Unmethylated Genes Research Articles

Natural methylation epialleles correlate with gene expression in maize.

DNA methylation in plants is depleted from cis regulatory elements in and near genes but is present in some gene bodies, including exons. Methylation in exons solely in the CG context is called gene body methylation (gbM). Methylation in exons in both CG and non-CG contexts is called TE-like methylation (teM). Assigning functions to both forms of methylation in genes has proven to be challenging. Toward that end, we utilized recent genome assemblies, gene annotations, transcription data, and methylome data to quantify common patterns of gene methylation and their relations to gene expression in maize. We found that gbM genes exist in a continuum of CG methylation levels without a clear demarcation between unmethylated genes and gbM genes. Analysis of expression levels across diverse maize stocks and tissues revealed a weak but highly significant positive correlation between gbM and gene expression except in endosperm. gbM epialleles were associated with an approximately 3% increase in steady-state expression level relative to unmethylated epialleles. In contrast to gbM genes, which were conserved and were broadly expressed across tissues, we found that teM genes, which make up about 12% of genes, are mainly silent, are poorly conserved, and exhibit evidence of annotation errors. We used these data to flag teM genes in the 26 NAM founder genome assemblies. While some teM genes are likely functional, these data suggest that the majority are not, and their inclusion can confound interpretation of whole-genome studies.

Methylation specific enzyme-linked oligonucleotide assays (MS-ELONA) for ultrasensitive DNA methylation analysis

Methylation of the promoter region of cancer related genes plays a crucial role in the occurrence and development of cancer, and the degree of methylation has great potential for the early cancer diagnosis. At present, the technology used to quantify DNA methylation is mainly based on the DNA sequencing which are time-consuming and high-cost in the relating application. We have developed an ultrasensitive method of methylation specific enzyme-linked oligonucleotide assays (MS-ELONA) to detect and quantify the level of DNA methylation. We could detect as little as 2 pg of methylated DNA in the 100000-fold excess of unmethylated genes, and discriminate prostate cancer from benign prostatic hyperplasia (BPH) and control with serum samples. We also demonstrate the reversibility of DNA methylation modification by treatment with demethylation drugs. With 16-channel electrochemical work station, our research reveals a simple and inexpensive method to quantify the methylation level of specially appointed genes, and have the potential to be applied in the clinical research.

DNA methylation signatures of duplicate gene evolution in angiosperms.

Gene duplication is a source of evolutionary novelty. DNA methylation may play a role in the evolution of duplicate genes (paralogs) through its association with gene expression. While this relationship has been examined to varying extents in a few individual species, the generalizability of these results at either a broad phylogenetic scale with species of differing duplication histories or across a population remains unknown. We applied a comparative epigenomics approach to 43 angiosperm species across the phylogeny and a population of 928 Arabidopsis (Arabidopsis thaliana) accessions, examining the association of DNA methylation with paralog evolution. Genic DNA methylation was differentially associated with duplication type, the age of duplication, sequence evolution, and gene expression. Whole genome duplicates were typically enriched for CG-only gene-body methylated or unmethylated genes, while single-gene duplications were typically enriched for non-CG methylated or unmethylated genes. Non-CG methylation, in particular, was characteristic of more recent single-gene duplicates. Core angiosperm gene families differentiated into those which preferentially retain paralogs and 'duplication-resistant' families, which convergently reverted to singletons following duplication. Duplication-resistant families that still have paralogous copies were, uncharacteristically for core angiosperm genes, enriched for non-CG methylation. Non-CG methylated paralogs had higher rates of sequence evolution, higher frequency of presence-absence variation, and more limited expression. This suggests that silencing by non-CG methylation may be important to maintaining dosage following duplication and be a precursor to fractionation. Our results indicate that genic methylation marks differing evolutionary trajectories and fates between paralogous genes and have a role in maintaining dosage following duplication.

Final data from the phase 2a single-arm trial of SurVaxM for newly diagnosed glioblastoma.

