Variation In Methylation Levels Research Articles

Tethered particle motion of the adaptation enzyme CheR in bacterial chemotaxis

Bacteria perform chemotactic adaptation by sequential modification of multiple modifiable sites on chemoreceptors through stochastic action of tethered adaptation enzymes (CheR and CheB). To study the molecular kinetics of this process, we measured the response to different concentrations of MeAsp for the Tar-only Escherichia coli strain. We found a strong dependence of the methylation rate on the methylation level and established a new mechanism of adaptation kinetics due to tethered particle motion of the methylation enzyme CheR. Experiments with various lengths of the C-terminal flexible chain in the Tar receptor further validated this mechanism. The tethered particle motion resulted in a CheR concentration gradient that ensures encounter-rate matching of the sequential modifiable sites. An analytical model of multisite catalytic reaction showed that this enables robustness of methylation to fluctuations in receptor activity or cell-to-cell variations in the expression of adaptation enzymes and reduces the variation in methylation level among individual receptors.

5-methylcytosine turnover: Mechanisms and therapeutic implications in cancer.

DNA methylation at the fifth position of cytosine (5mC) is one of the most studied epigenetic mechanisms essential for the control of gene expression and for many other biological processes including genomic imprinting, X chromosome inactivation and genome stability. Over the last years, accumulating evidence suggest that DNA methylation is a highly dynamic mechanism driven by a balance between methylation by DNMTs and TET-mediated demethylation processes. However, one of the main challenges is to understand the dynamics underlying steady state DNA methylation levels. In this review article, we give an overview of the latest advances highlighting DNA methylation as a dynamic cycling process with a continuous turnover of cytosine modifications. We describe the cooperative actions of DNMT and TET enzymes which combine with many additional parameters including chromatin environment and protein partners to govern 5mC turnover. We also discuss how mathematical models can be used to address variable methylation levels during development and explain cell-type epigenetic heterogeneity locally but also at the genome scale. Finally, we review the therapeutic implications of these discoveries with the use of both epigenetic clocks as predictors and the development of epidrugs that target the DNA methylation/demethylation machinery. Together, these discoveries unveil with unprecedented detail how dynamic is DNA methylation during development, underlying the establishment of heterogeneous DNA methylation landscapes which could be altered in aging, diseases and cancer.

Evaluating methylation of human ribosomal DNA at each CpG site reveals its utility for cancer detection using cell-free DNA.

Ribosomal deoxyribonucleic acid (DNA) (rDNA) repeats are tandemly located on five acrocentric chromosomes with up to hundreds of copies in the human genome. DNA methylation, the most well-studied epigenetic mechanism, has been characterized for most genomic regions across various biological contexts. However, rDNA methylation patterns remain largely unexplored due to the repetitive structure. In this study, we designed a specific mapping strategy to investigate rDNA methylation patterns at each CpG site across various physiological and pathological processes. We found that CpG sites on rDNA could be categorized into two types. One is within or adjacent to transcribed regions; the other is distal to transcribed regions. The former shows highly variable methylation levels across samples, while the latter shows stable high methylation levels in normal tissues but severe hypomethylation in tumors. We further showed that rDNA methylation profiles in plasma cell-free DNA could be used as a biomarker for cancer detection. It shows good performances on public datasets, including colorectal cancer [area under the curve (AUC) = 0.85], lung cancer (AUC = 0.84), hepatocellular carcinoma (AUC = 0.91) and in-house generated hepatocellular carcinoma dataset (AUC = 0.96) even at low genome coverage (<1×). Taken together, these findings broaden our understanding of rDNA regulation and suggest the potential utility of rDNA methylation features as disease biomarkers.

