Cytosine Methylation Profiling Research Articles

Frequency of cytosine methylation in the adjacent regions of soybean retrotransposon SORE-1 depends on chromosomal location.

Mobilization of transposable elements (TEs) is suppressed by epigenetic mechanisms involving cytosine methylation. However, few studies have focused on clarifying relationships between epigenetic influences of TEs on the adjacent DNA regions and time after insertion of TEs into the genome and/or their chromosomal location. Here we addressed these issues using soybean retrotransposon SORE-1. We analyzed SORE-1, inserted in exon 1 of the GmphyA2 gene, one of the newest insertions in this family so far identified. Cytosine methylation was detected in this element but was barely present in the adjacent regions. These results were correlated, respectively, with the presence and absence of the production of short interfering RNAs. Cytosine methylation profiles of 74 SORE-1 elements in the Williams 82 reference genome indicated that methylation frequency in the adjacent regions of SORE-1 was profoundly higher in pericentromeric regions than in euchromatic chromosome arms and was only weakly correlated with the length of time after insertion into the genome. Notably, the higher level of methylation in the 5' adjacent regions of SORE-1 coincided with the presence of repetitive elements in pericentromeric regions. Together, these results suggest that epigenetic influence of SORE-1 on the adjacent regions is influenced by its location on the chromosome.

Comparative DNA Methylome of Phytoplasma Associated Retrograde Metamorphosis in Sesame (Sesamum indicum L.).

Simple SummarySesame (Sesamum indicum L.) is an important oilseed crop that is well known for its highly nutritious oil content. Due to its high oil content and nutritional properties, sesame is also known as the ‘queen of oilseeds’. Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. Sesame phyllody is the leading biotic constraint drastically affecting sesame productivity and resulting in yield losses of up to 80% in major sesame-producing countries in the world. In this study, we explored the role of DNA methylation in phytoplasma infection. Whole Genome Bisulfite Sequencing and a Quantitative analysis of DNA methylation using real-time PCR (qAMP) revealed an alteration of methylation pattern upon phytoplasma infection. Few selected development and defense-related loci were either hypo- or hypermethylated. We hereby report the first methylome study in healthy and phytoplasma-infected sesame. Our study provides fundamental insights into the role of DNA methylation in the symptom development of phytoplasma infection in sesame plants.Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. The mechanism of the dramatic conversion of flowers to leafy structures leading to yield losses and the drastic reduction in leaf size due to Phytoplasma infection remains yet to be identified. Cytosine methylation profiles of healthy and infected sesame plants studied using Whole Genome Bisulfite Sequencing (WGBS) and Quantitative analysis of DNA methylation with the real-time PCR (qAMP) technique revealed altered DNA methylation patterns upon infection. Phyllody was associated with global cytosine hypomethylation, though predominantly in the CHH (where H = A, T or C) context. Interestingly, comparable cytosine methylation levels were observed between healthy and little leaf-affected plant samples in CG, CHG and CHH contexts. Among the different genomic fractions, the highest number of differentially methylated Cytosines was found in the intergenic regions, followed by promoter, exonic and intronic regions in decreasing order. Further, most of the differentially methylated genes were hypomethylated and were mainly associated with development and defense-related processes. Loci for STOREKEEPER protein-like, a DNA-binding protein and PP2-B15, an F-Box protein, responsible for plugging sieve plates to maintain turgor pressure within the sieve tubes were found to be hypomethylated by WGBS, which was confirmed by methylation-dependent restriction digestion and qPCR. Likewise, serine/threonine-protein phosphatase-7 homolog, a positive regulator of cryptochrome signaling involved in hypocotyl and cotyledon growth and probable O-methyltransferase 3 locus were determined to be hypermethylated. Phytoplasma infection-associated global differential methylation as well as the defense and development-related loci reported here for the first time significantly elucidate the mechanism of phytoplasma-associated disease development.

Leukemia stemness and co-occurring mutations drive resistance to IDH inhibitors in acute myeloid leukemia

Allosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we perform multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 60 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis reveals that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1/CEBPA or RAS-RTK pathway genes is the main driver of acquired resistance to IDHi, along with BCOR, homologous IDH gene, and TET2. These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML.

TET1 upregulation drives cancer cell growth through aberrant enhancer hydroxymethylation of HMGA2 in hepatocellular carcinoma

Ten‐eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1‐upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast‐like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5‐hmC) profiling and found that 5‐hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4–monomethylated, where the H3K27‐acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast‐like HCC.

The genome of the Cauliflower mosaic virus, a plant pararetrovirus, is highly methylated in the nucleus.