2037 Background: Newly diagnosed glioblastoma (nGBM) routinely treated with surgery, radiation, and temozolomide (TMZ), still result in early progression and near-universal lethality within 5 years. Tumor associated “survivin” is expressed in >95% of nGBM and targetable by SurVaxM immunotherapy. Results from the recently completed multi-center phase 2a trial of SurVaxM in nGBM are presented. Methods: nGBM patients (pts) were enrolled at 5 trial sites. Eligibility criteria included: age ≥ 18, Karnofsky performance status ≥70, IHC confirmation of survivin expression, expression of HLA-A*02, A*03, A*11 or A*24 MHC-I alleles and residual contrast enhancement of ≤1 cm3 by MRI within 72h post-resection. Pts received standard TMZ chemoradiation followed by initiation of 4 priming doses of SurVaxM (500 mcg in emulsion with Montanide ISA 51, every 2 weeks) with 100 mcg sargramostim. Maintenance doses of SurVaxM-Montanide plus sargramostim were thereafter administered every 12 weeks. Adjuvant TMZ was administered for at least 6 cycles, after at least the first dose of SurVaxM and beginning no sooner than 28 days after completion of chemoradiation. Pts were monitored by MRI every 8 weeks, and progression was assessed using modified RANO criteria. The primary endpoint was 70% progression free survival (PFS) at 6 mos. Primary analyses of median PFS (mPFS) and median overall survival (OS) were measured from first immunization. Safety, tolerability, and immune responsiveness were also determined. Results: 63 pts with nGBM were enrolled, comprised of 38 males and 25 females with a median age of 60 years. The cohort was consistent with the 4 commonly observed primary molecular GBM subtypes (classical, mesenchymal, neural and proneural). SurVaxM was well tolerated, with no serious adverse events. A strong positive correlation, accounting for censoring, was observed between PFS and OS of all pts (r = 0.79; 95% CI (0.66,0.87)). SurVaxM was immunogenic and produced survivin-specific CD8+ T-cells and antibody (IgG) titers in both methylated and unmethylated MGMT pts and both groups showed clinical benefit. Conclusions: SurVaxM appeared to be safe and well-tolerated in pts with nGBM. SurVaxM was effective at stimulating survivin-specific immune responses and the primary endpoint was met. SurVaxM represents a promising therapy for nGBM, including for those pts with unmethylated MGMT genes. For pts treated with SurVaxM, PFS may be an acceptable surrogate for OS. Clinical trial information: NCT02455557. [Table: see text]

Gene body methylation is under selection in Arabidopsis thaliana.

In plants, mammals and insects, some genes are methylated in the CG dinucleotide context, a phenomenon called gene body methylation (gbM). It has been controversial whether this phenomenon has any functional role. Here, we took advantage of the availability of 876 leaf methylomes in Arabidopsis thaliana to characterize the population frequency of methylation at the gene level and to estimate the site-frequency spectrum of allelic states. Using a population genetics model specifically designed for epigenetic data, we found that genes with ancestral gbM are under significant selection to remain methylated. Conversely, ancestrally unmethylated genes were under selection to remain unmethylated. Repeating the analyses at the level of individual cytosines confirmed these results. Estimated selection coefficients were small, on the order of 4 Nes = 1.4, which is similar to the magnitude of selection acting on codon usage. We also estimated that A. thaliana is losing gbM threefold more rapidly than gaining it, which could be due to a recent reduction in the efficacy of selection after a switch to selfing. Finally, we investigated the potential function of gbM through its link with gene expression. Across genes with polymorphic methylation states, the expression of gene body methylated alleles was consistently and significantly higher than unmethylated alleles. Although it is difficult to disentangle genetic from epigenetic effects, our work suggests that gbM has a small but measurable effect on fitness, perhaps due to its association to a phenotype-like gene expression.

