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:
- 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
- Page et al. Hepatic Stellate Cell Transdifferentiation Involves Genome-Wide Remodeling of the DNA Methylation Landscape. Journal of Hepatology 2015
- Moran et al. Validation of a DNA methylation microarray for 850,000 CpG sites of the human genome enriched in enhancer sequences. Epigenomics 2015
- Mendioroz et al. Trans effect of chromosome aneuploidies on DNA methylation patterns in human Down syndrome and mouse models. Genome Biology 2015, 16:263
- Sellars et al. Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages. Nature immunology (July 2015)
- 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
- Matsubara et al. Exploration of hydroxymethylation in Kagami-Ogata syndrome caused by hypermethylation of imprinting control regions. Clinical Epigenetics. (2015)7:90
- 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
2014 oxBS papers:
- Stewart S et al. oxBS:-450K: A method for analysing hydroxymethylation using 450K Bead Chips. Methods. 2014 Aug 28
- Booth MJ et al. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nature Chemistry. 2014 Mar 23
- 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.
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.