Patterns of DNA methylation in the normal colon vary by anatomical location,gender, and age

Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Genomic organisation of DNA segments containing foldback sequences

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hydridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.     

DNA methylation variation of human-specific Alu repeats

DNA methylation is the major repression mechanism for human retrotransposons, such as the Alu family. Here, we have determined the methylation levels associated with 5238 loci belonging to 2 Alu subfamilies, AluYa5 and AluYb8, using high-throughput targeted repeat element bisulfite sequencing (HT-TREBS). The results indicate that ～90% of loci are repressed by high methylation levels. Of the remaining loci, many of the hypomethylated elements are found near gene promoters and show high levels of DNA methylation variation. We have characterized this variation in the context of tumorigenesis and interindividual differences. Comparison of a primary breast tumor and its matched normal tissue revealed early DNA methylation changes in ～1% of AluYb8 elements in response to tumorigenesis. Simultaneously, AluYa5/Yb8 elements proximal to promoters also showed differences in methylation of up to one order of magnitude, even between normal individuals. Overall, the current study demonstrates that early loss of methylation occurs during tumorigenesis in a subset of young Alu elements, suggesting their potential clinical relevance. However, approaches such as deep-bisulfite-sequencing of individual loci using HT-TREBS are required to distinguish clinically relevant loci from the background observed for AluYa5/Yb8 elements in general with regard to high levels of interindividual variation in DNA methylation.     

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small,intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.     

MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles

Aberrant DNA methylation is a common epigenetic alteration found in colorectal adenomas and cancers and plays a role in cancer initiation and progression. Aberrantly methylated DNA loci can also be found infrequently present in normal colon tissue, where they seem to have potential to be used as colorectal cancer (CRC) risk biomarkers. However, detection and precise quantification of the infrequent methylation events seen in normal colon is likely beyond the capability of commonly used PCR technologies. To determine the potential for methylated DNA loci as CRC risk biomarkers, we developed MethyLight droplet digital PCR (ddPCR) assays and compared their performance to the widely used conventional MethyLight PCR. Our analyses demonstrated the capacity of MethyLight ddPCR to detect a single methylated NTRK3 allele from among more than 3125 unmethylated alleles, 25-fold more sensitive than conventional MethyLight PCR. The MethyLight ddPCR assay detected as little as 19 and 38 haploid genome equivalents of methylated EVL and methylated NTRK3, respectively, which far exceeded conventional MethyLight PCR (379 haploid genome equivalents for both genes). When assessing methylated EVL levels in CRC tissue samples, MethyLight ddPCR reduced coefficients of variation (CV) to 6–65% of CVs seen with conventional MethyLight PCR. Importantly, we showed the ability of MethyLight ddPCR to detect infrequently methylated EVL alleles in normal colon mucosa samples that could not be detected by conventional MethyLight PCR. This study suggests that the sensitivity and precision of methylation detection by MethyLight ddPCR enhances the potential of methylated alleles for use as CRC risk biomarkers.     

DNA methylation aberrancies as a guide for surveillance and treatment of human cancers

DNA methylation aberrancies are hallmarks of human cancers and are characterized by global DNA hypomethylation of repetitive elements and non-CpG rich regions concomitant with locus-specific DNA hypermethylation. DNA methylation changes may result in altered gene expression profiles, most notably the silencing of tumor suppressors, microRNAs, endogenous retorviruses and tumor antigens due to promoter DNA hypermethylation, as well as oncogene upregulation due to gene-body DNA hypermethylation. Here, we review DNA methylation aberrancies in human cancers, their use in cancer surveillance and the interplay between DNA methylation and histone modifications in gene regulation. We also summarize DNA methylation inhibitors and their therapeutic effects in cancer treatment. In this context, we describe the integration of DNA methylation inhibitors with conventional chemotherapies, DNA repair inhibitors and immune-based therapies, to bring the epigenome closer to its normal state and increase sensitivity to other therapeutic agents to improve patient outcome and survival.     