Chromatin remodeling by the histone methyltransferase EZH2 drives lung pre-malignancy and is a target for cancer prevention

BackgroundTrimethylation of lysine 27 and dimethylation of lysine 9 of histone-H3 catalyzed by the histone methyltransferases EZH2 and G9a impede gene transcription in cancer. Our human bronchial epithelial (HBEC) pre-malignancy model studied the role of these histone modifications in transformation. Tobacco carcinogen transformed HBEC lines were characterized for cytosine DNA methylation, transcriptome reprogramming, and the effect of inhibiting EZH2 and G9a on the transformed phenotype. The effects of targeting EZH2 and G9a on lung cancer prevention was assessed in the A/J mouse lung tumor model.ResultsCarcinogen exposure induced transformation and DNA methylation of 12–96 genes in the four HBEC transformed (T) lines that was perpetuated in malignant tumors. In contrast, 506 unmethylated genes showed reduced expression in one or more HBECTs with many becoming methylated in tumors. ChIP-on-chip for HBEC2T identified 327 and 143 genes enriched for H3K27me3 and H3K9me2. Treatment of HBEC2T and HBEC13T with DZNep, a lysine methyltransferase inhibitor depleted EZH2, reversed transformation, and induced transcriptional reprogramming. The EZH2 small molecule inhibitor EPZ6438 also affected transformation and expression in HBEC2T, while a G9a inhibitor, UNC0642 was ineffective. Genetic knock down of EZH2 dramatically reduced carcinogen-induced transformation of HBEC2. Only DZNep treatment prevented progression of hyperplasia to adenomas in the NNK mouse lung tumor model through reducing EZH2 and affecting the expression of genes regulating cell growth and invasion.ConclusionThese studies demonstrate a critical role for EZH2 catalyzed histone modifications for premalignancy and its potential as a target for chemoprevention of lung carcinogenesis.

DNA methylation changes in genes involved in inflammation and depression in fibromyalgia: a pilot study.

The present pilot study aims to investigate DNA methylation changes of genes related to fibromyalgia (FM) development and its main comorbid symptoms, including sleep impairment, inflammation, depression and other psychiatric disorders. Epigenetic modifications might trigger or perpetuate complex interplay between pain transduction/transmission, central pain processing and experienced stressors in vulnerable individuals. We conducted DNA methylation analysis by targeted bisulfite NGS sequencing testing differential methylation in 112 genomic regions from leukocytes of eight women with FM and their eight healthy sisters as controls. Tests for differentially methylated regions and cytosines brought focus on the GRM2 gene, encoding the metabotropic glutamate receptor2. The slightly increased DNA methylation observed in the GRM2 region of FM patients may confirm the involvement of the glutamate pathway in this pathological condition. Logistic regression highlighted the simultaneous association of methylation levels of depression and inflammation-related genes withFM. Altogether, the results evidence the glutamate pathway involvement in FM and support the idea that a combination of methylated and unmethylated genes could represent a risk factor to FM or its consequence, more than single genes. Further studies on the identified biomarkers could contribute to unravel the causative underlying FM mechanisms, giving reliable directions to research, improving the diagnosis and effective therapies.

DNMT1 Promotes Genome Methylation and Early Embryo Development in Cockroaches.

SummaryThe influence of DNA methylation on gene behavior and its consequent phenotypic effects appear to be very important, but the details are not well understood. Insects offer a diversity of DNA methylation modes, making them an excellent lineage for comparative analyses. However, functional studies have tended to focus on quite specialized holometabolan species, such as wasps, bees, beetles, and flies. Here, we have studied DNA methylation in the hemimetabolan insect Blattella germanica. In this cockroach, a gene involved in DNA methylation, DNA methyltransferase 1 (DNMT1), is expressed in early embryogenesis. In our experiments, RNAi of DNMT1 reduces DNA methylation and impairs blastoderm formation. Using reduced representation bisulfite sequencing and transcriptome analyses, we observed that methylated genes are associated with metabolism and are highly expressed, whereas unmethylated genes are related to signaling and show low expression. Moreover, methylated genes show greater expression change and less expression variability than unmethylated genes.

