Publications utilising CEGX technology
Staying up to date with Epigenetics and DNA methylation literature is one of our most important duties to make sure that we can develop cutting edge tools for scientists to continue progressing with their scientific discoveries. The following is a list of high impact publications utilising oxidative bisulfite to study DNA (hydroxy)methylation in various fields including cancer biology, development, stem cell research, and neuroscience.
2017 oxBS papers:
- Raiber et al. Base resolution maps reveal the importance of 5-hydroxymethylcytosine in a human glioblastoma. npj Genome Medicine. 2017; doi:10.1038/s41525-017-0007-6
Importance of studying 5hmC and 5mC revealed in human glioblastoma
Using single base resolution maps of whole genomes, methylomes and hydroxylmethylomes for matched human glioblastoma and tumour margin samples delivers new insights into the interrelation of genetics and epigenetic variations. Previous studies using bisulfite converted DNA to measure combined levels of 5mC and 5hmC have led to genome-wide hypomethylation being regarded as an epigenetic hallmark of tumorigenesis. However, by using both bisulfite and oxidative bisulfite (oxBS) converted DNA to discriminate between 5mC and 5hmC, the authors identified global hypermethylation in the tumour. This result suggests that data obtained from bisulfite-only approaches need to be re-interpreted. Notably, the authors also observed that cells neighbouring tumour cells display epigenetic alterations characteristic of the tumour itself although genetically they appear “normal”. This shows the potential transfer of epigenetic information between cells that contributes to the intratumour heterogeneity of glioblastoma.
- Zhang et al. Acetylation Enhances TET2 Function in Protecting against Abnormal DNA Methylation during Oxidative Stress. Molecular Cell. 2017; 65;2 -323
Acetylation of TET2 and binding to DMNT1 enhances protection against abnormal DNA methylation
Previous studies have revealed the function of the ten-eleven translocation (TET) family of proteins in DNA demethylation, now researchers from the Johns Hopkins University School of Medicine have further elucidated the mechanism by which the TET2 protein protects against abnormal DNA methylation in cancer cells. The researchers demonstrated that acetylation of TET2 increases enzymatic activity of the protein and enhances its interaction with DNA (cytosine-5)-methyltransferase 1 (DNMT1). The binding with DNMT1 further increases the stability of the TET2 protein and localises the protein to chromatin where it can prevent accumulation of DNA methylation by converting 5mC to 5hmC.
- Chen et al. Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine. BMC Genomics. 2017; 18:96
A low-cost, adaptable method to quantify 5hmC and 5mC modifications
This study is a proof-of-principle which describes a novel, low-cost, versatile methodology whereby bisulfite and oxidative bisulfite conversion of DNA can be used to quantify 5mC or 5hmC modifications at targeted genomic regions from a variety of samples. Samples were analysed from multiple types of disease and tissue, including post-mortem brain tissue and cancerous tumours. Using the TrueMethyl kit, the authors were able to analyse 5mC and 5hmC content in the samples in parallel. Bisulfite or oxidative bisulfite converted DNA was used in multiplex PCR reactions to amplify targeted genomic regions prior to next-generation sequencing. This approach could be widely applicable to, for example, validation of whole-genome studies, or to clinical or candidate gene studies.
- Yotova et al. Epigenetic Alterations Affecting Transcription Factors and Signaling Pathways in Stromal Cells of Endometriosis. PLOS One. 2017 e0170859
Disease-associated changes in 5hmC modifications occur in genomic regions with transcriptional regulatory activity
This study investigated the epigenetic changes associated with endometriosis in human samples. Whole genome 5mC and 5hmC profiles were compared in healthy and diseased endometrial cells and stromal cells. 5hmC modifications were significantly reduced in the epithelial cells of diseased samples compared to normal samples; however 5hmC levels were comparable between diseased and normal stromal cells. Furthermore, no difference in 5mC was witnessed. This indicates that 5hmC changes, but not 5mC, are specific to the affected cell type in diseased samples. Targeted oxidative bisulfite sequencing, using the TrueMethyl kit, revealed that 5hmC modifications largely mirrored 5mC, and that these modifications reside within functional genetic loci and in regions controlling the expression of genes associated with endometriosis lesion formation.