Measurements of DNA Methylation at Seven Loci in Various Tissues of CD1 Mice

In humans, considerable variation in methylation at single loci and repetitive elements in various cells and tissues is observed. Recently, several inter- and intra-tissue correlations for DNA methylation have been reported. To investigate the extent and reproducibility of such correlations, we investigated inter- and intra-tissue methylation correlations among seven different loci in 9 different tissues in a population of 100 healthy seven-week-old CD1 outbred mice. We used a highly quantitative approach to measure methylation levels to high accuracy at two single loci in the alpha-actin and myosine light chain promoters, at three differentially methylated regions of the Peg3, Snrpn and Lit1 genes associated with imprinted loci, and at two repetitive elements in the Line-1 and IAP-LTR genes in the various tissues. In this population of mice, methylation at several loci was sex-associated and intra-tissue correlations among the studied loci were observed for brain and spleen. Inter-tissue correlations were rarely observed. To investigate method-dependent experimental variability, we re-analyzed the same spleen and tongue samples using SIRPH and pyrosequencing methods and reconfirmed intra-tissue correlations for spleen and sex-associated correlations for DNA methylation for tongue. When we repeated DNA methylation measurements for a second mouse population raised under similar conditions three months later, we did not detect sex-associated or intra-tissues correlations. Additional studies that examine large numbers of loci may be required to further understand the factors that influence stability of DNA methylation.     

Variable maternal methylation overlapping the nc886/vtRNA2-1 locus is locked between hypermethylated repeats and is frequently altered in cancer

Cancer is as much an epigenetic disease as a genetic one; however, the interplay between these two processes is unclear. Recently, it has been shown that a large proportion of DNA methylation variability can be explained by allele-specific methylation (ASM), either at classical imprinted loci or those regulated by underlying genetic variants. During a recent screen for imprinted differentially methylated regions, we identified the genomic interval overlapping the non-coding nc886 RNA (previously known as vtRNA2-1) as an atypical ASM that shows variable levels of methylation, predominantly on the maternal allele in many tissues. Here we show that the nc886 interval is the first example of a polymorphic imprinted DMR in humans. Further analysis of the region suggests that the interval subjected to ASM is approximately 2 kb in size and somatically acquired. An in depth analysis of this region in primary cancer samples with matching normal adjacent tissue from the Cancer Genome Atlas revealed that aberrant methylation in bladder, breast, colon and lung tumors occurred in approximately 27% of cases. Hypermethylation occurred more frequently than hypomethylation. Using additional normal-tumor paired samples we show that on rare occasions the aberrant methylation profile is due to loss-of-heterozygosity. This work therefore suggests that the nc886 locus is subject to variable allelic methylation that undergoes cancer-associated epigenetic changes in solid tumors.     

Association between hypermethylation of DNA repetitive elements in white blood cell DNA and early-onset colorectal cancer

Changes in the methylation levels of DNA from white blood cells (WBCs) are putatively associated with an elevated risk for several cancers. The aim of this study was to investigate the association between colorectal cancer (CRC) and the methylation status of three DNA repetitive elements in DNA from peripheral blood. WBC DNA from 539 CRC cases diagnosed before 60 years of age and 242 sex and age frequency-matched healthy controls from the Australasian Colorectal Cancer Family Registry were assessed for methylation across DNA repetitive elements Alu, LINE-1 and Sat2 using MethyLight. The percentage of methylated reference (PMR) of cases and controls was calculated for each marker. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multivariable logistic regression adjusted for potential confounders. CRC cases demonstrated a significantly higher median PMR for LINE-1 (p < 0.001), Sat2 (p < 0.001) and Alu repeats (p = 0.02) when compared with controls. For each of the DNA repetitive elements, individuals with PMR values in the highest quartile were significantly more likely to have CRC compared with those in the lowest quartile (LINE-1 OR = 2.34, 95%CI = 1.48–3.70; p < 0.001, Alu OR = 1.83, 95%CI = 1.17–2.86; p = 0.01, Sat2 OR = 1.72, 95%CI = 1.10–2.71; p = 0.02). When comparing the OR for the PMR of each marker across subgroups of CRC, only the Alu marker showed a significant difference in the 5-fluoruracil treated and nodal involvement subgroups (both p = 0.002). This association between increasing methylation levels of three DNA repetitive elements in WBC DNA and early-onset CRC is novel and may represent a potential epigenetic biomarker for early CRC detection.     