Chromosome X aneusomy and androgen receptor gene copy number aberrations in apocrine carcinoma of the breast

Carcinomas with apocrine differentiation (CAD) of the breast are rare tumours typically presenting high immunohistochemical expression of androgen receptor (AR) which is a target molecule for personalised therapy. To date, no studies have evaluated the genetic changes that are associated with AR immunohistochemical expression in CADs. The present work aims to characterise AR status in CADs. Twenty CAD tumours were studied with immunohistochemistry, in situ fluorescence hybridization and DNA methylation analysis, to evaluate AR expression and its regulator status. All tumours demonstrated high AR immunohistochemical expression, with over 95% of the neoplastic cells showing AR positivity in 19/20 cases. CADs showed AR gene copy loss in a percentage of neoplastic cells ranging from 5 to 84% (mean 48.93%). AR regulator genes, including the MAGE family, UXT and FLNA, presented variable methylation levels, but were mainly hypomethylated and therefore all transcriptionally active. The results of this study indicate that CADs present AR monosomy, paralleled by higher transcriptional activity of the gene with potential to influence response to AR deprivation therapy.

KRAB zinc finger protein diversification drives mammalian interindividual methylation variability

Most transposable elements (TEs) in the mouse genome are heavily modified by DNA methylation and repressive histone modifications. However, a subset of TEs exhibit variable methylation levels in genetically identical individuals, and this is associated with epigenetically conferred phenotypic differences, environmental adaptability, and transgenerational epigenetic inheritance. The evolutionary origins and molecular mechanisms underlying interindividual epigenetic variability remain unknown. Using a repertoire of murine variably methylated intracisternal A-particle (VM-IAP) epialleles as a model, we demonstrate that variable DNA methylation states at TEs are highly susceptible to genetic background effects. Taking a classical genetics approach coupled with genome-wide analysis, we harness these effects and identify a cluster of KRAB zinc finger protein (KZFP) genes that modifies VM-IAPs in trans in a sequence-specific manner. Deletion of the cluster results in decreased DNA methylation levels and altered histone modifications at the targeted VM-IAPs. In some cases, these effects are accompanied by dysregulation of neighboring genes. We find that VM-IAPs cluster together phylogenetically and that this is linked to differential KZFP binding, suggestive of an ongoing evolutionary arms race between TEs and this large family of epigenetic regulators. These findings indicate that KZFP divergence and concomitant evolution of DNA binding capabilities are mechanistically linked to methylation variability in mammals, with implications for phenotypic variation and putative paradigms of mammalian epigenetic inheritance.

The assessment of epigenetic diversity, differentiation, and structure in the 'Fuji' mutation line implicates roles of epigenetic modification in the occurrence of different mutant groups as well as spontaneous mutants.

The ‘Fuji’ line includes many varieties with a similar genetic background and consistent inducement factors with epigenetic occurrence, thus it may be considered an ideal candidate for epigenetic research. In this study, 91 bud mutations of ‘Fuji’ apple were used as the test materials. Using the genetic variation within ‘Fuji’ as the control, the characteristics of epigenetic variation at different levels in both varieties and mutant groups were examined. The results showed that: (1) the global genomic DNA methylation level of the 91 bud mutants of ‘Fuji’ ranged from 29.120%-45.084%, with an average of 35.910%. Internal cytosine methylation was the main DNA methylation pattern. Regarding the variation of methylation patterns of ‘Fuji’ mutants, the vast majority of loci maintained the original methylation pattern existed in ‘Fuji’. CHG methylation variation was the main type of variation; (2) the variation in methylation patterns between the mutant groups was greater than that of methylation levels. Among these patterns, the variation in CHG methylation patterns (including CHG hypermethylation and CHG demethylation) was expected to be dominant. The observed variation in methylation levels was more important in the Color mutant group; however, the variation in methylation patterns was more obvious in both the early maturation and Spur mutant groups. Moreover, the range of variation in the Early-maturation group was much wider than that in the Spur mutant group; (3) epigenetic diversity and genetic diversity were both low between the mutant groups. In the ‘Fuji’ mutant groups, there was few correlation between genetic and epigenetic variation, and epigenetic differentiation resulted in more loci with moderate or greater differentiation; (4) the purifying selection seemed to play a major role in the differentiation of different groups of ‘Fuji’ mutants (65.618%), but epigenetic diversity selection still occurred at nearly 35% of loci. Sixteen epigenetic outlier loci were detected.

Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs.

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs invivo.

Detailed methylation map of LINE-1 5'-promoter region reveals hypomethylated CpG hotspots associated with tumor tissue specificity.

