DNA methylation: its contribution to systemic lupus erythematosus

Abstract Recent studies on epigenetics, including the methylation of DNA and the enzymes regulating methylation, seem likely to contribute to understanding the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). In fact, the relationship between DNA methylation and SLE has long been the subject of investigation. To obtain a deeper understanding of the role of DNA methylation in the onset of SLE, we reviewed the findings reported in the literature and our own data about DNA methylation and SLE. Various studies have indicated the possible importance of DNA methylation, especially hypomethylation, in the aetiology of SLE. Epigenetic studies may provide clues for elucidating the pathogenesis of SLE and for developing new strategies to treat this disease.     

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance

Differences in the global methylation pattern, ie hyper‐ as well as hypo‐methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5‐ cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam‐2 before and after demethylation using 5‐azacytidine. Exposure to 5‐azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell‐specific marker VASA, showed increased expression. Following treatment with 5‐azacytidine, TCam‐2 cells were analysed using a high‐throughput methylation screen for changes in the methylation sites of 14 000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

DNA methylation is required for the control of stem cell differentiation in the small intestine

The mammalian intestinal epithelium has a unique organization in which crypts harboring stem cells produce progenitors and finally clonal populations of differentiated cells. Remarkably, the epithelium is replaced every 3–5 d throughout adult life. Disrupted maintenance of the intricate balance of proliferation and differentiation leads to loss of epithelial integrity or barrier function or to cancer. There is a tight correlation between the epigenetic status of genes and expression changes during differentiation; however, the mechanism of how changes in DNA methylation direct gene expression and the progression from stem cells to their differentiated descendants is unclear. Using conditional gene ablation of the maintenance methyltransferase Dnmt1, we demonstrate that reducing DNA methylation causes intestinal crypt expansion in vivo. Determination of the base-resolution DNA methylome in intestinal stem cells and their differentiated descendants shows that DNA methylation is dynamic at enhancers, which are often associated with genes important for both stem cell maintenance and differentiation. We establish that the loss of DNA methylation at intestinal stem cell gene enhancers causes inappropriate gene expression and delayed differentiation.     

DNA甲基化与系统性红斑狼疮

系统性红斑狼疮是一种针对自身抗原产生自身抗体从而形成免疫复合物为特征的慢性炎症性的自身免疫性疾病,能够累及机体的多个脏器(以皮肤、关节、肾脏受累为多见).系统性红斑狼疮的发病机制涉及到环境、遗传、性别多个因素的相互作用,对于哪个因素在其中起着关键性的作用,是如何诱发疾病的发生,以及在发病过程中的具体作用机制到目前来说还没有一个统一认识.目前许多研究表明,DNA的甲基化可以作为环境、性别等因素的共同作用通路在狼疮的发病中起到一定的作用,现就关于DNA的甲基化与系统性红斑狼疮的关系做一综述.     

The DMM complex prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa

Transposable elements are common in genomes and must be controlled. Many organisms use DNA methylation to silence such selfish DNA, but the mechanisms that restrict the methylation to appropriate regions are largely unknown. We identified a JmjC domain protein in Neurospora, DNA METHYLATION MODULATOR-1 (DMM-1), that prevents aberrant spreading of DNA and histone H3K9 methylation from inactivated transposons into nearby genes. Mutation of a conserved residue within the JmjC Fe(II)-binding site abolished dmm-1 function, as did mutations in conserved cysteine-rich domains. Mutants defective only in dmm-1 mutants grow poorly, but growth is restored by reduction or elimination of DNA methylation using the drug 5-azacytosine or by mutation of the DNA methyltransferase gene dim-2. DMM-1 relies on an associated protein, DMM-2, which bears a DNA-binding motif, for localization and proper function. HP1 is required to recruit the DMM complex to the edges of methylated regions.     

Comparison of CpG island hypermethylation and repetitive DNA hypomethylation in premalignant stages of gastric cancer,stratified for Helicobacter pylori infection