Cytosine methylation is an important defense against invasive DNAs. Here, cytosine methylation profiles of a plant pararetrovirus, Cauliflower mosaic virus (CaMV), were investigated. Nuclear CaMV DNA is highly methylated throughout the genome including at transcription regulatory regions, but the virion DNA is unmethylated. Invitro CG methylation of the viral 35S promoter reduces transcription from the downstream gene. Although nuclear CaMV DNA is highly methylated, its transcripts are accumulated in the nucleus. The data suggest that a small population of unmethylated viral genomes produced through reverse transcription are constantly delivered back to the nucleus. Small RNA profiles suggest that methylation of the CaMV DNA may be due to denovo methylation through 21-, 22-, and 24-nt small RNAs with adenines at their 5' terminus.

Plant-RRBS: DNA Methylome Profiling Adjusted to Plant Genomes, Utilizing Efficient Endonuclease Combinations, for Multi-Sample Studies.

In plants, methylation at cytosines often leads to changes in gene expression and inactivation of transposable elements. Changes in cytosine methylation (epimutations) might produce epialleles with distinct phenotypes. We present a genome-wide cytosine methylation profiling method based on bisulfite conversion and next-generation sequencing, which is applicable for plant species with available reference genomes. This so-called plant-RRBS profiling method reproducibly covers specific genomic regions and enriches for coverage of cytosine positions that are suitable for comparative analyses in multi-sample studies in basic biology and breeding studies. The plant-RRBS workflow consists of genomic DNA digestion with coverage-efficient restriction endonuclease combinations followed by a performant library generation and next-generation sequencing and a straightforward, publically available methylation data processing pipeline. Plant-RRBS has a twofold higher ratio of cytosine coverage per coveredgenome as compared to whole-genome bisulfite sequencing, covering tens of millions of cytosine positions, and allows detection of differential cytosine methylation, which was evaluated using rice epilines.

DNMT1 in Six2 Progenitor Cells Is Essential for Transposable Element Silencing and Kidney Development.

Cytosine methylation of regulatory regions, such as promoters and enhancers, plays a key role in regulating gene expression, however, its role in kidney development has not been analyzed. To identify functionally important epigenome-modifying enzymes and genome regions where methylation modifications are functionally important for kidney development, we performed genome-wide methylation analysis, expression profiling, and systematic genetic targeting of DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) and Ten-eleven translocation methylcytosine hydroxylases (Tet2) in nephron progenitor cells (Six2Cre) in mice. Genome-wide methylome analysis indicated dynamic changes on promoters and enhancers during development. Six2CreDnmt3af/f, Six2CreDnmt3bf/f, and Six2CreTet2f/f mice showed no significant structural or functional renal abnormalities. In contrast, Six2CreDnmt1f/f mice died within 24 hours of birth, from a severe kidney developmental defect. Genome-wide methylation analysis indicated a marked loss of methylation of transposable elements. RNA sequencing detected endogenous retroviral transcripts. Expression of intracellular viral sensing pathways (RIG-I), early embryonic, nonrenal lineage genes and increased cell death contributed to the phenotype development. In podocytes, loss of Dnmt1, Dnmt3a, Dnmt3b, or Tet2 did not lead to functional or structural differences at baseline or after toxic injury. Genome-wide cytosine methylation and gene expression profiling showed that by silencing embryonic, nonrenal lineage genes and transposable elements, DNMT1-mediated cytosine methylation is essential for kidney development.

Advanced aging phenotype is revealed by epigenetic modifications in rat liver after in utero malnutrition.

SummaryAdverse environmental exposures of mothers during fetal period predispose offspring to a range of age‐related diseases earlier in life. Here, we set to determine whether a deregulated epigenetic pattern is similar in young animals whose mothers’ nutrition was modulated during fetal growth to that acquired during normal aging in animals. Using a rodent model of maternal undernutrition (UN) or overnutrition (ON), we examined cytosine methylation profiles of liver from young female offspring and compared them to age‐matched young controls and aged (20‐month‐old) animals. HELP‐tagging, a genomewide restriction enzyme and sequencing assay demonstrates that fetal exposure to two different maternal diets is associated with nonrandom dysregulation of methylation levels with profiles similar to those seen in normal aging animals and occur in regions mapped to genes relevant to metabolic diseases and aging. Functional consequences were assessed by gene expression at 9 weeks old with more significant changes at 6 months of age. Early developmental exposures to unfavorable maternal diets result in altered methylation profiles and transcriptional dysregulation in Prkcb, Pc, Ncor2, and Smad3 that is also seen with normal aging. These Notch pathway and lipogenesis genes may be useful for prediction of later susceptibility to chronic disease.

