Nightshift work,chronotype, and genome-wide DNA methylation in blood

Molecular mechanisms underlying the negative health effects of shift work are poorly understood, which remains a barrier to developing intervention strategies to protect the long-term health of shift workers. We evaluated genome-wide differences in DNA methylation (measured in blood) between 111 actively employed female nightshift and 86 actively employed female dayshift workers from the Seattle metropolitan area. We also explored the effect of chronotype (i.e., measure of preference for activity earlier or later in the day) on DNA methylation among 110 of the female nightshift workers and an additional group of 131 male nightshift workers. Methylation data were generated using the Illumina Infinium HumanMethylation450 BeadChip (450K) Array. After applying the latest methylation data processing methods, we compared methylation levels at 361,210 CpG loci between the groups using linear regression models adjusted for potential confounders and applied the false-discovery rate (FDR) ≤ 0.05 to account for multiple comparisons. No statistically significant associations at the genome-wide level were observed with shift work or chronotype, though based on raw P values and absolute effect sizes, there were suggestive associations in genes that have been previously linked with cancer (e.g., BACH2, JRK, RPS6KA2) and type-2 diabetes (e.g., KCNQ1). Given that our study was underpowered to detect moderate effects, examining these suggestive results in well-powered independent studies or in pooled data sets may improve our understanding of the pathways underlying the negative health effects of shift work and the influence of personal factors such as chronotype. Such an approach may help identify potential interventions that can be used to protect the long-term health of shift workers.     

Circadian gene methylation in rotating-shift nurses: a cross-sectional study

Investigating the methylation status of the circadian genes may contribute to a better understanding of the shift work-related circadian disruption in individuals exposed to artificial light at night. In the present study, we determined the methylation status of the circadian genes associated with a shift work pattern among nurses and midwives participating in a cross-sectional study in Lodz, Poland.

Quantitative methylation polymerase chain reaction assays were used to assess promoter CpG methylation in PER1, PER2, PER3, CRY1, CRY2, BMAL1, CLOCK, and NPAS2 in genomic DNA from whole blood of 347 women having a rotating-shift work schedule and 363 women working days only. The percentage of methylated reference (PMR) was assessed using fluorescent probes for PER1, PER2, PER3, CRY1, and NPAS2, and the percentage of gene methylation, as the methylation index (MI), using two sets of primers for BMAL1, CLOCK, and CRY2.

We tested the possible association between current and lifetime rotating night-shift work characteristics and circadian gene methylation by using proportional odds regression model with blood DNA methylation, categorized into tertiles, and adjusted for age, current smoking status, folate intake and blood collection time. The findings indicated that CpG methylation in PER2 promoter was significantly decreased (P < 0.004) among nurses and midwives currently working rotating shifts, as compared with day-working nurses and midwives. The lower percentage of PER2 methylation was associated with a higher monthly frequency of current night duties (2–7 night shifts, and eight or more night shifts per month) (P = 0.012) and was associated at borderline significance (P = 0.092) with the lifetime duration of shift work (>10 ≤ 20 years and >20 ≤ 43 years of rotating-shift work) among nurses and midwives (N = 710). Moreover, women with a longer lifetime duration of shift work presented a lower status of PER1 methylation (P = 0.040) than did the women with up to 10 years of rotating-shift work. Long lifetime duration of shift work (> 10 years) among current rotating night-shift workers (N = 347) was associated with BMAL1 hypomethylation (P = 0.013).

Among eight of the investigated circadian genes, only PER1, PER2, and BMAL1 showed differential methylation attributable to the rotating-shift work of nurses and midwives. The findings on blood-based DNA methylation in the circadian genes may provide a better insight into the mechanistic principles underlying the possible health effects of night-shift work but these should be verified in further studies recruiting larger populations of shift workers.     

Semiconductor-based sequencing of genome-wide DNA methylation states

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we developed a MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is low cost, rapid, and scalable. We applied this protocol to demonstrate MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450 BeadChip array, and accurately identifies sites of differential DNA methylation. Furthermore, we applied an integrative approach to further investigate and confirm the role of DNA methylation in alternative splicing and to profile 5mC and 5hmC variants of DNA methylation in normal human brain tissue that is localized over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions.     

