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Concept: Bisulfite sequencing


BACKGROUND: DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection. RESULTS: We characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125. CONCLUSIONS: We have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.

Concepts: DNA, Archaea, Bacteria, Organism, Eukaryote, Prokaryote, DNA methylation, Bisulfite sequencing


Molecular abnormalities in the 11p15.5 imprinted gene cluster lead to two different growth diseases: Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). They are mainly caused by epigenetic alterations in one of the two imprinting 11p15 control regions (ICR1 and ICR2). These CpG-rich regions are differentially methylated on the maternally and paternally derived chromosomes. We report four different methylation patterns along the BWS/SRS critical region, clarified by methylation-specific multiplex ligation-dependent probe amplification. The mathematical processing of the data provides information about alterations in the methylation status: from hypo- to almost complete demethylation of KvDMR, hypo- and hypermethylation of H19DMR and combined results from both regions provide information on paternal uniparental disomy (patUPD). The study concerns two BWS cases with KvDMR hypomethylation and almost complete loss of methylation, respectively; two patUPD11p15 cases with H19DMR hypermethylation/KvDMR hypomethylation, and one SRS case with H19DMR demethylation. In some cases KvDMR hypomethylation in patUPD11p15 can be difficult to assess, which requires combination with STR analysis or alternative method. The STR analysis provides also information on complete or segmental coverage and iso- or heterodisomy. Following this systematic approach, the precise diagnosis can be clarified in a few days and different methylation patterns could be detected.

Concepts: DNA, Gene expression, Histone, Epigenetics, DNA methylation, Methylation, Bisulfite sequencing, H19


The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation beyond CpG sites and CpG islands. These technologies have opened new avenues to understand the interplay between epigenetic events, chromatin plasticity and gene regulation. However, the processing, managing and mining of this huge volume of data require specialized computational tools and statistical methods that are yet to be standardized. Here, we describe a complete bisulfite sequencing analysis workflow, including recently developed programs, highlighting each of the crucial analysis steps required, i.e. sequencing quality control, reads alignment, methylation scoring, methylation heterogeneity assessment, genomic features annotation, data visualization and determination of differentially methylated cytosines. Moreover, we discuss the limitations of these technologies and considerations to perform suitable analyses.

Concepts: DNA, Gene expression, Histone, Epigenetics, DNA methylation, Methylation, Molecular genetics, Bisulfite sequencing


We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16-17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.

Concepts: DNA, Histone, Epigenetics, Diabetes mellitus, DNA methylation, Methylation, DNA methyltransferase, Bisulfite sequencing


The nucleoside adenosine is a potent regulator of vascular homeostasis, but it remains unclear how expression or function of the adenosine-metabolizing enzyme adenosine kinase (ADK) and the intracellular adenosine levels influence angiogenesis. We show here that hypoxia lowered the expression of ADK and increased the levels of intracellular adenosine in human endothelial cells. Knockdown (KD) of ADK elevated intracellular adenosine, promoted proliferation, migration, and angiogenic sprouting in human endothelial cells. Additionally, mice deficient in endothelial ADK displayed increased angiogenesis as evidenced by the rapid development of the retinal and hindbrain vasculature, increased healing of skin wounds, and prompt recovery of arterial blood flow in the ischemic hindlimb. Mechanistically, hypomethylation of the promoters of a series of pro-angiogenic genes, especially for VEGFR2 in ADK KD cells, was demonstrated by the Infinium methylation assay. Methylation-specific PCR, bisulfite sequencing, and methylated DNA immunoprecipitation further confirmed hypomethylation in the promoter region of VEGFR2 in ADK-deficient endothelial cells. Accordingly, loss or inactivation of ADK increased VEGFR2 expression and signaling in endothelial cells. Based on these findings, we propose that ADK downregulation-induced elevation of intracellular adenosine levels in endothelial cells in the setting of hypoxia is one of the crucial intrinsic mechanisms that promote angiogenesis.

Concepts: DNA, Gene, Molecular biology, Epigenetics, Blood vessel, DNA methylation, Methylation, Bisulfite sequencing


Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.

Concepts: DNA, Gene expression, Molecular biology, Epigenetics, Methylation, DNA polymerase, Thermus aquaticus, Bisulfite sequencing


The mechanism of action of olanzapine in treating schizophrenia is not clear. This research reports the effects of a therapeutic equivalent treatment of olanzapine on DNA methylation in a rat model in vivo.Genome-wide DNA methylation was assessed using a MeDIP-chip analysis. All methylated DNA immunoprecipitation (MeDIP), sample labelling, hybridization and processing were performed by Arraystar Inc (Rockville, MD, USA). The identified gene promoters showing significant alterations to DNA methylation were then subjected to Ingenuity Pathway Analysis (Ingenuity System Inc, CA, USA).

Concepts: DNA, Gene expression, Promoter, Histone, Epigenetics, DNA methylation, Maryland, Bisulfite sequencing


The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

Concepts: DNA, Gene, Gene expression, Cancer, Epigenetics, DNA methylation, Methylation, Bisulfite sequencing


Genome-wide characterization by next-generation sequencing has greatly improved our understanding of the landscape of epigenetic modifications. Since 2008, whole-genome bisulfite sequencing (WGBS) has become the gold standard for DNA methylation analysis, and a tremendous amount of WGBS data has been generated by the research community. However, the systematic comparison of DNA methylation profiles to identify regulatory mechanisms has yet to be fully explored. Here we reprocessed the raw data of over 500 publicly available Arabidopsis WGBS libraries from various mutant backgrounds, tissue types, and stress treatments and also filtered them based on sequencing depth and efficiency of bisulfite conversion. This enabled us to identify high-confidence differentially methylated regions (hcDMRs) by comparing each test library to over 50 high-quality wild-type controls. We developed statistical and quantitative measurements to analyze the overlapping of DMRs and to cluster libraries based on their effect on DNA methylation. In addition to confirming existing relationships, we revealed unanticipated connections between well-known genes. For instance, MET1 and CMT3 were found to be required for the maintenance of asymmetric CHH methylation at nonoverlapping regions of CMT2 targeted heterochromatin. Our comparative methylome approach has established a framework for extracting biological insights via large-scale comparison of methylomes and can also be adopted for other genomics datasets.

Concepts: DNA, Gene, Gene expression, Histone, Epigenetics, DNA methylation, Methylation, Bisulfite sequencing


Aberrant DNA methylation is an epigenetic hallmark of melanoma, known to play important roles in melanoma formation and progression. Recent advances in genome-wide methylation methods have provided the means to identify differentially methylated genes, methylation signatures, and potential biomarkers. However, despite considerable effort and advances in cataloging methylation changes in melanoma, many questions remain unanswered. The aim of this review is to summarize recent developments, emerging trends, and important unresolved questions in the field of aberrant DNA methylation in melanoma. In addition to reviewing recent developments, we carefully synthesize the findings in an effort to provide a framework for understanding the current state and direction of the field. To facilitate clarity, we divided the review into DNA methylation changes in melanoma, biomarker opportunities, and therapeutic developments. We hope this review contributes to accelerating the utilization of the diagnostic, prognostic, and therapeutic potential of DNA methylation for the benefit of melanoma patients.

Concepts: DNA, Gene expression, Restriction enzyme, Histone, Epigenetics, DNA methylation, Methylation, Bisulfite sequencing