Methylation-sensitive amplified polymorphism-based genome-wide analysis of cytosine methylation profiles in Nicotiana tabacum cultivars.

This study aimed to investigate cytosine methylation profiles in different tobacco (Nicotiana tabacum) cultivars grown in China. Methylation-sensitive amplified polymorphism was used to analyze genome-wide global methylation profiles in four tobacco cultivars (Yunyan 85, NC89, K326, and Yunyan 87). Amplicons with methylated C motifs were cloned by reamplified polymerase chain reaction, sequenced, and analyzed. The results show that geographical location had a greater effect on methylation patterns in the tobacco genome than did sampling time. Analysis of the CG dinucleotide distribution in methylation-sensitive polymorphic restriction fragments suggested that a CpG dinucleotide cluster-enriched area is a possible site of cytosine methylation in the tobacco genome. The sequence alignments of the Nia1 gene (that encodes nitrate reductase) in Yunyan 87 in different regions indicate that a C-T transition might be responsible for the tobacco phenotype. T-C nucleotide replacement might also be responsible for the tobacco phenotype and may be influenced by geographical location.

HSC commitment–associated epigenetic signature is prognostic in acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by disruption of HSC and progenitor cell differentiation. Frequently, AML is associated with mutations in genes encoding epigenetic modifiers. We hypothesized that analysis of alterations in DNA methylation patterns during healthy HSC commitment and differentiation would yield epigenetic signatures that could be used to identify stage-specific prognostic subgroups of AML. We performed a nano HpaII-tiny-fragment-enrichment-by-ligation-mediated-PCR (nanoHELP) assay to compare genome-wide cytosine methylation profiles between highly purified human long-term HSC, short-term HSC, common myeloid progenitors, and megakaryocyte-erythrocyte progenitors. We observed that the most striking epigenetic changes occurred during the commitment of short-term HSC to common myeloid progenitors and these alterations were predominantly characterized by loss of methylation. We developed a metric of the HSC commitment–associated methylation pattern that proved to be highly prognostic of overall survival in 3 independent large AML patient cohorts, regardless of patient treatment and epigenetic mutations. Application of the epigenetic signature metric for AML prognosis was superior to evaluation of commitment-based gene expression signatures. Together, our data define a stem cell commitment–associated methylome that is independently prognostic of poorer overall survival in AML.

Epigenetic Deregulation In Relapsed Acute Myeloid Leukemia

Treatment failure in Acute Myeloid Leukemia (AML) is attributed in many cases to relapsed disease. Relapsed AML is a fundamental clinical challenge since most patients have poor clinical outcomes. The exact biological basis of AML relapse remains unclear. Genetic clonal evolution is widely believed to underlie the emergence of chemotherapy resistant clones. However, only limited, predominantly non-overlapping, somatic mutations and copy number aberrations were found to occur upon AML relapse. Furthermore, in a subset of cases, no relapse specific somatic mutations or copy number aberrations were identified. This suggests a role for other mechanisms in relapsed AML. We hypothesize that epigenetic plasticity and deregulation contributes to the pathogenesis of relapse in AML.To explore this notion, we performed a genome scale epigenetic and genetic analysis of thirty-nine paired diagnosis and relapsed AML human patient samples using exome capture, RNA-seq and ERRBS for DNA methylation sequencing. Exome capture was performed on each patient's germline DNA as well. Exome capture revealed only a limited number of known recurrent somatic mutations acquired upon disease relapse, in agreement with previous reports. In contrast, upon disease relapse we identified thousands of statistically significant changes in cytosine methylation patterns. Globally, the majority of patients (85%) displayed striking predominance of DNA hypermethylation (p= 1.00433e-05, binomial test for equality of proportions) upon disease relapse. Notably a smaller set of patients displayed the opposite epigenetic phenotype with prominent loss of cytosine methylation. While differential methylation in the hypermethylated group of patients localized predominantly to CpG islands, the majority of differential methylation in the hypomethylated group localized to regions lacking both CpG islands and shores. In spite of these two distinct overall cytosine methylation patterns, the majority of differentially methylated cytosines are located in intergenic regions in all cases, and a subset of promoters were hypermethylated in almost all patients at relapse. A pathway analysis indicated that the commonly hypermethylated gene promoters at relapse are involved in the Hedghog, Wnt and calcium signaling pathways (p<0.05, modified Fisher Exact test). Integration of these findings with mutational and transcriptional profiles is underway.In order to determine whether epigenetic events linked to AML relapse could be modeled experimentally we performed a pilot study of a human AML xenograft in immunocompromised mice. Engrafted mice were treated with Ara-C at a clinically relevant dose (60mg/Kg; n=2) or vehicle alone (n=3) for five consecutive days. Human AML cells were collected at various timepoints including 28 days after Ara-C treatment where the AML had frankly relapsed in mice. Cytosine methylation profiles obtained through ERRBS revealed predominantly hypermethylated cytosines when compared to the xenotransplanted diagnostic sample (72% hypermethylated versus 28% hypomethylated). Remarkably, there was a strong overlap with gene promoters that are also aberrantly methylated in relapsed AML patients (p<0.01, hypergeometric test), including members of the Wnt signaling pathway.We conclude that there are epigenetically distinct forms of relapsed AML. Nonetheless, there is convergent epigenetic regulation of specific gene pathways that may contribute to relapsed AML pathogenesis and xenotransplanted AML mice can serve as experimental models for further study. Finally, the genomic distribution of reprogrammed methylation suggests a role for epigenetic plasticity at distal regulatory elements. Whereas it remains unclear whether these changes represent clonal selection, their extensive and dynamic range suggest that exposure to chemotherapy may alter the fidelity of mechanisms that control cytosine methylation distribution thus permitting widespread and distant epigenetic reprogramming and contributing to disease relapse. Disclosures:No relevant conflicts of interest to declare.