CpG island hypermethylation and genomic DNA hypomethylation are found not only in gastric cancers but also in associated premalignant lesions. Helicobacter pylori infection induces aberrant CpG island hypermethylation in gastric mucosae. However, little is known about the relationship between H. pylori infection and aberrant methylation in premalignant lesions. The present study characterized methylation changes in a subset of genes and repetitive DNA elements (ALU, LINE‐1, SAT2) and examined their relationship with H. pylori infection in premalignant lesions of gastric cancers. We performed MethyLight analysis of 25 genes and SAT2 and COBRA analysis of ALU and LINE‐1 in 212 gastric tissue samples. H. pylori infection was closely associated with enhanced hypermethylation of CpG island loci in chronic gastritis samples, but this association was not found among intestinal metaplasias, gastric adenomas and gastric cancers. The number of methylated genes was greater in intestinal metaplasia and gastric adenoma samples than in chronic gastritis samples, regardless of H. pylori infection. Methylation of repetitive DNA elements in gastric lesions generally decreased with progression of the gastric lesion along the multistep carcinogenesis. No difference was noted in the number of methylated genes in chronic gastritis or intestinal metaplasia between gastric cancer patients and non‐cancer subjects. In conclusion, we found that there was no enhanced CpG island hypermethylation in gastric cancer and premalignant lesions in association with H. pylori infection and our findings suggest that CpG island hypermethylation and repetitive DNA hypomethylation are enhanced with progression of the gastric lesion through the multistep carcinogenesis, regardless of the status of H. pylori infection. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells

The mechanisms by which arsenic‐induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT‐PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose‐dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome‐wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long‐lasting arsenic‐induced genomic instability that manifests in several ways, including aberrant chromosomal effects. Environ. Mol. Mutagen. 57:137–150, 2016. © 2015 Wiley Periodicals, Inc.     

DNA methylation in genes associated with the evolution of ageing and disease: A critical review

Ageing is characterised by a physical decline in biological functioning which results in a progressive risk of mortality with time. As a biological phenomenon, it is underpinned by the dysregulation of a myriad of complex processes. Recently, however, ever-increasing evidence has associated epigenetic mechanisms, such as DNA methylation (DNAm) with age-onset pathologies, including cancer, cardiovascular disease, and Alzheimer’s disease. These diseases compromise healthspan. Consequently, there is a medical imperative to understand the link between epigenetic ageing, and healthspan. Evolutionary theory provides a unique way to gain new insights into epigenetic ageing and health. This review will: (1) provide a brief overview of the main evolutionary theories of ageing; (2) discuss recent genetic evidence which has revealed alleles that have pleiotropic effects on fitness at different ages in humans; (3) consider the effects of DNAm on pleiotropic alleles, which are associated with age related disease; (4) discuss how age related DNAm changes resonate with the mutation accumulation, disposable soma and programmed theories of ageing; (5) discuss how DNAm changes associated with caloric restriction intersect with the evolution of ageing; and (6) conclude by discussing how evolutionary theory can be used to inform investigations which quantify age-related DNAm changes which are linked to age onset pathology.     

急性白血病细胞系中SFRP基因启动子甲基化及去甲基化诱导表达的研究

 目的：探讨分泌型卷曲相关蛋白（SFRP）基因家族启动子CpG岛异常甲基化状态与急性白血病（AL）发生发展的关系,以及DNA甲基转移酶（DNMT）抑制剂5-氮杂-2-脱氧胞苷酸（5-Aza-CdR）去甲基化诱导SFRP基因表达作用的可能机制。方法：采用甲基化特异性PCR检测不同AL细胞系（Molt-4、Jurkat、HL-60和NB4）和不同浓度5-Aza-CdR作用下Jurkat细胞系中SFRP1、SFRP2、SFRP4和SFRP5基因启动子区的甲基化状态,采用实时荧光定量RT-PCR检测SFRP1、SFRP2、SFRP4和SFRP5 mRNA表达,采用半定量RT-PCR检测DNMT1、DNMT3A和DNMT3B mRNA表达。结果：在正常人细胞中不存在SFRP基因的甲基化。SFRP1、SFRP2和SFRP5基因在HL-60、NB4、Molt-4和Jurkat细胞系中均呈完全甲基化状态;SFRP4基因在NB4、Molt-4和Jurkat细胞系中完全甲基化,在HL-60细胞系中则部分甲基化。5-Aza-CdR可逆转SFRP1、SFRP2、SFRP4和SFRP5基因的高甲基化状态,并能够下调DNMT1、DNMT3A和DNMT3B mRNA水平,诱导SFRP基因恢复表达。结论：在AL细胞系中,SFRP1、SFRP2、SFRP4和SFRP5基因出现高甲基化,与AL的发生密切相关,可能成为AL新的基因标志物。5-Aza-CdR通过抑制DNMT表达,逆转SFRP基因的甲基化状态,恢复其表达。     

Methylome-wide comparison of human genomic DNA extracted from whole blood and from EBV-transformed lymphocyte cell lines