Identification of distinct loci for de novo DNA methylation by DNMT3A and DNMT3B during mammalian development

De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.

Standard for characteristics of adipose derived stem cells cultivated with serum free defined media

Background & Aim Stem cells are regarded as hopeful source for the regeneration of tissue and organ systems. The potential utility of mesenchymal stem cells (MSCs) has been heavily investigated in the fields or regenerative medicine and cell therapy. For the successful development of production effective stem cell therapy, mass production of consistent quality cells is required. Excellent culture medium is important in mass production of quality cells. Classically, fetal bovine serum (FBS) has been used as culture supplement for MSCs. However, due to the undefined and heterologous composition of FBS, researchers have transitioned to serum-free media. However, there has not been a comparision of the cells cultured in FBS containing media and serum free defined media (SFDM). In this study, we aimed to compare the stem cell characteristics and in-vivo efficacy cultured in both media and to suggest the standard of MSCs cultivated with serum free defined media. Methods, Results & Conclusion Comparision of adipose derived mesenchymal stem cells (ADSCs) was performed based on cellular characteristics, mRNA expression change, protein expression change and in-vivo efficacy using acute pancreatitis mouse model. The epigenetic profiles were checked through sequencing, we identified 49 methylated genes related to the negative system development. It also showed differences in 33 unmethylated genes involved in negative regulation of neurogenesis, negative regulation of cell development, neurogenesis and positive regulation of RNA metabolic processes. The mRNA expression levels of ADSCs cultured in FBS containing media were significantly increased for classification of aging, apoptotic process, cell death, immune response and inflammatory response compared to SFDM. Protein differential expression analysis were investigated LC-MS/MS about 11% and 16% of protein were upregulated in SFDM group and FBS group, respectively. Classification of apoptosis, cardiovascular disease, developmental disorder, skelectal and muscular disorder, inflammatory disease, inflammatory response, immune cell trafficking, antigen presentation, and cell-mediated immune response were up significant ate FBS group. To determine in-vivo efficacy of ADSCs cultured in SFDM, cells of SFDM group were administered to acute pancreatitis mouse model and the severity of disease were then evaluated. This study provides comprehensive characteristics of MSC derived from different cultured media condition to better understand their cellular and molecular biology.

Sex-specific changes in the aphid DNA methylation landscape.

Aphids present an ideal system to study epigenetics as they can produce diverse, but genetically identical, morphs in response to environmental stimuli. Here, using whole genome bisulphite sequencing and transcriptome sequencing of the green peach aphid (Myzus persicae), we present the first detailed analysis of cytosine methylation in an aphid and investigate differences in the methylation and transcriptional landscapes of male and asexual female morphs. We found that methylation primarily occurs in a CG dinucleotide (CpG) context and that exons are highly enriched for methylated CpGs, particularly at the 3′ end of genes. Methylation is positively associated with gene expression, and methylated genes are more stably expressed than unmethylated genes. Male and asexual female morphs have distinct methylation profiles. Strikingly, these profiles are divergent between the sex chromosome and the autosomes; autosomal genes are hypomethylated in males compared to asexual females, whereas genes belonging to the sex chromosome, which is haploid in males, are hypermethylated. Overall, we found correlated changes in methylation and gene expression between males and asexual females, and this correlation was particularly strong for genes located on the sex chromosome. Our results suggest that differential methylation of sex‐biased genes plays a role in aphid sexual differentiation.

DNA Methylation Patterns in the Social Spider, Stegodyphus dumicola.