Temperature-independent genome-wide DNA methylation profile in turbot post-embryonic development

The morphological and biological characteristics of ectothermic vertebrates are known to be strongly influenced by environmental conditions, particularly temperature. Epigenetic mechanisms such as DNA methylation have been reported to contribute to the phenotypic plasticity observed in vertebrates in response to environmental changes. Additionally, DNA methylation is a dynamic process that occurs throughout vertebrate ontogeny and it has been associated with the activation and silencing of gene expression during post-embryonic development and metamorphosis. In this study, we investigated genome-wide DNA methylation profiles during turbot metamorphosis, as well as the epigenetic effects of temperature on turbot post-embryonic development. Fish growth and rates of development were greatly affected by rearing temperature. Thus, turbot raised at ambient temperature (18 °C) achieved greater body weights and progressed through development more quickly than those reared at a colder temperature (14 °C). Genome-wide DNA methylation dynamics analyzed via a methylation-sensitive amplified polymorphism (MSAP) technique were not significantly different between animals reared within the two different thermal environments. Furthermore, comparisons between phenotypically similar fish revealed that genome-wide DNA methylation profiles do not necessarily correlate with specific developmental stages in turbot.     

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA

Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.     

Controlling for conservation in genome-wide DNA methylation studies

Background

A commonplace analysis in high-throughput DNA methylation studies is the comparison of methylation extent between different functional regions, computed by averaging methylation states within region types and then comparing averages between regions. For example, it has been reported that methylation is more prevalent in coding regions as compared to their neighboring introns or UTRs, leading to hypotheses about novel forms of epigenetic regulation.

Results

We have identified and characterized a bias present in these seemingly straightforward comparisons that results in the false detection of differences in methylation intensities across region types. This bias arises due to differences in conservation rates, rather than methylation rates, and is broadly present in the published literature. When controlling for conservation at coding start sites the differences in DNA methylation rates disappear. Moreover, a re-evaluation of methylation rates at intronexon junctions reveals that the magnitude of previously reported differences is greatly exaggerated. We introduce two correction methods to address this bias, an inferencebased matrix completion algorithm and an averaging approach, tailored to address different underlying biological questions. We evaluate how analysis using these corrections affects the detection of differences in DNA methylation across functional boundaries.

Conclusions

We report here on a bias in DNA methylation comparative studies that originates in conservation rate differences and manifests itself in the false discovery of differences in DNA methylation intensities and their extents. We have characterized this bias and its broad implications, and show how to control for it so as to enable the study of a variety of biological questions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1604-3) contains supplementary material, which is available to authorized users.     

Sequential changes in genome-wide DNA methylation status during adipocyte differentiation

DNA methylation is an epigenetic mark on the mammalian genome. There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the unique sequences distributed throughout the genome. To determine the epigenetic changes during adipocyte differentiation, we investigated the sequential changes in DNA methylation status of 3T3-L1 cells at the growing, confluent, postconfluent and mature adipocyte cell stages. Treatment of 3T3-L1 cells with 5-aza-2′-deoxycytidine inhibited differentiation in a stage-dependent manner, supporting the idea that formation of accurate DNA methylation profile, consisting of methylated and unmethylated T-DMRs, may be involved in differentiation. Analysis by methylation-sensitive quantitative real-time PCR of the 65 known T-DMRs which contain NotI sites detected 8 methylations that changed during differentiation, and the changes in the patterns of these methylations were diverse, confirming that the differentiation process involves epigenetic alteration at the T-DMRs. Intriguingly, the dynamics of the methylation change vary depending on the T-DMRs and differentiation stages. Restriction landmark genomic scanning detected 32 novel T-DMRs, demonstrating that differentiation of 3T3-L1 cells involves genome-wide epigenetic changes by temporal methylation/demethylation, in addition to maintenance of a static methylated/demethylated state, and both depend on differentiation stage.     

MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes

Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the ‘perfect solution’, and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost.     

Identification of DNA methylation signature to predict prognosis in gastric adenocarcinoma