DNA from Epstein–Barr virus-transformed lymphocyte cell lines (LCLs) has proven useful for studies of genetic sequence polymorphisms. Whether LCL DNA is suitable for methylation studies is less clear. We conduct a genome-wide methylation investigation using an array set with 45 million probes to investigate the methylome of LCL DNA and technical duplicates of WB DNA from the same 10 individuals. We focus specifically on methylation sites that show variation between individuals and, therefore, are potentially useful as biomarkers. The sample correlations for the methylation variable probes ranged from 0.69 to 0.78 for the WB duplicates and from 0.27 to 0.72 for WB vs LCL. To compare the pattern of the methylation signals, we grouped adjacent probes based on their inter-correlations. These analyses showed ∼29 000 and ∼14 000 blocks in WB and LCL, respectively. Merely 31% of the methylated regions detected in WB were detectable in LCLs. Furthermore, we observed significant differences in mean difference between WB and LCL as compared with duplicates of WB (P-value =2.2 × 10⁻¹⁶). Our study shows that there are substantial differences in the DNA methylation patterns between LCL and WB. Thus, LCL DNA should not be used as a proxy for WB DNA in methylome-wide studies.     

DNA methylation alterations in response to pesticide exposure in vitro

Although pesticides are subject to extensive carcinogenicity testing before regulatory approval, pesticide exposure has repeatedly been associated with various cancers. This suggests that pesticides may cause cancer via nonmutagenicity mechanisms. The present study provides evidence to support the hypothesis that pesticide‐induced cancer may be mediated in part by epigenetic mechanisms. We examined whether exposure to seven commonly used pesticides (i.e., fonofos, parathion, terbufos, chlorpyrifos, diazinon, malathion, and phorate) induces DNA methylation alterations in vitro. We conducted genome‐wide DNA methylation analyses on DNA samples obtained from the human hematopoietic K562 cell line exposed to ethanol (control) and several organophosphate pesticides (OPs) using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian‐adjusted t‐tests were used to identify differentially methylated gene promoter CpG sites. In this report, we present our results on three pesticides (fonofos, parathion, and terbufos) that clustered together based on principle component analysis and hierarchical clustering. These three pesticides induced similar methylation changes in the promoter regions of 712 genes, while also exhibiting their own OP‐specific methylation alterations. Functional analysis of methylation changes specific to each OP, or common to all three OPs, revealed that differential methylation was associated with numerous genes that are involved in carcinogenesis‐related processes. Our results provide experimental evidence that pesticides may modify gene promoter DNA methylation levels, suggesting that epigenetic mechanisms may contribute to pesticide‐induced carcinogenesis. Further studies in other cell types and human samples are required, as well as determining the impact of these methylation changes on gene expression. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

DNA methylation of the extraembryonic tissues: an in situ study on human metaphase chromosomes

DNA methylation level and pattern of human metaphase chromosomes from extraembryonic tissues (chorionic villi and placental fibroblasts) were analysed in situ. The DNA methylation global level of these tissues was studied by comparing them with the one observed in fetal fibroblasts and adult lymphocytes. In order to assess the tissue specificity and significance of the observed differences, chromosomal preparations were then treated in parallel. They were first stained with distamycin A/DAPI and pictured, then treated with immunofluorescent staining using monoclonal antibodies raised against 5-methylcytosine. Compared with metaphases from lymphocytes or placental and fetal fibroblasts, distamycin-A/DAPI stained metaphases and constitutive heterochromatic regions with very similar intensities. In contrast, in chorionic villi, the immunofluorescent intensities revealing the presence of 5-methylcytosine was much duller than in the other tissues. In addition, in both chorionic villi and placental fibroblasts, large differences were observed between various chromosome structures within individual metaphases. In particular, the secondary constriction of chromosome 9, the distal segment of chromosome Y and the short arms of acrocentric chromosomes exhibited a much lower staining than the one observed for the secondary constrictions of chromosome 1 and 16 of the same metaphases. Because all these structures are known to be deeply methylated in other somatic tissues, this suggests that in extraembryonic tissues DNA methylation level remained hypomethylated and the pattern is under precise control.     