Variable DNA methylation of transposable elements: The case study of mouse Early Transposons

Phenotypic variation stems from both genetic and epigenetic differences between individuals. In order to elucidate how phenotypes are determined, it is necessary to understand the forces that generate variation in genome sequence as well as its epigenetic state. In both contexts, transposable elements (TEs) may play an important role. It is well established that TE activity is a major generator of genetic variation, but recent research also suggests that TEs contribute to epigenetic variation. Stochastic epigenetic silencing of some TE insertions in mice has been shown to cause phenotypic variability between individuals. However, the prevalence of this phenomenon has never been evaluated. Here, we use 18 insertions of a mouse Endogenous Retrovirus (ERV) family, the early transposons (ETns), to detect insertion-dependent determinants of DNA methylation levels and variability between both cells and individuals. We show that the structure and age of insertions influence methylation levels and variability, resulting in a subgroup of loci that displays unexpectedly high variability in methylation and suggesting stochastic events during methylation establishment. Despite variation in methylation according to the age and structure of each locus, homologous CpG sites show similar tendencies in methylation levels across loci, emphasizing the role of the insertion’s sequence in methylation determination. Our results show that differences in methylation of ETns between individuals is not a sporadic phenomenon and support the hypothesis that ERVs contribute to phenotypic variability through their stochastic silencing.     

Hypermethylation of tobacco heterochromatic loci in response to osmotic stress

 Plants have to cope with a number of envi-ronmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methylation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension culture, and the stress was elicited with NaCl or D-mannitol. Using the restriction enzymes MspI/HpaII and MboI/Sau3AI in combination with Southern hydridization we observed a reversible hypermethylation of the external cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucleotides within the CCGG motifs (HpaII sites) appeared to be fully methylated in tobacco DNA repetitive sequences under normal physiological conditions. The data suggest epigenetic changes in the plant genome based on de novo methylation of DNA in response to environmental stress. Received: 26 November 1996/Accepted: 20 December 1996     

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.     

Genome-wide methylation analysis identifies differentially methylated CpG loci associated with severe obesity in childhood

Childhood obesity is a major public health issue. Here we investigated whether differential DNA methylation was associated with childhood obesity. We studied DNA methylation profiles in whole blood from 78 obese children (mean BMI Z-score: 2.6) and 71 age- and sex-matched controls (mean BMI Z-score: 0.1). DNA samples from obese and control groups were pooled and analyzed using the Infinium HumanMethylation450 BeadChip array. Comparison of the methylation profiles between obese and control subjects revealed 129 differentially methylated CpG (DMCpG) loci associated with 80 unique genes that had a greater than 10% difference in methylation (P-value < 0.05). The top pathways enriched among the DMCpGs included developmental processes, immune system regulation, regulation of cell signaling, and small GTPase-mediated signal transduction. The associations between the methylation of selected DMCpGs with childhood obesity were validated using sodium bisulfite pyrosequencing across loci within the FYN, PIWIL4, and TAOK3 genes in individual subjects. Three CpG loci within FYN were hypermethylated in obese individuals (all P < 0.01), while obesity was associated with lower methylation of CpG loci within PIWIL4 (P = 0.003) and TAOK3 (P = 0.001). After building logistic regression models, we determined that a 1% increase in methylation in TAOK3, multiplicatively decreased the odds of being obese by 0.91 (95% CI: 0.86 – 0.97), and an increase of 1% methylation in FYN CpG3, multiplicatively increased the odds of being obese by 1.03 (95% CI: 0.99 – 1.07). In conclusion, these findings provide evidence that childhood obesity is associated with specific DNA methylation changes in whole blood, which may have utility as biomarkers of obesity risk.     

DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease

Genetic risk factors for chronic kidney disease (CKD) are being identified through international collaborations. By comparison, epigenetic risk factors for CKD have only recently been considered using population-based approaches. DNA methylation is a major epigenetic modification that is associated with complex diseases, so we investigated methylome-wide loci for association with CKD. A total of 485,577 unique features were evaluated in 255 individuals with CKD (cases) and 152 individuals without evidence of renal disease (controls). Following stringent quality control, raw data were quantile normalized and β values calculated to reflect the methylation status at each site. The difference in methylation status was evaluated between cases and controls with resultant P values adjusted for multiple testing. Genes with significantly increased and decreased levels of DNA methylation were considered for biological relevance by functional enrichment analysis using KEGG pathways in Partek Genomics Suite. Twenty-three genes, where more than one CpG per loci was identified with P_adjusted < 10⁻⁸, demonstrated significant methylation changes associated with CKD and additional support for these associated loci was sought from published literature. Strong biological candidates for CKD that showed statistically significant differential methylation include CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2, and PRKAG2 genes; several genes are differentially methylated in kidney tissue and RNA-seq supports a functional role for differential methylation in ELMO1 and PRKAG2 genes. This study reports the largest, most comprehensive, genome-wide quantitative evaluation of DNA methylation for association with CKD. Evidence confirming methylation sites influence development of CKD would stimulate research to identify epigenetic therapies that might be clinically useful for CKD.     

Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements

Close to 50% of the human genome harbors repetitive sequences originally derived from mobile DNA elements, and in normal cells, this sequence compartment is tightly regulated by epigenetic silencing mechanisms involving chromatin-mediated repression. In cancer cells, repetitive DNA elements suffer abnormal demethylation, with potential loss of silencing. We used a genome-wide microarray approach to measure DNA methylation changes in cancers of the head and neck and to compare these changes to alterations found in adjacent non-tumor tissues. We observed specific alterations at thousands of small clusters of CpG dinucleotides associated with DNA repeats. Among the 257,599 repetitive elements probed, 5% to 8% showed disease-related DNA methylation alterations. In dysplasia, a large number of local events of loss of methylation appear in apparently stochastic fashion. Loss of DNA methylation is most pronounced for certain members of the SVA, HERV, LINE-1P, AluY, and MaLR families. The methylation levels of retrotransposons are discretely stratified, with younger elements being highly methylated in healthy tissues, while in tumors, these young elements suffer the most dramatic loss of methylation. Wilcoxon test statistics reveals that a subset of primate LINE-1 elements is demethylated preferentially in tumors, as compared to non-tumoral adjacent tissue. Sequence analysis of these strongly demethylated elements reveals genomic loci harboring full length, as opposed to truncated elements, while possible enrichment for functional LINE-1 ORFs is weaker. Our analysis suggests that, in non-tumor adjacent tissues, there is generalized and highly variable disruption of epigenetic control across the repetitive DNA compartment, while in tumor cells, a specific subset of LINE-1 retrotransposons that arose during primate evolution suffers the most dramatic DNA methylation alterations.     

Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study

Many epidemiologic studies of environmental exposures and disease susceptibility measure DNA methylation in white blood cells (WBC). Some studies are also starting to use saliva DNA as it is usually more readily available in large epidemiologic studies. However, little is known about the correlation of methylation between WBC and saliva DNA. We examined DNA methylation in three repetitive elements, Sat2, Alu, and LINE-1, and in four CpG sites, including AHRR (cg23576855, cg05575921), cg05951221 at 2q37.1, and cg11924019 at CYP1A1, in 57 girls aged 6–15 years with blood and saliva collected on the same day. We measured all DNA methylation markers by bisulfite-pyrosequencing, except for Sat2 and Alu, which were measured by the MethyLight assay. Methylation levels measured in saliva DNA were lower than those in WBC DNA, with differences ranging from 2.8% for Alu to 14.1% for cg05575921. Methylation levels for the three repetitive elements measured in saliva DNA were all positively correlated with those in WBC DNA. However, there was a wide range in the Spearman correlations, with the smallest correlation found for Alu (0.24) and the strongest correlation found for LINE-1 (0.73). Spearman correlations for cg05575921, cg05951221, and cg11924019 were 0.33, 0.42, and 0.79, respectively. If these findings are replicated in larger studies, they suggest that, for selected methylation markers (e.g., LINE-1), methylation levels may be highly correlated between blood and saliva, while for others methylation markers, the levels may be more tissue specific. Thus, in studies that differ by DNA source, each interrogated site should be separately examined in order to evaluate the correlation in DNA methylation levels across DNA sources.