2016 oxBS papers:
- Gross et al. Variations in 5-methylcytosine and 5-hydroxymethylcytosine among human brain, blood, and saliva using oxBS and the Infinium MethylationEPIC array Biology Methods and Protocols 1 (1) 2016
CEGX True Methyl kit is compatible with the Illumina Infinium MethylationEPIC array and 5hmC accounts for approximately one-third of methylation events in bisulfite sequencing
DNA was extracted from human blood, brain and saliva and subject to bisulfite or oxidative bisulfite conversion, before analysis of 5mC and 5hmC modifications using the Illumina Infinium MethylationEPIC array. The TrueMethyl kit was used for oxidative bisulfite conversion and efficiency was directly comparable to that of the Zymo EZ DNA Methylation bisulfite conversion kit. 5mC and 5hmC modifications were distributed throughout the genome including regions with transcriptional regulatory activity. 5hmC accounts for approximately one-third of the total signal for bisulfite converted data; therefore it is crucial that this be considered for future work.
- Solvsten et al. Voluntary Physical Exercise Induces Expression and Epigenetic Remodeling of VegfA in the Rat Hippocampus Molecular Neurobiology 1-16. 2016
5hmC modifications are detected in rat brain at promoter regions of key growth factors
In this study, the aim was to characterise gene expression and accompanying epigenetic changes in rat hippocampus and frontal cortex in response to exercise over a period of two weeks. Expression of a panel of growth factor genes was analysed; several were upregulated in one or both of the brain areas. VegfA was upregulated in active rats in the hippocampus only, and this was mirrored by hypomethylation of CpG sites in the promoter region of the gene. Specifically, hypomethylation was observed in predicted Sp1/Sp3 transcription factor binding sites. Using oxidative bisulfite sequencing, 5hmC modifications were observed at a higher frequency than 5mC, but 5hmC levels were unaffected by exercise. This may suggest that 5hmC modifications are less dynamic than 5mC modifications in response to environmental factors such as exercise.
- Johnson et al. 5-Hydroxymethylcytosine localizes to enhancer elements and is associated with survival in glioblastoma patients Nature Communications. 7, Article number: 13177. 2016
Functional implications of 5hmC on transcriptional regulation and glioblastoma patient survival
Parallel bisulfite and oxidative bisulfite sequencing was performed in glioblastoma and prefrontal cortex tissue, and an algorithm utilised to estimate levels of 5mC, 5hmC or unmodified sites genome-wide. 5hmC modifications were significantly lower in glioblastoma tissue compared to normal tissue. 5hmC modifications were enriched in regions associated with transcription factor binding, in enhancer or super-enhancer elements, alternative mRNA splicing and in genes commonly mutated in glioblastoma. Furthermore, 5hmC modifications were enriched in actively transcribed regions indicating a positive correlation with expression. Patients who had tumours with lower 5hmC levels had a poorer prognosis, therefore 5hmC levels may have direct functional implications on survival.
- Wallner et al. Epigenetic dynamics of monocyte-to-macrophage differentiation. Epigenetics & Chromatin. 2016; 9:33.
De-repression of a phagocytic gene network after onset of macrophage differentiation
The elucidation of monocyte to macrophage differentiation can be demonstrated by high-throughput sequencing and the investigation of DNA demethylation is an important part of this. DNA demethylation in monocytes affects only a small number of genes important in macrophage structure and function and is repressed in somatic tissue. TrueMethyl ox-BS sequencing kit was employed to differentiate 5mC from 5hmC and thus ascertain the level of 5hmC in monocytes. Levels of 5hmC are increased during the first 12 hours of differentiation. These increased levels are an indicator of TET enzyme activity which appears to be targeted to specific genomic regions unique to macrophages upon induction of differentiation by CSF1.
- Vento-Tomo DNA demethylation of inflammasome-associated genes is enhanced in patients with cryopyrin-associated periodic syndromes Journal of Allergy and Clinical Immunology 139 (1) 2017
Disease-associated demethylation of CpG sites is coupled to an increase in 5hmC and expression of inflammasome-related genes.