Gastric adenocarcinoma is an important death-related cancer. To find factors related to survival and prognosis, and thus improve recovery prospects, a powerful signature is needed. DNA methylation plays an important role in gastric adenocarcinoma processes and development, and here we report on the search for a significant DNA methylation gene to aid with the earlier diagnosis of gastric adenocarcinoma patients. A Cox proportional risk regression analysis and random survival forest algorithm were used to analyze gastric adenocarcinoma patients’ DNA methylation data from The Cancer Genome Atlas, a public database. DNA methylation gene signature consisting of five genes (SERPINA3, AP000357.4, GZMA, AC004702.2, and GREB1L) were selected. As the most accurate predictor, the area under the curve in the training and test group were 0.72 and 0.61, respectively. The signature was able to sort patients into high- and low-risk groups with meaningful overall survival rates (median: 18.36 vs 72.23 months, log-rank test, P < 0.001) in the training group, which predictive ability was validated in a test data set (median: 25.56 vs 58.80 months, log-rank test, P < 0.016). A multivariate Cox regression analysis showed the significant DNA methylation was an independent prediction prognostic factor for gastric adenocarcinoma patients. Functional analysis suggests that these signature genes may be related to pathways and biological processes associated with tumorigenesis. The significant DNA methylation gene could be a novel prediction and prognostic biomarker that both aids in the treatment and predicts the overall survival likelihoods of gastric adenocarcinoma patients.     

DNA methylation variation in cloned mice

Summary: Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue‐specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue‐specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue‐specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned. genesis 30:45–50, 2001. © 2001 Wiley‐Liss, Inc.     

Sleep quality and methylation status of core circadian rhythm genes among nurses and midwives

Poor sleep quality or sleep restriction is associated with sleepiness and concentration problems. Moreover, chronic sleep restriction may affect metabolism, hormone secretion patterns and inflammatory responses. Limited recent reports suggest a potential link between sleep deprivation and epigenetic effects such as changes in DNA methylation profiles. The aim of the present study was to assess the potential association between poor sleep quality or sleep duration and the levels of 5-methylcytosine in the promoter regions of PER1, PER2, PER3, BMAL1, CLOCK, CRY1 CRY2 and NPAS2 genes, taking into account rotating night work and chronotype as potential confounders or modifiers. A cross-sectional study was conducted on 710 nurses and midwives (347 working on rotating nights and 363 working only during the day) aged 40–60 years. Data from in-person interviews about sleep quality, chronotype and potential confounders were used. Sleep quality and chronotype were assessed using Pittsburgh Sleep Quality Questionnaire (PSQI) and Morningness–Eveningness Questionnaire (MEQ), respectively. Morning blood samples were collected. The methylation status of the circadian rhythm genes was determined via quantitative methylation-specific real-time PCR assays (qMSP) reactions using DNA samples derived from leucocytes. The proportional odds regression model was fitted to quantify the relationship between methylation index (MI) as the dependent variable and sleep quality or sleep duration as the explanatory variable. Analyses were carried out for the total population as well as for subgroups of women stratified by the current system of work (rotating night shift/day work) and chronotype (morning type/intermediate type/evening type). A potential modifying effect of the system of work or the chronotype was examined using the likelihood ratio test. No significant findings were observed in the total study population. Subgroup analyses revealed two statistically significant associations between a shorter sleep duration and 1) methylation level in PER2 among day workers, especially those with the morning chronotype (OR = 2.31, 95%CI:1.24–4.33), and 2) methylation level in CRY2 among subjects with the intermediate chronotype, particularly among day workers (OR = 0.52, 95%CI:0.28–0.96). The study results demonstrated a positive association between average sleep duration of less than 6 hours and the methylation level of PER2 among morning chronotype subjects, and an inverse association for CRY2 among intermediate chronotype subjects, but only among day workers. Both the system of work and the chronotype turned out to be important confounders and modifiers in a number of analyses, making it necessary to consider them as potential covariates in future research on sleep deficiency outcomes. Further studies are warranted to explore this under-investigated topic.     

Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.     

Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.     

线粒体DNA与DNA甲基化的关系

线粒体DNA（mitochondrial DNA,mtDNA）遗传信息量虽小,却控制着线粒体一些最基本的性质,对细胞及其功能有着重要影响。mtDNA的损伤与衰老、肿瘤等疾病的发生有关。DNA甲基化是调节基因表达的重要方式之一。mtDNA基因的表达受核DNA（nuclear DNA,nDNA）的调控,mtDNA和nDNA协同作用参与机体代谢调节和发病。本文就近年来mtDNA与DNA甲基化的关系作一综述。     

Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells

The development of germ cells is a highly ordered process that begins during fetal growth and is completed in the adult. Epigenetic modifications that occur in germ cells are important for germ cell function and for post-fertilization embryonic development. We have previously shown that male germ cells in the adult mouse have a highly distinct epigenetic state, as revealed by a unique genome-wide pattern of DNA methylation. Although it is known that these patterns begin to be established during fetal life, it is not known to what extent DNA methylation is modified during spermatogenesis. We have used restriction landmark genomic scanning (RLGS) and other techniques to examine DNA methylation at multiple sites across the genome during postnatal germ cell development in the mouse. Although a significant proportion of the distinct germ cell pattern is acquired prior to the type A spermatogonial stage, we find that both de novo methylation and demethylation occur during spermatogenesis, mainly in spermatogonia and spermatocytes in early meiotic prophase I. Alterations include predominantly non-CpG island sequences from both unique loci and repetitive elements. These modifications are progressive and are almost exclusively completed by the end of the pachytene spermatocyte stage. These studies better define the developmental timing of genome-wide DNA methylation pattern acquisition during male germ cell development.     

MicroRNA mediates DNA methylation of target genes

Small RNAs represented by microRNA (miRNA) plays important roles in plant development and responds to biotic and abiotic stresses. Previous studies have placed special emphasis on gene-repression mediated by miRNA. In this work, the DNA methylation pattern of microRNA genes (MIRs) was interrogated. Full-length cDNA and EST were used to confirm the entity of pri-miRNA. In parallel, miRNA in 24 nucleotides (nt) was pooled to detect chromatin modification effect by using bisulfite sequencing data. 97 MIRs were supported by full-length cDNA and 30 more were hit by EST. Notably, methylation levels of conserved MIRs were significantly lower than the non-conserved at all contexts (CG, CHG, and CHH). Additionally, a substantial part of 24-nt miRNA was able to induce target site methylation, providing a broader perspective for researchers.     

Exploring genome-wide DNA methylation profiles altered in hepatocellular carcinoma using Infinium HumanMethylation 450 BeadChips

Hepatocellular carcinoma (HCC) incidence has increased in the US and also has one of the fastest growing death rates of any cancer. The purpose of the current study was to discover novel genome-wide aberrant DNA methylation patterns in HCC tumors that are predominantly HCV-related. Infinium HumanMethylation 450K BeadChip arrays were used to examine genome-wide DNA methylation profiles in 66 pairs of HCC tumor and adjacent non-tumor tissues. After Bonferroni adjustment, a total of 130,512 CpG sites significantly differed in methylation level in tumor compared with non-tumor tissues, with 28,017 CpG sites hypermethylated and 102,495 hypomethylated in tumor tissues. Absolute tumor/non-tumor methylation differences ≥ 20% were found in 24.9% of the hypermethylated and 43.1% of the hypomethylated CpG sites; almost 10,000 CpG sites have ≥ 30% DNA methylation differences. Most (60.1%) significantly hypermethylated CpG sites are located in CpG islands, with 21.6% in CpG shores and 3.6% in shelves. In contrast, only a small proportion (8.2%) of significantly hypomethylated CpG sites are situated in islands, while most are found in open sea (60.2%), shore (17.3%) or shelf (14.3%) regions. A total of 2,568 significant CpG sites (2,441 hypermethylated and 127 hypomethylated) covering 589 genes are located within 684 differentially methylated regions defined as regions with at least two significant CpG sites displaying > 20% methylation differences in the same direction within 250-bp. The top 500 significant CpG sites can significantly distinguish HCC tumor from adjacent tissues with one misclassification. Within adjacent non-tumor tissues, we also identified 75 CpG sites significantly associated with gender, 228 with HCV infection, 17,207 with cirrhosis, and 56 with both HCV infection and cirrhosis after multiple comparisons adjustment. Aberrant DNA methylation profiles across the genome were identified in tumor tissues from US HCC cases that are predominantly related to HCV infection. These results demonstrate the significance of aberrant DNA methylation in HCC tumorigenesis.     

哺乳动物DNA甲基化修饰的动态调控机制

DNA甲基化是最主要的表观遗传修饰之一,主要发生在胞嘧啶第五位碳原子上,称为5-甲基胞嘧啶。哺乳动物DNA甲基化由从头DNA甲基转移酶DNMT3A/3B在胚胎发育早期建立。细胞分裂过程中甲基化模式的维持由DNA甲基转移酶DNMT1实现。TET家族蛋白氧化5-甲基胞嘧啶成为5-羟甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶,从而起始DNA的去甲基化过程。这些DNA甲基化修饰酶精确调节DNA甲基化的动态过程,在整个生命发育过程中发挥重要作用,其失调也与多种疾病发生密切相关。本文对近年来DNA甲基化修饰酶的结构与功能研究进行讨论。