time PCR of Syncytin-1 and ERV-3 RNA with fractionated OvCa ascites cells demonstrated high amounts of molecules. ELISA results confirmed high levels of Syncytin-1 (98.6 - 525.8 microg/ml) in ascites of primary OvCa and in one relapse patient (413.4 microg/ml), but were virtually undetectable in an ascites from a gastro-intestinal carcinoma and control peritoneal fluids (2.7 microg/ml). Conclusions: Our findings that various immune checkpoints are differentially regulated following ERV activation in serous OvCa cell lines endorses that analyses of immune checkpoints prior to treatment could help stratify patient therapy. ERVs like exogenous retroviruses not only play an essential functional role at the RNA level via TLR3, but also possibly at the protein level via TLR4 modulation of immune / inflammatory responses. Citation Format: Pamela L. Strissel, Katherine B. Chiappinelli, Johanna Strehl, Franziska MB Würfel, Matthias W. Beckmann, Stephen B. Baylin, Reiner Strick. Human endogenous retroviruses differentially regulate immune checkpoints and modulation of immune responses in serous ovarian carcinoma.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A55.