Autoinflammatory syndromes can be caused by increased inflammasome activity due to gene mutations; however, the same mutations in different individuals do not necessarily result in similar phenotypes. During differentiation of macrophages from monocytes, or when monocytes were stimulated with pro-inflammatory cytokines, enzymatic demethylation of CpG sites was coupled to an increase in 5hmC at the same sites, as well as an increase in proximal gene expression. The CEGX TrueMethyl kit was used for oxidative bisulfite conversion of DNA. Demethylation was catalysed by TET2, and was found to be dependent on NFκB activity in this context. These changes in DNA methylation were also present in cryopyrin-associated periodic syndromes (CAPS) ex vivo patient samples, regardless of gene mutational status, and could be reversed by anti-IL-1 therapy.
- Green et al. Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression. FASEB J. 2016.
5hmC modifications in placental tissue are associated with dynamically-expressed genes throughout placental maturation and development.
The CEGX TrueMethyl kit was used to convert DNA in parallel from 23 placental tissue samples in order to quantify genomewide 5mC or 5hmC modifications using the Illumina HumanMethylation450 BeadChip. 19 of these samples were also used for RNA-seq analysis, before combining the data to measure the relationship between 5mC, 5hmC and gene expression. Although mean 5hmC levels were lower than 5mC, a high number of probes showed robust levels of 5hmC across samples. CpG islands were distinctly devoid of 5hmC, regions with H3K27Ac were depleted of 5hmC, but poised enhancer regions were enriched in 5hmC. 5hmC modifications correlated significantly with the expression of transitional genes important for placental maturation and development.
- Houseman et al. OxyBS: estimation of 5-methylcytosine and 5-hydroxymethylcytosine from tandem-treated oxidative bisulfite and bisulfite DNA. Bioinformatics, 2016, 1-3.
- Hadad et al. Absence of genomic hypomethylation or regulation of cytosine-modifying enzymes with aging in male and female mice. Epigenetics & Chromatin. 2016; 9:30
Contradicting the genomic hypomethylation theory of aging
This study aimed to investigate aging associated DNA methylation. Total levels of 5mC or 5hmC in mouse brains did not change with aging in either male or females; however, oxBS-Seq using the TrueMethyl kit allowed more localised methylation changes to specific genomic loci to be detected. Differential methylation levels were also reported between the sexes. This study demonstrates the need for careful study design ensuring base-specific analysis of specific tissue and sexes.
- Toraño et al. Age-associated hydroxymethylation in human bone-marrow mesechymal stem cells. J. Transl Med. 2016; 14:207
Epigenetic modifications in bone-marrow suggest that the age of donors should be considered
This study aimed to investigate age-associated DNA hydroxymethylation.
Levels of 5hmC in mesechymal stem cells, determined using the MethylBlue ox-BS protocol, indicated that CpG sites gained hydroxymethylation and lost 5mC in the advanced age group. This suggests a role for 5hmC during aging. This study also noted that there were age-associated patterns of 5hmC changes in different age groups but none were locus-specific. This research demonstrates that 5hmC should be considered with 5mC as an important mark in the study of DNA methylation and aging. These marks may also affect the potential of the bone-marrow for therapeutic use.
- Lunnon et al. Variation in 5-hydroxymethylcytosine across human cortex and cerebellum. Genome Biol. 2016; 17:27
The incorporation of oxidative bisulfite treatment helps quantify 5hmC in the human cortex and cerebellum
This study was designed to quantify and compare DNA methylation in human cortex and cerebellum tissue. The treatment of samples using TrueMethyl oxBS in conjunction with standard BS treatment allowed discrimination between 5mC and 5hmC. This enabled the accurate measurement of 5hmC at functional sites within the tissue, the demonstration of differences between the prefrontal cortex and the cerebellum and to show that there is considerable inter-individual variation of 5hmC at some sites. It is possible that these new data could confound previous findings attributed to DNA variation in methylation using cumulative 5mC and 5hmC values.
It is anticipated that these data could be used to investigate the role of 5hmC in neurological disorders.