DNA methylation profiling of human placentas reveals promoter hypomethylation of multiple genes in early-onset preeclampsia

Preeclampsia and intrauterine growth restriction (IUGR) are two of the most common adverse pregnancy outcomes, but their underlying causes are mostly unknown. Although multiple studies have investigated gene expression changes in these disorders, few studies have examined epigenetic changes. Analysis of the DNA methylation pattern associated with such pregnancies provides an alternative approach to identifying cellular changes involved in these disorders. We analyzed methylation of 1505 CpG sites associated with 807 genes in 26 placentas from early-onset preeclampsia (EOPET), late-onset preeclampsia, IUGR and control subjects using an Illumina GoldenGate Methylation panel. Thirty-four loci were hypomethylated (false discovery rate <10% and methylation difference >10%) in the early-onset preeclamptic placentas while no and only five differentially methylated loci were found in late-onset preeclamptic and IUGR placentas, respectively. Hypomethylation of 4 loci in EOPET was further confirmed by bisulfite pyrosequencing of 26 independent placental samples. The promoter of TIMP3 was confirmed to be significantly hypomethylated in EOPET placentas (P=0.00001). Our results suggest that gene-specific hypomethylation may be a common phenomenon in EOPET placentas, and that TIMP3 could serve as a potential prenatal diagnostic marker for EOPET.     

Clinicopathological features of CpG island methylator phenotype-positive colorectal cancer and its adverse prognosis in relation to KRAS/BRAF mutation

CpG island methylator phenotype (CIMP) is a recently described subset of colorectal cancers (CRC) with widespread methylation of multiple promoter CpG islands. But the prognostic implication of CIMP in CRC has not been clarified. Thus, the aim of the present study was to differentiate the unique characteristics of CIMP from those of microsatellite instability (MSI)-high CRC, especially with regard to prognosis. CIMP, MSI, and mutations of KRAS codons 12 and 13 and of BRAF codon 600 were evaluated in 134 sporadic CRC. Patient survival and other clinicopathological variables were correlated with CIMP or genetic changes. High CIMP, high MSI, and mutations in KRAS or BRAF were detected in 31.3%, 14.2%, 33.6%, and 4.5% of overall CRC, respectively. High CIMP was closely associated with MSI and BRAF mutation but not with KRAS mutation. CIMP-high, microsatellite-stable (MSS) CRC were significantly associated with proximal location and nodal metastasis and had close but non-significant associations with liver metastasis. A worse clinical outcome was found for CIMP-high, MSS CRC with KRAS/BRAF mutation but not for those lacking KRAS/BRAF mutation. The findings support the contention that CIMP-high CRC have distinct clinicopathological and epidemiological features and suggest that the alleged poor clinical outcome of CIMP-high CRC patients is closely associated with the presence of KRAS/BRAF mutation.     

Combined therapy with cisplatin and 5-AZA-2CdR modifies methylation and expression of DNA repair genes in oral squamous cell carcinoma

The methylation and expression of DNA repair system genes has been studied in several tumor types. These genes have been associated with resistance to chemotherapy treatments by epigenetic regulation. Studies have yet to show the effects of combined therapy using an epigenetic drug (5-aza-2CdR) and cisplatin (CDDP) on DNA repair genes in oral squamous cell carcinoma (OSCC). This study proposed to investigate the effects of CDDP in combination with 5-aza-2CdR on the methylation of MGMT and MLH1 genes in oral cancer cells. Oral squamous cell carcinoma cell lineages (SCC-9, SCC-15, and SCC-25) were submitted to 72 hours of treatment: 0.1 μM CDDP (or 4.44 μM SCC-9), 0.1 μM and 0.3 μM 5-aza-2CdR (or 1 μM and 3 μM SCC-9), and the drugs in combination. Cell viability was assessed by MTT, DNA methylation of MGMT and MLH1 genes by Methylation Sensitivity High-Resolution Melting (MS-HRM), and the relative expression of the genes by RT-qPCR. The results show that all treatments reduced cell viability; however, in SCC-15 and SCC-9 (IC₅₀ value), 5-aza-2CdR promotes cell sensitization to cytotoxic effect of cisplatin. The MGMT promoter region was 100% demethylated in the SCC-15 and SCC-25 cells but partially (50%) methylated in SCC-9 before drug treatment. Treatment with IC₅₀ CDDP value kept the methylation status and decreased MGMT expression in SCC-9; MGMT gene in SCC-15 and SCC-25 cells became downregulated after treatment with 5-aza-2CdR. MLH1 was demethylated, but the treatments with low-doses and combined drugs decreased the expression in SCC-9 and SCC-25; however high doses of 5-aza-2CdR and drug combination with IC₅₀ value CDDP increased expression of MLH1 in SCC-9. The data presented suggest that epigenetic drugs associated with chemotherapy have clinical translational potential as a therapy strategy to avoid or reverse cancer resistance, requiring further investigation.