Integrated genetic and epigenetic analysis of childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.

Variation of cytosine methylation in response to water availability in two contrasting growth types of an amphibious plant Alternanthera philoxeroides

We used methylation-sensitive amplified polymorphisms (MSAP) to examine the alterations of cytosine methylation in two contrasting growth types of an amphibious plant Alternanthera philoxeroides in response to change of water availability. Using 34 pairs of selective primer combinations, we amplified 1026 and 1128 clear and reproducible bands in root and leaf of A. philoxeroides, respectively. When the aquatic types of plants were transplanted into drought culture, we found a decrease in the overall DNA methylation. When the terrestrial types of plants were transferred into flood culture, we detected a higher frequency of methylation than demethylation events. Alterations of DNA methylation were more evident in root than in leaf in response to change of water availability. When the confounding effects of variable environmental factors were removed, differences of cytosine methylation profiles were observed between two growth types of plants under common growth conditions.

Fingerprinting Acute Leukemia: DNA Methylation Profiling of B-Acute Lymphoblastic Leukemia

In this issue of Cancer Discovery, Geng and colleagues report on their use of a combination of promoter cytosine methylation profiling with gene expression and ChIP sequencing to elucidate molecular signatures of adult B-acute lymphoblastic leukemia patient samples with BCR-ABL1, E2A-PBX1, and MLL rearrangements. The unique epigenetic and gene expression signatures of these clinically unfavorable B-ALL subtypes identify novel biomarkers and provide a strong rationale for repurposing existing therapies to treat these molecularly distinct diseases.

Alterations in inheritance pattern and level of cytosine DNA methylation, and their relationship with heterosis in rice

Novel hybrid-specific/heterotic gene expression patterns observed from expression studies suggest the need to characterize the underlying regulatory mechanism(s) to reveal the biological basis of heterosis in crop plants. To gain an insight into the molecular basis of heterosis in rice, we investigated the inheritance pattern and level of cytosine methylation, a major epigenetic regulatory mechanism, in the leaf tissue of an elite Indian rice hybrid KRH2 and its parents at three stages (15 day-old, 35 day-old seedling and flag leaf) and their correlation with heterosis using methylation-sensitive amplification polymorphism (MSAP) technique. In contrast to parents, the level of methylation was high during initial growth stages and reduced as the hybrid grew. Even though, a majority of cytosine methylation profiles were transmitted to hybrid at all stages, a considerable level (25.4%) of cytosine methylation pattern was observed to be novel in hybrid and some of these altered loci were identified to code for known/hypothetical proteins. It was also observed that demethylation events occurred more predominantly during early stages than hypermethylation while the frequency of demethylation events decreased in flag leaf with a remarkable increase in hypermethylation in the hybrid. These bi-directional events in methylation in KRH2 were validated in six hybrids with different levels of grain yield heterosis and the level of cytosine methylation in the hybrid was observed to be significantly correlated with heterosis.