- Heyn et al. Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer. Genome Biol. 2016; 17:11
Super-enhancers targeted by aberrant DNA methylation
This study investigated the epigenic alterations caused by aberrant DNA methylation of super-enhancers associated with cancer. The examination of DNA methylation as a chemical mark in gene regulation showed disease-associated variation at super-enhancer sites when compared to normal tissue. Comparison of oxBS and BS treated samples showed that 5hmC was not responsible for the increase in DNA methylation observed at these sites in any of the cancer samples. Super-enhancer DNA methylation profiles appear to be altered, possibly by changes in transcription factor binding, with subsequent effects on gene expression. There is a need for more extensive catalogues of human DNA methylomes to improve the understanding of DNA methylation at multiple sites.
- Yue et al. Control of Foxp3 stability through modulation of TET activity. J Exp Med 2016; Vol 213 (3) 377-397
Regulatory T cell development is controlled by methylation status at key regulatory regions and can be enhanced by TET activating agents.
Foxp3 expression is regulated by methylation status at intragenic regulatory elements and is an essential regulator of regulatory T cell (Treg) development and function: key to preventing autoimmunity and maintaining immune homeostasis. Two TET isoforms (Tet2/Tet3) are responsible for demethylation of the Foxp3 regulatory locus which results in stabilisation of Foxp3 expression and maintenance of Treg cell identity. Bisulfite and oxidative bisulfite sequencing, using the CEGX TrueMethyl kit, revealed loss of 5mC and an increase in 5hmC at the regulatory locus during Treg development which was impaired in Tet2/Tet3 double knockout mice. 5hmC was enriched in Treg precursor cells. Vitamin C, an agonist of the TET proteins, was able to enhance demethylation and stabilise Foxp3 expression during Treg cell development thus potentially providing a novel therapeutic strategy to combat immune system dysfunctions.
- Vento-Tormo et al. IL-4 orchestrates STAT6-mediated DNA demethylation leading to dentritic cell differentiation.Genome Bioloy 2016; 17:4
Functional implications of 5mC loss, via the 5hmC intermediate, on the monocyte differentiation process in response to stimulation.
Differentiation of innate immune system component cells from progenitors is a tightly regulated process, with epigenetic modifications playing a central role in translating extracellular signals into control of cell identity and fate. In an in vitro model of monocyte differentiation to macrophages or dendritic cells, the differentiation process induced demethylation of several thousand CpG sites which were cell-type specific and enriched in enhancer regions. Changes in methylation status had functional consequences: several thousand genes were either up- or down-regulated. TET-mediated demethylation events were confirmed by the formation of the 5hmC intermediate as determined using the CEGX TrueMethyl kit. Changes in the epigenome were reliant upon an intact JAK2-STAT6 signalling pathway, thus demethylation is a crucial component of the cellular response to differentiation signals.
2015 oxBS papers:
- Wille et al. 5-hydroxymethylation of the EBV genome regulates the latent to lytic switch. PNAS 2015
5hmC modification of epithelial cell and Epstein-Barr virus genomes can regulate lytic viral activation and tumour formation
Epstein-Barr virus has been associated with the formation of undifferentiated nasopharyngeal carcinoma (NPC) from undifferentiated epithelial cells, and the EBV viral genome can undergo methylation changes which determine the type of infection (either lytic or latent). Using oxidative bisulfite sequencing, 5hmC modification of the viral genome was detected and shown to accompany lytic reactivation of the virus. Furthermore, 5hmC modification accumulated during normal epithelial cell differentiation but not in EBV infected NPC cells. Increasing or decreasing TET activity resulted in increased 5hmC modification, or increased 5mC modification, of lytic EBV promoter regions in EBV infected cell lines, respectively. 5hmC modification of the promoters altered binding of key transcription factors which regulate the EBV switch from latent to lytic infection. Altered levels of 5hmC in the host and viral genome may contribute to the formation of NPC tumour cells.
- Page et al. Hepatic Stellate Cell Transdifferentiation Involves Genome-Wide Remodeling of the DNA Methylation Landscape. Journal of Hepatology 2015
Disease-specific modifications in 5mC/5hmC drive hepatocyte differentiation and fibrosis
Liver fibrosis results from a phenotypic change from hepatic stellate cells to a pro-fibrogenic myofibroblast-like cell, a transdifferentiation accompanied by a huge change in the transcriptome and thought to be regulated by epigenetic changes. Using the CEGX TrueMethyl oxidative bisulfite kit, liver fibrosis was shown to be accompanied by a stabilisation of 5mC, depletion of global 5hmC levels and altered expression of the TET and DNMT enzymes. 5hmC modifications were altered across the genome between normal and disease-like cells, however a particular enrichment in 5hmC modification was found on chromosome 9. Depletion of Dnmt3a resulted in decreased expression of collagen 1A1 and α-smooth muscle actin; thus changes in the epigenetic landscape, including disease-specific 5mC/5hmC modifications, control differentiation of hepatocytes and drive fibrogenesis.