Variation in DNA methylation patterns among genes, individuals, and populations appears to be highly variable among taxa, but our understanding of the functional significance of this variation is still incomplete. We here present the first whole genome bisulfite sequencing of a chelicerate species, the social spider Stegodyphus dumicola. We show that DNA methylation occurs mainly in CpG context and is concentrated in genes. This is a pattern also documented in other invertebrates. We present RNA sequence data to investigate the role of DNA methylation in gene regulation and show that, within individuals, methylated genes are more expressed than genes that are not methylated and that methylated genes are more stably expressed across individuals than unmethylated genes. Although no causal association is shown, this lends support for the implication of DNA CpG methylation in regulating gene expression in invertebrates. Differential DNA methylation between populations showed a small but significant correlation with differential gene expression. This is consistent with a possible role of DNA methylation in local adaptation. Based on indirect inference of the presence and pattern of DNA methylation in chelicerate species whose genomes have been sequenced, we performed a comparative phylogenetic analysis. We found strong evidence for exon DNA methylation in the horseshoe crab Limulus polyphemus and in all spider and scorpion species, while most Parasitiformes and Acariformes species seem to have lost DNA methylation.

Single-cell expression noise and gene-body methylation in Arabidopsis thaliana

Gene-body methylation (gbM) refers to an increased level of methylated cytosines specifically in a CG sequence context within genes. gbM is found in plant genes with intermediate expression level, which evolve slowly, and is often broadly conserved across millions of years of evolution. Intriguingly however, some plants lack gbM, and thus it remains unclear whether gbM has a function. In animals, there is support for a role of gbM in reducing erroneous transcription and transcription noise, but so far most studies in plants have tested for an effect of gbM on expression level, not noise. Here, we therefore tested whether gbM was associated with reduced expression noise in Arabidopsis thaliana, using single-cell transcriptome sequencing data from root quiescent centre cells. We find that gbM genes have lower expression noise levels than unmethylated genes. However, an analysis of covariance revealed that, if other genomic features are taken into account, this association disappears. Nonetheless, gbM genes were more consistently expressed across single-cell samples, supporting previous inference that gbM genes are constitutively expressed. Finally, we observed that fewer RNAseq reads map to introns of gbM genes than to introns of unmethylated genes, which indicates that gbM might be involved in reducing erroneous transcription by reducing intron retention.

A silver(I) doped bud-like DNA nanostructure as a dual-functional nanolabel for voltammetric discrimination of methylated from unmethylated genes.

A small DNA structure, referred to as DNA nanobud (NB), was used for the first time to design a dual-functional nanolabel in order to recognize a particular oligonucleotide sequence, generate and amplify the electrochemical analytical signal. NBs containing numerous repetitive desired sequences were prepared through self-assembly of 8-h rolling circle amplification. Then, redox-active silver ions were loaded onto the NBs by over-night incubation with a solution of AgNO3. The incorporation of Ag+ into NBs was confirmed by field emission scanning electron microscopy, dynamic light scattering, UV-Vis spectroscopy, zeta potential measurements, and energy-dispersive X-ray spectroscopy. A DNA sandwich complex was created after hybridization of Ag+-NB with target sequence, which was captured by immobilized probe on a gold electrode. Cyclic voltammetry was applied to measure the redox signal of silver ions produced typically at a potential around 0.02V vs. Ag/AgCl. The label can specifically discriminate fully methylated BMP3 gene from fully unmethylated bisulfate-converted part of the gene. The electrochemical signal produced by DNA sandwich complex of gold/probe/BMP3/Ag+-NB was linear toward BMP3 concentration from 100 pM to 100nM. The method has a 100 pM BMP3 detection limit. Conceivably, this nanolabel can be designed and modified such that it may also be used to detect other sequences with lower detection limits. Graphical abstract Ag+-NB as a new nanolabel for genosensing was formed by loading Ag+ on a spherical DNA nanostructure, nanobud (NB), synthesized by rolling circle amplification process. By using a gold electrode (AuE), Ag+-NB with numerous electroactive cations and binding sites can detect targets and generate amplified electrochemical signals.