Inheritance of DNA Methylation in Two Cotton Hybrid Derived from CRI-12

DNA methylation in the form of cytosine methylation is proposed as an evolutionary event, which contributes to genome evolution and plays an important role in maintaining genome integrity and controlling dynamics of gene activity. Hybridization and polyploidization play significant roles in the evolution of higher plants. Compared with animals, cytosine methylation is more abundant in plants, particularly at genomic regions containing transposons and their derivatives. Accumulated evidence had suggested that DNA methylation plays important roles in normal plant development. In different stages of plant growth, the changes in DNA methylation level play an important role in response to variations of its heredity and the environment. Notwithstanding these interesting findings, the causing factors for, and generality of, methylation dynamics in plants, particularly with hybrid formation and trans-generational heritability, remained largely vague. However, there is a paucity of evidence to support any direct link between the epigenetic phenomena of cytosine methylation alteration and gene silencing following hybridization and polyploidization. In this study, MSAP (methylation-sensitive amplified fragment length polymorphism) was used in this study to detect the DNA methylation patterns in the 5′-CCGG sites of two cotton hybrids derived from CRI-12 and their parents for understanding developmental stability and inheritance of cytosine methylation. It was found MSAP ratios, which were the ratios of MSAP type in the two cotton hybrids were 12.41–20.05%, cytosine methylation profiles were variable, from increase to decrease during plant growth and development. Full methylation of internal cytosine (6.90–11.47%) was the dominant in two cotton hybrids. Meanwhile, the MSAP profiles enable the monitoring of inheritance or variation of parental methylation patterns in hybrid progenies. It was found that a great majority (from 96.61% to 98.86%, depending on crosses) of the methylation profiles in cotton inbred lines transmitted to the inter-strain hybrids; however, from 1.14% to 3.39% of the profiles in the hybrids exhibited variation from the expected parental additivity. Both inherited and altered methylation profiles can be divided into distinct groups, and their frequencies are variable among the cross-combinations, and during plant growth and development. Bands in hybrids that appeared in both groups digested by Hpa II-EcoR I and Msp I-EcoR I, which were inherited from either or both of the maternal and paternal parent—this type apparently comprised the greatest majority, as all monomorphic bands belong to this type. In addition, sequencing of differentially methylated fragments and subsequent homology analysis of isolated bands that showed variation in hybrids indicated that diverse sequences were involved, including known-function cellular genes and mobile elements, Such as leucine-rich repeat family protein, PDR-like ABC-transporter, putative oligopeptide transporter, GTP-binding protein, similar to pathogenesis-related protein, DOMON domain-containing protein, putative adenosine phosphosulfate kinase, putative protein, RNA-directed DNA polymerase. The remaining 14 bands showed no homology to the database sequences. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the cotton genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be induced by interspecific hybridization between two cotton hybrids, and indicated a direct relationship between cytosine methylation alteration and gene expression variation.

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.

Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells.

Cytosine methylation, an epigenetic modification of DNA, is a target of growing interest for developing high throughput profiling technologies. Here we introduce two new, complementary techniques for cytosine methylation profiling utilizing next generation sequencing technology: bisulfite padlock probes (BSPPs) and methyl sensitive cut counting (MSCC). In the first method, we designed a set of ~10,000 BSPPs distributed over the ENCODE pilot project regions to take advantage of existing expression and chromatin immunoprecipitation data. We observed a pattern of low promoter methylation coupled with high gene body methylation in highly expressed genes. Using the second method, MSCC, we gathered genome-scale data for 1.4 million HpaII sites and confirmed that gene body methylation in highly expressed genes is a consistent phenomenon over the entire genome. Our observations highlight the usefulness of techniques which are not inherently or intentionally biased in favor of only profiling particular subsets like CpG islands or promoter regions.

Cytosine methylation profiling of cancer cell lines

DNA-methylation changes in human cancer are complex and vary between the different types of cancer. Capturing this epigenetic variability in an atlas of DNA-methylation changes will be beneficial for basic research as well as translational medicine. Hypothesis-free approaches that interrogate methylation patterns genome-wide have already generated promising results. However, these methods are still limited by their quantitative accuracy and the number of CpG sites that can be assessed individually. Here, we use a unique approach to measure quantitative methylation patterns in a set of >400 candidate genes. In this high-resolution study, we employed a cell-line model consisting of 59 cancer cell lines provided by the National Cancer Institute and six healthy control tissues for discovery of methylation differences in cancer-related genes. To assess the effect of cell culturing, we validated the results from colon cancer cell lines by using clinical colon cancer specimens. Our results show that a large proportion of genes (78 of 400 genes) are epigenetically altered in cancer. Although most genes show methylation changes in only one tumor type (35 genes), we also found a set of genes that changed in many different forms of cancer (seven genes). This dataset can easily be expanded to develop a more comprehensive and ultimately complete map of quantitative methylation changes. Our methylation data also provide an ideal starting point for further translational research where the results can be combined with existing large-scale datasets to develop an approach that integrates epigenetic, transcriptional, and mutational findings.

Epigenetics

Epigenetics