BackgroundLong interspersed nuclear elements (LINE‐1) sequences constitute a substantial portion of the human genome, and their methylation often correlating with global genomic methylation. Previous studies have highlighted the feasibility of using LINE‐1 methylation to discriminate tumors from healthy tissues. However, most studies are based on only a few specific LINE‐1 CpG sites.MethodsHerein, we have performed a systematic fine‐scale analysis of methylation at 14 CpGs located in the 5′‐region of consensus LINE‐1, in bladder, colon, prostate, and gastric tumor tissues using a global degenerate approach.ResultsOur results reveal variable methylation levels between different CpGs, as well as some tissue‐specific differences. Trends toward hypomethylation were observed in all tumors types to certain degrees, showing statistically significance in bladder and prostate tumors. Our data points toward the presence of unique LINE‐1 DNA methylation patterns for each tumor type and tissue, indicating that not the same CpGs will be informative for testing in all tumor types.ConclusionThis study provides an accurate guide that will help to design further assays that could avoid artifacts and explain the variability of obtained LINE‐1 methylation values between different studies.

Identifying fenofibrate responsive CpG sites

As part of GAW20, we analyzed the familiality and variability of methylation to identify cytosine-phosphate-guanine (CpG) sites responsive to treatment with fenofibrate. Methylation was measured at approximately 450,000 sites in pedigree members, prior to and after 3 weeks of treatment. Initially, we aimed to identify responsive sites by analyzing the pre- and posttreatment methylation changes within individuals, but these data exhibited a confounding treatment/batch effect. We applied an alternative indirect approach by searching for CpG sites whose methylation levels exhibit a genetic response to the drug. We reasoned that these sites would exhibit highly familial and variable methylation levels posttreatment, but not pretreatment. Using a 0.1% threshold, posttreatment sibling correlation (scor) and standard deviation (SD) distributions share 16 outliers, while the corresponding pretreatment distributions share none. Comparing the pre- and posttreatment CpG outliers, 36 (8%) of SD distributions, and 449/450 (nearly 100%) of scor distributions differ. Combined, these results identify methylation sites within the KIAA1804 and ANAPC2 genes. Each gene also has a highly significant methylation quantitative trait locus (meQTL) (KIAA1804: p < 1e-200; ANAPC2: p < 3e-248), indicating that methylation levels at these CpG sites are driven by meQTL and fenofibrate.

Convergence of evidence from a methylome-wide CpG-SNP association study and GWAS of major depressive disorder

DNA methylation is an epigenetic modification that provides stability and diversity to the cellular phenotype. It is influenced by both genetic sequence variation and environmental factors, and can therefore potentially account for variation of heritable phenotypes and disorders. Therefore, methylome-wide association studies (MWAS) are promising complements to genome-wide association studies (GWAS) of genetic variants. Of particular interest are methylation sites (CpGs) that are created or destroyed by the alleles of single-nucleotide polymorphisms (SNPs), as these so-called CpG-SNPs may show variation in methylation levels on top of what can be explained by the sequence variation. Using sequencing-based data from 1132 major depressive disorder (MDD) cases and controls, we performed a MWAS of 970,414 common CpG-SNPs. The analysis identified 27 suggestively significant (P < 1.00 × 10−5) CpG-SNPs associations. Furthermore, the MWAS results were over-represented (odds ratios ranging 1.36–5.00; P ranging 4.9 × 10−3–8.1 × 10−2) among findings from three recent GWAS for MDD-related phenotypes. Overlapping loci included, e.g., ROBO2, ASIC2, and DCC. As the CpG-SNP analysis accounts for the number of alleles that creates CpGs, the methylation differences could not be explained by differences in allele frequencies. Thus, the results show that the MWAS and GWASs provide independent lines of evidence for the involvement of these loci in MDD. In conclusion, our methylation study of MDD contributes novel information about loci of relevance that complements previous findings and generates new hypothesis about MDD etiology, such as that the functional effects of genetic association may be partly mediated and/or enhanced by the methylation status in these loci.

Signatures of DNA Methylation across Insects Suggest Reduced DNA Methylation Levels in Holometabola.