- Moran et al. Validation of a DNA methylation microarray for 850,000 CpG sites of the human genome enriched in enhancer sequences. Epigenomics 2015
The MethylationEPIC BeadChipInfinium 850K array is compatible with the CEGX TrueMethyl kit for genome wide detection of 5hmC
5mC is the most well-characterised epigenetic modification and its importance in the regulation of gene expression is highlighted by the distorted DNA methylation patterns witnessed in numerous diseases. The Illumina Infinium HumanMethylation450 BeadChip was previously the most widely used platform to interrogate the DNA methylome; 5mC and 5hmC patterns could be mapped at 450,000 CpG sites across the genome. In this study, the 450K and the new MethylationEPIC BeadChip Infinium 850K arrays from Illumina were compared, with special focus on the 333,265 CpG sites located in predicted or experimentally determined enhancer regions. The 850K array can reproduce the 450K data reliably, with Pearson correlation coefficients of > 0.99, and can be used for assessment of modifications from fixed and embedded samples. Ability to detect 5hmC levels across the genome was confirmed using the CEGX TrueMethyl kit.
- Mendioroz et al. Trans effect of chromosome aneuploidies on DNA methylation patterns in human Down syndrome and mouse models. Genome Biology 2015, 16:263
Altered transcription factor binding due to epigenetic changes in brain cells and T cells; associated with trisomy 21 and with functional implications on gene expression
Epigenetic alterations have recently been proposed as a mechanism controlling the spectrum of phenotypes associated with chromosomal aneuploidy and Down syndrome, where the additional chromosome may impact upon cell networks in trans, and lead to epigenetic alterations. DNA 5mC and 5hmC content was quantified using the Illumina 450K Methylation BeadChip and validated using bisulfite sequencing, using the CEGX TrueMethyl kit for oxidative bisulfite conversion of DNA. Gene- and tissue-specific alterations in DNA methylation were identified in Down syndrome brain cells and T cells, where some differentially methylated regions harboured 5mC and 5hmC changes, and some had primarily 5hmC changes (enrichment in genes associated with brain development and function or T cell development). Altered DNA methylation patterns affecting developmentally-regulated genes were present in foetal samples, indicating early onset of changes, and a subset of these genes displayed altered expression in Down syndrome brain samples which was likely due to altered transcription factor binding.
- Sellars et al. Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages. Nature immunology (July 2015)
T cell fate is determined by the methylation or demethylation, via 5hmC, of a CD4 enhancer element which regulates gene expression and differentiation
The process of T cell development is highly regulated and involves heritable epigenetic programing of lineage specific genes in order to silence or enhance their expression accordingly. A shRNA screen revealed that Dnmt1 played a critical role in the regulation of CD4 expression and maintained effective silencing of the gene in cytotoxic T cells, and DNA methylation was confirmed as crucial to this process as Dnmt-depleted cells transferred to mice lost their CD4-silencing ability and expressed both CD4 and CD8 markers. A differentially methylated locus was established proximal to the CD4 transcription start site and was hypermethylated in CD8 cytotoxic T cells relative to CD4 helper T cells, and was present in T cell progenitors thus methylation at this locus determines cell fate. The TET enzymes mediate demethylation of this region at specific CpG sites in order to enhance CD4 expression, which was confirmed by the presence of 5hmC modifications. T cell differentiation is therefore dependent on epigenetic modification of the CD4 locus to modulate lineage-specific gene expression.
