High-resolution mapping of DNA methylation in human genome using oligonucleotide tiling array

DNA methylation is an epigenetic mark crucial in regulation of gene expression. Aberrant DNA methylation causes silencing of tumor suppressor genes and promotes chromosomal instability in human cancers. Most of previous studies for DNA methylation have focused on limited genomic regions, such as selected genes or promoter CpG islands (CGIs) containing recognition sites of methylation-sensitive restriction enzymes. Here, we describe a method for high-resolution analysis of DNA methylation using oligonucleotide tiling arrays. The input material is methylated DNA immunoprecipitated with anti-methylcytosine antibodies. We examined the ENCODE region (∼1% of human genome) in three human colorectal cancer cell lines and identified over 700 candidate methylated sites (CMS), where 24 of 25 CMS selected randomly were subsequently verified by bisulfite sequencing. CMS were enriched in the 5′ regulatory regions and the 3′ regions of genes. We also compared DNA methylation patterns with histone H3 and H4 acetylation patterns in the HOXA cluster region. Our analysis revealed no acetylated histones in the hypermethylated region, demonstrating reciprocal relationship between DNA methylation and histone H3 and H4 acetylation. Our method recognizes DNA methylation with little bias by genomic location and, therefore, is useful for comprehensive high-resolution analysis of DNA methylation providing new findings in the epigenomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Breast cancer family history and allele-specific DNA methylation in the legacy girls study

Family history, a well-established risk factor for breast cancer, can have both genetic and environmental contributions. Shared environment in families as well as epigenetic changes that also may be influenced by shared genetics and environment may also explain familial clustering of cancers. Epigenetic regulation, such as DNA methylation, can change the activity of a DNA segment without a change in the sequence; environmental exposures experienced across the life course can induce such changes. However, genetic-epigenetic interactions, detected as methylation quantitative trait loci (mQTLs; a.k.a. meQTLs) and haplotype-dependent allele-specific methylation (hap-ASM), can also contribute to inter-individual differences in DNA methylation patterns. To identify differentially methylated regions (DMRs) associated with breast cancer susceptibility, we examined differences in white blood cell DNA methylation in 29 candidate genes in 426 girls (ages 6–13 years) from the LEGACY Girls Study, 239 with and 187 without a breast cancer family history (BCFH). We measured methylation by targeted massively parallel bisulfite sequencing (bis-seq) and observed BCFH DMRs in two genes: ESR1 (Δ4.9%, P = 0.003) and SEC16B (Δ3.6%, P = 0.026), each of which has been previously implicated in breast cancer susceptibility and pubertal development. These DMRs showed high inter-individual variability in methylation, suggesting the presence of mQTLs/hap-ASM. Using single nucleotide polymorphisms data in the bis-seq amplicon, we found strong hap-ASM in SEC16B (with allele specific-differences ranging from 42% to 74%). These findings suggest that differential methylation in genes relevant to breast cancer susceptibility may be present early in life, and that inherited genetic factors underlie some of these epigenetic differences.     

Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing

Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation.     

Epigenetic regulation of microRNAs in cancer: an integrated review of literature

MicroRNAs (miRNAs) belong to the heterogeneous class of non-coding RNAs (ncRNAs) that regulate the translation and degradation of target mRNAs, and control approximately 30% of human genes. MiRNA genes might be silenced in human tumors (oncomiRs) by aberrant hypermethylation of CpG islands that encompass or lie adjacent to miRNA genes and/or by histone modifications. We performed literature search for research articles describing epigenetically regulated miRNAs in cancer and identified 45 studies that were published between 2006 and 7/2010. The data from those papers are fragmented and methodologically heterogeneous and our work represents first systematic review towards to integration of diverse sets of information. We reviewed the methods used for detection of miRNA epigenetic regulation, which comprise bisulfite genomic sequencing PCR (BSP), bisulfite pyrosequencing, methylation specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation sensitive single nucleotide primer extension (Ms-SNuPE), MassARRAY technique and some modifications of those methods. This integrative study revealed 122 miRNAs that were reported to be epigenetically regulated in 23 cancer types. Compared to protein coding genes, human oncomiRs showed an order of magnitude higher methylation frequency (11.6%; 122/1048 known miRNAs). Nearly half, (45%; 55/122) epigenetically regulated miRNAs were associated with different cancer types, but other 55% (67/122) miRNAs were present in only one cancer type and therefore representing cancer-specific biomarker potential. The data integration revealed miRNA epigenomic hot spots on the chromosomes 1q, 7q, 11q, 14q and 19q. CpG island analysis of corresponding miRNA precursors (pre-miRNAs) revealed that 20% (26/133) of epigenetically regulated miRNAs had a CpG island within the range of 5kb upstream, among them 14% (19/133) of miRNAs resided within the CpG island. Our integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.     