It has been experimentally shown that DNA methylation is involved in the regulation of gene expression and the silencing of transposable element activity in eukaryotes. The variable levels of DNA methylation among different insect species indicate an evolutionarily flexible role of DNA methylation in insects, which due to a lack of comparative data is not yet well-substantiated. Here, we use computational methods to trace signatures of DNA methylation across insects by analyzing transcriptomic and genomic sequence data from all currently recognized insect orders. We conclude that: 1) a functional methylation system relying exclusively on DNA methyltransferase 1 is widespread across insects. 2) DNA methylation has potentially been lost or extremely reduced in species belonging to springtails (Collembola), flies and relatives (Diptera), and twisted-winged parasites (Strepsiptera). 3) Holometabolous insects display signs of reduced DNA methylation levels in protein-coding sequences compared with hemimetabolous insects. 4) Evolutionarily conserved insect genes associated with housekeeping functions tend to display signs of heavier DNA methylation in comparison to the genomic/transcriptomic background. With this comparative study, we provide the much needed basis for experimental and detailed comparative analyses required to gain a deeper understanding on the evolution and function of DNA methylation in insects.

Alterations in sperm DNA methylation patterns of oligospermic males

Aberrant in sperm DNA methylation patterns and histone modification play a role in male fecundity decline. This study was prepared to determine whether sperm DNA methylation at CpG dinucleotides is different in oligospermic males compared to proven fertile males and then to evaluate the correlation between the changes in sperm DNA methylation patterns and semen parameters of oligospermic males. A total of 165 males (64 proven fertile males “controls” and 101 oligospermic males “cases”) were included in the study. Three CpG sites have the highest difference in methylation levels (cg23081194, cg25750688, and cg04807108) were underwent to further analysis using deep bisulfite sequencing in 125 samples (44 controls and 81 cases). The results of a validation study showed that variation in methylation levels was found in more than one CpG site: there was a significant alteration in methylation levels at all CpGs tested within the UBE2G2 and cg25750688 site related amplicon (p≤0.0001), and at eight CpGs (CpG1, CpG3, CpG6, CpG8, CpG11, CpG13, CpG14, and CpG15) within the cg04807108 site related amplicon (p≤0.0001) in cases compared to controls. Besides, a significant correlation was found between the changes in the methylation levels at different CpGs and semen parameters of case group. In conclusion, this study showed that these sites have a significant alteration in sperm DNA methylation levels in oligospermic males compared to proven fertile males, and these changes correlated with semen parameters.

Aberrations in sperm dna methylation patterns of males suffer from infertility problems

To identify the aberrations in sperm DNA methylation patterns of males who have been unable to have children after ten years of attempting childbearing compared to males who have been proven fertile, and to assess the relationship between alterations in sperm DNA methylation levels and sperm parameters. A total of 108 semen samples were collected (65 male with infertility problems as cases and 43 proven fertile males as a control), during the period between May 2016 to August 2016. Thirty samples were subjected to 450K arrays as a screening study to identified the differentially methylated CpGs between case and control groups, and then three CpG sites have the highest difference in the methylation level (cg19169023, cg22963027, and cg21575308) which are located in TYRO3, CGβ, and FAM189A1 genes, respectively were selected to validate on 78 samples using deep bisulfite sequencing. The result of screening study showed that 15 CpG have significantly and consistently different in the DNA methylation level between cases and controls males. Only 8 of those (DNA methylation difference between 33.34% and 80.31%) do not overlap common annotated SNPs, and all of them linked directly with the genes body and CpG Island (TYRO3, CGβ, FAM189A1, GAA, PRRC2A, MAPK8IP3, PDE11A, and ANXA2). Whereas, the result of validation study showed that variation in methylation levels was found in more than one CpG: the DNA methylation levels at all CpGs tested within the TYRO3 gene-related amplicon showed significant differences in the case group compared to the control group (CpG1, P < 0.003, CpG2, P < 0.0001, CpG3, P < 0.003, and CpG4, P < 0.030). Moreover, one CpGs of the two CpGs tested within the CGβ gene-related amplicon was significantly different (CpG1, P < 0.0001) in the case group compared to the control group. In addition, a significant difference was found at two CpGs tested within the FAM189A1 gene-related amplicon between case and control groups (CpG1, P < 0.004, and CpG2, P < 0.002). A negative significant correlation was found between the percentages of total sperm motility, percentages of sperm progressive motility, percentages of sperm non-progressive motility, and percentages of sperm normal form and the methylation levels at CpG1 of the FAM189A1 gene-related amplicon (P < 0.0001, P < 0.002, P < 0.0001, and P < 0.001, respectively). This study identified CpGs related to TYRO3, CGβ, and FAM189A1 genes have alterations at methylation levels in sperm DNA from males, who have been unable to have children after ten years of attempting childbearing compared to proven fertile males. In addition, an association between changes in the methylation level at CpGs in FAM189A1 gene-related amplicon and sperm motility was found.