The frequency of promoter DNA hypermethylation is decreased in colorectal neoplasms of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an inherited disorder characterized by numerous colorectal adenomatous polyps with predisposition to the development of colorectal cancer (CRC). Here, we conducted genome-wide DNA methylation analysis of FAP neoplasms, including seven cancer samples and 16 adenoma samples, using an Infinium 450K BeadArray. As controls for sporadic colorectal neoplasms and mucosae, we used Infinium 450k data from 297 CRC samples, 45 colorectal adenoma samples, and 37 normal mucosa samples with reference to The Cancer Genome Atlas and other databases. Unsupervised two-way hierarchical clustering analysis of FAP and sporadic CRC/adenoma revealed that CRC was classified into four DNA methylation epigenotypes (MEs): high-ME (HME), intermediate-ME (IME), low-ME (LME), and normal-like ME (NME). Five FAP neoplasms (two cancer and three adenoma) were clustered with IME, whereas 18 FAP neoplasms (five cancer and 13 adenoma) were clustered into NME. IME FAP neoplasms significantly correlated with KRAS mutations, similar to sporadic CRC. Within IME cases, however, aberrant DNA methylation was significantly less frequent in FAP neoplasms than sporadic neoplasms, and these unmethylated genes included WNT family genes and several types of oncogenes. In summary, FAP neoplasms were classified into at least two molecular subtypes, i.e., NME in the majority of cases showing mostly no aberrant methylation and IME in some cases accompanied by KRAS mutations but less frequent aberrant DNA methylation than sporadic neoplasms, suggesting that FAP may follow a tumorigenesis pathway different from that of sporadic CRC.

The association between DNA methylation and exon expression in the Pacific oyster Crassostrea gigas.

BackgroundDNA methylation is one of the most important epigenetic modifications of eukaryotic genomes and is believed to play integral roles in diverse biological processes. Although DNA methylation has been well studied in mammals, data are limited in invertebrates, particularly Mollusca. The Pacific oyster Crassostrea gigas is an emerging genetic model for functional analysis of DNA methylation in Mollusca. Recent studies have shown that there is a positive association between methylation status and gene expression in C. gigas; however, whether this association exists at the exon level remains to be determined.ResultsIn this study, we characterized the genome-wide methylation pattern across two different tissues of C. gigas and found that methylated genes are expressed in more tissues and development stages than unmethylated genes. Furthermore, we found that different types of exons had different methylation levels, with the lowest methylation levels in the first exons, followed by the last exons, and the internal exons. We found that the exons included in the gene transcript contained significantly higher DNA methylation levels than skipped exons. We observed that the DNA methylation levels increased slowly after the start sites and end sites of exons seperately, and then decreased quickly towards the middle sites of exons. We also found that methylated exons were significantly longer than unmethylated exons.ConclusionThis study constitutes the first genome-wide analysis to show an association between exon-level DNA methylation and mRNA expression in the oyster. Our findings suggest that exon-level DNA methylation may play a role in the construction of alternative splicing by positively influencing exon inclusion during transcription.

The gene tweaks that let us speak

Our speech might have arisen from simple fiddles to existing genes

Epigenomic and functional analyses reveal roles of epialleles in the loss of photoperiod sensitivity during domestication of allotetraploid cottons

BackgroundPolyploidy is a pervasive evolutionary feature of all flowering plants and some animals, leading to genetic and epigenetic changes that affect gene expression and morphology. DNA methylation changes can produce meiotically stable epialleles, which are transmissible through selection and breeding. However, the relationship between DNA methylation and polyploid plant domestication remains elusive.ResultsWe report comprehensive epigenomic and functional analyses, including ~12 million differentially methylated cytosines in domesticated allotetraploid cottons and their tetraploid and diploid relatives. Methylated genes evolve faster than unmethylated genes; DNA methylation changes between homoeologous loci are associated with homoeolog-expression bias in the allotetraploids. Significantly, methylation changes induced in the interspecific hybrids are largely maintained in the allotetraploids. Among 519 differentially methylated genes identified between wild and cultivated cottons, some contribute to domestication traits, including flowering time and seed dormancy. CONSTANS (CO) and CO-LIKE (COL) genes regulate photoperiodicity in Arabidopsis. COL2 is an epiallele in allotetraploid cottons. COL2A is hypermethylated and silenced, while COL2D is repressed in wild cottons but highly expressed due to methylation loss in all domesticated cottons tested. Inhibiting DNA methylation activates COL2 expression, and repressing COL2 in cultivated cotton delays flowering.ConclusionsWe uncover epigenomic signatures of domestication traits during cotton evolution. Demethylation of COL2 increases its expression, inducing photoperiodic flowering, which could have contributed to the suitability of cotton for cultivation worldwide. These resources should facilitate epigenetic engineering, breeding, and improvement of polyploid crops.