- Yang et al. Hydrogen sulphide promotes Tet1- and Tet2-mediated Foxp3 demethylation to drive regulatory T cell differentiation and maintain immune homeostasis. Immunity 43, 251-263 (2015)
- Kubo N et al. DNA methylation and gene expression dynamics during spermatogonial stem cell differentiation in the early postnatal mouse testis. BMC Genomics. (2015)16:624
Dynamic DNA methylation and hydroxymethylation modifications describe the phases of male germ cell development
The epigenetic landscapes and gene expression profiles of the various stages of spermatogenesis have been largely understudied, thus whole-genome bisulfite sequencing and RNA-sequencing was carried out in prospermatogonia (PSG) and spermatogonia (SG). The CEGX TrueMethyl kit was used to analyse 5mC and 5hmC. Large differences in global 5mC levels were witnessed between the developmental stages, with large (<12 Mb) regions of hypomethylation detected in both stages. Quantification of 5hmC confirmed that satellite repeats at the centromeric and periocentromeric regions were enriched for this modification in PSG, however 5hmC was only found at 1.4% of CpGs across the PSG genome. 5hmC modification is absent after this developmental stage, which may suggest that 5hmC modification is involved in the transcriptional silencing of these regions at this specific stage of sperm development.
- Matsubara et al. Exploration of hydroxymethylation in Kagami-Ogata syndrome caused by hypermethylation of imprinting control regions. Clinical Epigenetics. (2015)7:90
5hmC is present at low levels in blood DNA extracted from Kagami-Ogata patients but may play a role in neurodevelopmental abnormalities
Kagami-Ogata syndrome is a human imprinting disease which has previously been described as a result of hypermethylation of two differentially methylated regions (DMRs) which act as imprinting control regions (ICRs): IG-DMR and MEG3-DMR. Using the CEGX TrueMethyl kit to bisulfite or oxidative bisulfite convert DNA, hypermethylation at the two DMRs was seen in DNA isolated from the blood of Kagami-Ogata syndrome patients but not in controls. This hypermethylation consisted almost exclusively of 5mC modifications rather than 5hmC. Higher levels of 5hmC were witnessed in neural tissue at both the genomic and DMR levels. Thus, 5hmC may play a role in the neurodevelopment problems of Kagami-Otaga patients and is consistent with 5hmC modifications being highly disease and tissue specific.
- Feng J et al. Role of Tet1 and 5-hydroxymethylcytosine in cocaine action. Nature Neuroscience. 2015 Apr;18(4):536-44
- Field SF et al. Accurate Measurement of 5-Methylcytosine and 5-Hydroxymethylcytosine in Human Cerebellum DNA by Oxidative Bisulfite on an Array (OxBS-Array). PLoS One. 2015 Feb 23;10(2):e0118202
Improving the sensitivity of 5hmC detection using multiple replicates and oxidative bisulfite conversion of DNA with the CEGX TrueMethyl kit
The established method of detecting DNA methylation (5mC) is to bisulfite convert samples prior to downstream analyses; however, this method cannot distinguish between 5hmC and 5mC and so bisulfite converted samples actually measure the combined modification of 5mC+5hmC. This study describes a method by which 5hmC modifications can be reproducibly detected using four replicates of bisulfite converted and four replicates of oxidative bisulfite converted cerebellum DNA. Four replicates allowed detection of 5hmC at over 114,000 probes on the Illumina 450K methylation array, however reduction of replicates to 2 allowed for reliable detection of 5hmC at only approx. 50,000 probes. Findings were validated using multiple approaches: LC-MS and qPCR with glucosylation of 5hmC sites and digestion with a restriction enzyme. This study highlights the importance of using multiple technical replicates to improve reproducibility and that 5hmC can be detected at extremely low levels.
2014 oxBS papers:
- Stewart S et al. oxBS:-450K: A method for analysing hydroxymethylation using 450K Bead Chips. Methods. 2014 Aug 28
Oxidative bisulfite conversion using the CEGX TrueMethyl kit can be combined with the Illumina Infinium HumanMethylation 450K BeadChip to quantify 5hmC levels
The Illumina Infinium HumanMethylation 450K BeadChip has been a well-used platform for detecting and quantifying DNA methylation levels, however the method of bisulfite conversion used in DNA preparation cannot distinguish between 5mC and 5hmC modifications. Using the CEGX TrueMethyl kit for oxidative bisulfite conversion of human brain DNA, 5hmC modified CpG sites can be differentiated from 5mC modified bases. 5hmC was detected in relatively high abundance in three brain DNA samples and at lower levels in blood DNA. 5hmC levels were enriched within gene bodies but depleted from promoter regions and CpG islands. 5hmC levels determined by the 450K BeadChip were validated using LC-MS and oxidative bisulfite pyrosequencing: 5hmC levels were found to vary by up to 10% between the array and pyrosequencing methods. Oxidative bisulfite conversion coupled with the 450K BeadChip array is therefore a reliable and accurate method to determine 5hmC levels across the genome.