Association between alterations in DNA methylation level of spermatozoa at CpGs dinucleotide and male subfertility problems.

The purpose of this study was to assess the relationship between alterations in sperm DNA methylation levels and sperm count and sperm motility. Five CpG sites underwent deep bisulphite sequencing to validate the observed methylation difference in 78 samples (28 proven fertile males "controls," and 50 subfertile males "cases"). The results showed that variation in methylation levels was found in more than one CpG: the DNA methylation levels in CpG1, CpG2 and CpG3 of the PRRC2A gene-related amplicon showed high significant differences in the case group compared to the control group (p≤.0001, p≤.003, and p≤.0001 respectively). Moreover, three CpGs of the four CpGs tested within the ANXA2 gene-related amplicon (CpG1, CpG3 and CpG4) were significantly different (p≤.002, p≤.001, and p≤.0001, respectively) in the case group compared to the control group. In addition, a significant difference was found in seven CpGs of the twenty-two CpGs tested within the MAPK8Ip3 gene-related amplicon, besides six CpGs of the ten CpGs tested within the GAA gene-related amplicon between case and control groups. In conclusion, this study identifies that CpGs have a significantly different in methylation levels of sperm DNA for subfertile males.

Abstract 4360: Epigenetic alteration at HBV integrants is associated with methylation at flanking human genome

Abstract Integration of DNA viruses into the human genome plays an important role in various types of tumors, including hepatitis B virus (HBV)-related hepatocellular carcinoma. However, the molecular details and clinical impact of HBV integration on either human or HBV epigenomes are unknown. Here, we show that methylation of the integrated HBV DNA is related to the methylation status of the flanking human genome. We developed a next-generation sequencing-based method for structural methylation analysis of integrated viral genomes (denoted G-NaVI). This method is a novel approach that enables enrichment of viral fragments for sequencing using unique baits based on the sequence of the HBV genome. We detected integrated HBV sequences in the genome of the PLC/PRF/5 cell line and found variable levels of methylation within the integrated HBV genomes. Allele-specific methylation analysis revealed that the HBV genome often became significantly methylated when integrated into highly methylated host sites. After integration into unmethylated human genome regions such as promoters, however, the HBV DNA remains unmethylated and may eventually play an important role in tumorigenesis. The observed dynamic changes in DNA methylation of the host and viral genomes may functionally affect the biological behavior of HBV. These findings may impact public health given that millions of people worldwide are carriers of HBV. We also believe our assay will be a powerful tool to increase our understanding of the various types of DNA virus-associated tumorigenesis. Citation Format: Yoshiyuki Watanabe, Ritsuko Oikawa, Hiroyuki Yamamoto, Fumio Itoh. Epigenetic alteration at HBV integrants is associated with methylation at flanking human genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4360. doi:10.1158/1538-7445.AM2017-4360

A DNA methylation map of human cancer at single base-pair resolution

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

Aberrations in sperm DNA methylation patterns are associated with abnormalities in semen parameters of subfertile males