Arsenic trioxide increases expression of secreted frizzled-related protein 1 gene and inhibits the WNT/β-catenin signaling pathway in Jurkat cells.

The aim of the present study was to investigate the demethylation effect of arsenic trioxide (As2O3) on the secreted frizzled-related protein 1 (SFRP1) gene and its ability to inhibit the Wingless-type MMTV integration site family (WNT) pathway in Jurkat cells. Methylation-specific polymerase chain reaction was used to examine the CpG island methylation status of the SFRP1 gene in leukemia cell lines. In addition, the effects on Jurkat cells of treatment with different concentrations of As2O3 for 48 h were investigated. Reverse transcription-quantitative polymerase chain reaction was employed to measure the expression of mRNAs, while western blot analysis was used to examine protein expression in cells. The SFRP1 gene was methylated in Jurkat cells. However, both methylated and unmethylated SFRP1 genes were detected in HL60 and K562 cells. In normal bone marrow mononuclear cells, the SFRP1 gene was unmethylated. Following treatment with As2O3 for 48 h, the SFRP1 gene was demethylated, and the mRNA and protein expression levels of the SFRP1 gene were increased. By contrast, the mRNA and protein expression levels of β-catenin and cyclin Dl were downregulated. The protein expression of c-myc was also downregulated, but As2O3 exhibited no significant effect on the mRNA expression of c-myc. Abnormal methylation of the SFRP1 gene was detected in Jurkat cells. These results suggest that As2O3 activates SFRP1 gene expression at the mRNA and protein levels in Jurkat cells by demethylation of the SFRP1 gene. Furthermore, they indicate that As2O3 regulates WNT target genes and controls the growth of Jurkat cells through the WNT/β-catenin signaling pathway.

Arsenic trioxide induces cell cycle arrest and alters DNA methylation patterns of cell cycle regulatory genes in colorectal cancer cells

AimsCell cycle dysregulation is important in tumorigenesis. Transcriptional silencing of cell cycle regulatory genes, due to DNA methylation, is a common epigenetic event in malignancies. As2O3 has been shown to induce cell cycle arrest and also to be a potential hypomethylating agent. Our study aimed to investigate DNA methylation patterns of cell cycle regulatory genes promoters, the effects of Arsenic trioxide (As2O3) on the methylated genes and cell cycle distribution in colorectal cancer (CRC) cell lines. Main methodsThe methylation-specific PCR (MSP) and/or restriction enzyme-based methods were used to study the promoter methylation patterns of 24 cell cycle regulatory genes in CRC cell lines. Gene expression level and cell cycle distribution were determined by Real-time PCR and flow cytometric analyses, respectively. Key findingsOur methylation analysis indicated that only promoters of RBL1 (p107), CHFR and p16 genes were aberrantly methylated in three cell lines. As2O3 significantly decreased DNA methylation in promoter regions of these genes and restored their expression. We found that As2O3 significantly reduced the expression of DNA methyltransferase 1 (DNMT1) and increased arsenic methyltransferase (AS3MT). Furthermore, As2O3 altered transcriptional activity of several unmethylated cell cycle regulatory genes including cyclin B1, E1, D1, GADD45A and p21. Cell cycle flow cytometry analysis showed As2O3 induced G2/M arrest in all three cell lines. SignificanceThese data suggest that demethylation and alteration in the expression level of the cell cycle-related genes may be possible mechanisms in As2O3-induced cell cycle arrest in colorectal cancer cells.