- Booth MJ et al. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nature Chemistry. 2014 Mar 23
An in-depth step-by-step guide to oxidative bisulfite sequencing for the quantitative detection of 5hmC at single-base resolution
This study provides an in-depth, technical and comprehensive guide to carrying out oxidative bisulfite sequencing, which is an improved method for detecting 5hmC levels in a quantitative manner at single-base resolution and distinguishes this modification from 5mC. Development, potential applications, comparisons with other techniques and limitations of oxidative bisulfite sequencing are discussed in this paper. Information relating to experimental design, reagents and materials and troubleshooting are included. A step-by-step protocol with critical tips and instructions are given along with estimates of time consumption and anticipated results. This protocol recommends the use of the Cambridge Epigenetix TrueMethyl kit during the oxidative bisulfite conversion of DNA due to the optimisation of conditions, consistency and reproducibility that non-experts would benefit from.
- Sun D et al. MOABS: model based analysis of bisulfite sequencing data. Genome Biology. 2014 Feb 24; ;15(2):R38
- Liu Y et al. Base-Resolution Maps of 5-Methylcytosine and 5-Hydroxymethylcytosine in Dahl S Rats: Effect of Salt and Genomic Sequence. Hypertension. 2014 Jan 13
2013 oxBS papers:
- Jeong M et al. Large conserved domains of low DNA methylation maintained by Dnmt3a. Nat Genet. 2013 Nov 24.
- Booth MJ et al. Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc. 2013 Oct;8(10):1841-51.
- Ficz G et al. FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell. 2013 Sep 5;13(3):351-9.
- Nakashima H et al. Effects of dppa3 on DNA methylation dynamics during primordial germ cell development in mice. Biol Reprod. 2013 May 23;88(5):125.
Imprinted genes and retrotransposons are protected from TET-mediated demethylation during germ cell development
Whole-genome demethylation is thought to occur at various stages during pre- and post-implantation development of the embryo, and is a highly dynamic process regulated by the TET enzymes and diluted during DNA replication. Dppa3 is thought to bind to and protect 5mC from conversion to 5hmC in the maternal genome; however the role of Dppa3 during further development is unknown. Using Dppa3-deficient mice and the CEGX TrueMethyl kit for oxidative bisulfite or bisulfite sequencing, DNA methylation patterns were shown to be altered at specific regions known to be important for silencing of imprinted genes and retrotransposon elements. Increased levels of 5mC were coupled to lower levels of 5hmC, thus Dppa3 likely plays a role in TET-mediated demethylation of these regions. These changes had no detectable impact on cell differentiation, however.
2012 oxBS papers:
- Booth MJ et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science. 2012 May 18;336(6083):934-7.
First description of how parallel sequencing can be used to quantify 5mC and 5hmC at single-base resolution
Many methods have been used to attempt to measure 5hmC modifications however no method described thus far can give quantitative and high-resolution information about this epigenetic mark. This study describes for the first time the application of bisulfite and oxidative bisulfite sequencing of samples in parallel before next generation sequencing in order to identify CpG sites which carry 5-methylcytosine or 5-hydroxymethylcytosine modifications. Oxidative bisulfite sequencing can specifically measure 5hmC, while bisulfite sequencing measures both 5mC and 5hmC combined. This novel approach was used to describe the epigenetic landscape in mouse embryonic stem cells and its sensitivity was verified using mass spectrometry. 5hmC modifications were enriched in the promoters of genes associated with transcription factor activity such as the Homeobox genes, and in intergenic regions with moderate levels of 5mC which may suggest that these regions are particularly susceptible to epigenetic modification and highlights the functional importance of 5hmC.