Infertility affects about 15% of couples worldwide, with approximately 7% of males suffering from infertility problems. This study was designed to assess the relationship between alterations in sperm DNA methylation patterns and semen parameters in subfertile males. Of a total of 108 males, 30 samples were subjected to 450K BeadChip arrays to evaluate the variation in DNA methylation level between cases and controls. Three CpG sites showed the highest difference in methylation levels (cg09737095, cg14271023, and cg17662493), which are located in the KCNJ5, MLPH, and SMC1β genes, respectively; these were selected for further analysis using deep bisulfite sequencing in 78 independent samples (21 proven fertile “controls”, and 57 subfertile “cases”). The results of a validation study showed that variation in methylation levels was found in more than one CpG site: there was a significant decrease in methylation levels at six CpGs (CpG1, CpG3, CpG4, CpG6, CpG7, and CpG8) in the KCNJ5 gene-related amplicon (p≤0.001, p≤0.009, p≤0.007, p≤0.007, p≤0.020, and p≤0.016, respectively), and at (CpG1, CpG2, and CpG4) in the MLPH gene-related amplicon (p≤0.003, p≤0.005, and p≤0.0001, respectively), while there was a significant increase in the methylation level at six out of eight CpGs in the SMC1β gene-related amplicon in cases compared to controls. Our results show that three CpGs have a significant difference in sperm DNA methylation levels in subfertile males compared to proven fertile males.

Epigenetic modifications at DMRs of placental genes are subjected to variations in normal gestation, pathological conditions and folate supplementation

Invasive placentation and cancer development shares many similar molecular and epigenetic pathways. Paternally expressed, growth promoting genes (SNRPN, PEG10 and MEST) which are known to play crucial role in tumorogenesis, are not well studied during placentation. This study reports for the first time of the impact of gestational-age, pathological conditions and folic acid supplementation on dynamic nature of DNA and histone methylation present at their differentially methylated regions (DMRs). Here, we reported the association between low DNA methylation/H3K27me3 and higher expression of SNRPN, PEG10 and MEST in highly proliferating normal early gestational placenta. Molar and preeclamptic placental villi, exhibited aberrant changes in methylation levels at DMRs of these genes, leading to higher and lower expression of these genes, respectively, in reference to their respective control groups. Moreover, folate supplementation could induce gene specific changes in mRNA expression in placental cell lines. Further, MEST and SNRPN DMRs were observed to show the potential to act as novel fetal DNA markers in maternal plasma. Thus, variation in methylation levels at these DMRs regulate normal placentation and placental disorders. Additionally, the methylation at these DMRs might also be susceptible to folic acid supplementation and has the potential to be utilized in clinical diagnosis.

Pan-cancer analysis of frequent DNA co-methylation patterns reveals consistent epigenetic landscape changes in multiple cancers

BackgroundDNA methylation is the major form of epigenetic modifications through which the cell regulates the gene expression and silencing. There have been extensive studies on the roles of DNA methylation in cancers, and several cancer drugs were developed targeting this process. However, DNA co-methylation cluster has not been examined in depth, and co-methylation in multiple cancer types has never been studied previously.ResultsIn this study, we applied newly developed lmQCM algorithm to mine co-methylation clusters using methylome data from 11 cancer types in TCGA database, and found frequent co-methylated gene clusters exist in these cancer types. Among the four identified frequent clusters, two of them separate the tumor sample from normal sample in 10 out of 11 cancer types, which indicates that consistent epigenetic landscape changes exist in multiple cancer types.ConclusionThis discovery provides new insight on the epigenetic regulation in cancers and leads to potential new direction for epigenetic biomarker and cancer drug discovery. We also found that genes commonly believed to be silenced via hypermethylation in cancers may still display highly variable methylation levels among cancer cells, and should be considered while using them as epigenetic biomarkers.

Discovery of potential DNA methylation markers for forensic tissue identification using bisulphite pyrosequencing.

The presence of specific body fluids at crime scenes could be linked with particular types of crime, therefore attributing a DNA profile to a specific tissue could increase the evidential significance of a match with a suspect. Current methodologies such as tissue-specific mRNA profiling are useful but drawbacks include low tissue specificity and applicability to degraded samples. In this study, the potential of 11 tissue-specific differentially methylated regions, initially identified following large-scale methylation analysis of whole blood, buccal cells and sperm, was explored in order to identify markers for blood, saliva and semen. Bisulphite pyrosequencing analysis supported previous findings, but tissue-specific differentially methylated regions for blood and buccal cells did not show enough specificity to be proposed as markers for blood and saliva, respectively. For some CpGs, a large inter-individual variation in methylation levels was also observed. Two of the semen markers (cg04382920 and cg11768416) were used for further validation on a large set of stains. These two semen-specific assays showed high sensitivity (as low as 50 pg) and stability. Future experiments will shed light on the usefulness of these markers in forensic casework.