Genome‐wide DNA methylation analysis using next‐generation sequencing to reveal candidate genes responsible for boar taint in pigs

Boar taint (BT) is an offensive flavor observed in non‐castrated male pigs that reduces the carcass price. Surgical castration effectively avoids the taint but is associated with animal welfare concerns. The functional annotation of farm animal genomes for understanding the biology of complex traits can be used in the selection of breeding animals to achieve favorable phenotypic outcomes. The characterization of pig epigenomes/methylation changes between animals with high and low BT and genome‐wide epigenetic markers that can predict BT are lacking. Reduced representation bisulfite sequencing of DNA methylation patterns based on next‐generation sequencing is an efficient technology to identify candidate epigenetic biomarkers associated with BT. Three different BT levels were analyzed using reduced representation bisulfite sequencing data to calculate the methylation levels of cytosine and guanine dinucleotide (CpG) sites. The co‐analysis of differentially methylated CpG sites identified by this study and differentially expressed genes identified by a previous study found 32 significant co‐located genes. The joint analysis of GO terms and pathways revealed that methylation and gene expression of seven candidate genes were associated with BT; in particular, FASN plays a key role in fatty acid biosynthesis, and PEMT might be involved in estrogen regulation and the development of BT. This study is the first to report the genome‐wide DNA methylation profiles of BT in pigs using next‐generation sequencing and summarize candidate genes associated with epigenetic markers of BT, which could contribute to the understanding of the functional biology of BT traits and selective breeding of pigs against BT based on epigenetic biomarkers.     

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations.     

Maternal cocaine administration in mice alters DNA methylation and gene expression in hippocampal neurons of neonatal and prepubertal offspring

Previous studies documented significant behavioral changes in the offspring of cocaine-exposed mothers. We now explore the hypothesis that maternal cocaine exposure could alter the fetal epigenetic machinery sufficiently to cause lasting neurochemical and functional changes in the offspring. Pregnant CD1 mice were administered either saline or 20 mg/kg cocaine twice daily on gestational days 8-19. Male pups from each of ten litters of the cocaine and control groups were analyzed at 3 (P3) or 30 (P30) days postnatum. Global DNA methylation, methylated DNA immunoprecipitation followed by CGI(2) microarray profiling and bisulfite sequencing, as well as quantitative real-time RT-PCR gene expression analysis, were evaluated in hippocampal pyramidal neurons excised by laser capture microdissection. Following maternal cocaine exposure, global DNA methylation was significantly decreased at P3 and increased at P30. Among the 492 CGIs whose methylation was significantly altered by cocaine at P3, 34% were hypermethylated while 66% were hypomethylated. Several of these CGIs contained promoter regions for genes implicated in crucial cellular functions. Endogenous expression of selected genes linked to the abnormally methylated CGIs was correspondingly decreased or increased by as much as 4-19-fold. By P30, some of the cocaine-associated effects at P3 endured, reversed to opposite directions, or disappeared. Further, additional sets of abnormally methylated targets emerged at P30 that were not observed at P3. Taken together, these observations indicate that maternal cocaine exposure during the second and third trimesters of gestation could produce potentially profound structural and functional modifications in the epigenomic programs of neonatal and prepubertal mice.     

Epigenetic suppression of the anti-aging gene KLOTHO in human prostate cancer cell lines

KLOTHO was originally identified as an aging-suppressor gene that causes a human aging-like phenotype when tested in KLOTHO-deficient-mice. Recent evidence suggests that KLOTHO functions as a tumor suppressor by inhibiting Wnt signaling. KLOTHO gene silencing, including DNA methylation, has been observed in some human cancers. Aberrant activation of Wnt signaling plays a significant role in aging, and its silencing may be related to prostate cancer and other types of cancers. Thus, we investigated whether the expression of the anti-aging gene KLOTHO was associated with epigenetic changes in prostate cancer cell lines. KLOTHO mRNA was detected in the 22Rv1 cell line while it was not detected in DU145 and PC-3 cell lines. The restoration of KLOTHO mRNA in the DU145 and PC-3 cell lines was induced with a DNA methyltransferase inhibitor. Methylation-specific PCR was performed to determine the specific CpG sites in the KLOTHO promoter responsible for expression. In addition, the level of methylation was assessed in each CpG by performing bisulfite sequencing and quantitative pyrosequencing analysis. The results suggested a remarkable inverse relationship between KLOTHO expression and promoter methylation in prostate cancer cell lines.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Development of a Novel Output Value for Quantitative Assessment in Methylated DNA Immunoprecipitation-CpG Island Microarray Analysis

In DNA methylation microarray analysis, quantitative assessment of intermediate methylation levels in samples with various global methylation levels is still difficult. Here, specifically for methylated DNA immunoprecipitation-CpG island (CGI) microarray analysis, we developed a new output value. The signal log ratio reflected the global methylation levels, but had only moderate linear correlation (r = 0.72) with the fraction of DNA molecules immunoprecipitated. By multiplying the signal log ratio using a coefficient obtained from the probability value that took account of signals in neighbouring probes, its linearity was markedly improved (r = 0.94). The new output value, Me value, reflected the global methylation level, had a strong correlation also with the fraction of methylated CpG sites obtained by bisulphite sequencing (r = 0.88), and had an accuracy of 71.8 and 83.8% in detecting completely methylated and unmethylated CGIs. Analysis of gastric cancer cell lines using the Me value showed that methylation of CGIs in promoters and gene bodies was associated with low and high, respectively, gene expression. The degree of demethylation of promoter CGIs after 5-aza-2''-deoxycytidine treatment had no association with that of induction of gene expression. The Me value was considered to be useful for analysis of intermediate methylation levels of CGIs.     

Unmethylated state of 5′ upstream CpG islands may be necessary but not sufficient for the testis-enriched expression of ZNF230/Znf230

The testis-enriched genes ZNF230/Znf230 are located on human chromosome 11p15/mouse chromosome 7 near conserved imprinting control regions. Typical CpG islands (CGIs) extend from the promoter to the first exon in each of these genes. To investigate the correlation between the methylation status of the above CGIs and the expression patterns of the two genes, we performed bisulfite genomic sequencing of genomic DNA from human and mouse tissues and cells. The results showed that the CGIs of ZNF230/Znf230 were completely unmethylated in all selected tissues and cells, regardless of the expression levels of the two genes. Further experiments using Znf230-second-exon-knockout mice to investigate the imprinting status of Znf230 showed that its expression was not affected by genomic imprinting. However, an in vitro methylation assay illustrated that the methylation of these CpG sites could repress the expression of the luciferase reporter gene. Furthermore, chromatin immunoprecipitation with anti-Specificity protein 1 (Sp1) antibody showed that Sp1 could bind to the CGIs in the ZNF230/Znf230 gene promoter. Thus, we propose that the unmethylated state of ZNF230/Znf230 CGIs may be a prerequisite for their expression but not sufficient for their abundant expression in the testis, and that Sp1 binding may be one factor involved in preserving the methylation-free state of ZNF230/Znf230 CGIs.     

Effects of genome architecture and epigenetic factors on susceptibility of promoter CpG islands to aberrant DNA methylation induction

Aberrant DNA methylation is induced at specific promoter CpG islands (CGIs) in contrast with mutations. The specificity is influenced by genome architecture and epigenetic factors, but their relationship is still unknown. In this study, we isolated promoter CGIs susceptible and resistant to aberrant methylation induction during prostate and breast carcinogenesis. The effect of genome architecture was more evident for promoter CGIs susceptible in both of the two tissues than for promoter CGIs susceptible only in one tissue. Multivariate analysis of promoter CGIs with tissue-nonspecific susceptibility showed that genome architecture, namely a remote location from SINE (OR = 5.98; 95% CI = 2.33-15.34) and from LINE (OR = 2.08; 95% CI = 1.03-4.21), was associated with increased susceptibility, independent of epigenetic factors such as the presence of RNA polymerase II (OR = 0.09; 95% CI = 0.02-0.48) and H3K27me3 (OR = 3.28; 95% CI = 1.17-9.21). These results showed that methylation susceptibility of promoter CGIs is determined both by genome architecture and epigenetic factors, independently.