5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes
- Maja Klug†1, 3,
- Sandra Schmidhofer†1,
- Claudia Gebhard1,
- Reinhard Andreesen2 and
- Michael Rehli1Email author
© Klug et al.; licensee BioMed Central Ltd. 2013
Received: 10 October 2012
Accepted: 26 May 2013
Published: 26 May 2013
Cytosine methylation is a frequent epigenetic modification restricting the activity of gene regulatory elements. Whereas DNA methylation patterns are generally inherited during replication, both embryonic and somatic differentiation processes require the removal of cytosine methylation at specific gene loci to activate lineage-restricted elements. However, the exact mechanisms facilitating the erasure of DNA methylation remain unclear in many cases.
We previously established human post-proliferative monocytes as a model to study active DNA demethylation. We now show, for several previously identified genomic sites, that the loss of DNA methylation during the differentiation of primary, post-proliferative human monocytes into dendritic cells is preceded by the local appearance of 5-hydroxymethylcytosine. Monocytes were found to express the methylcytosine dioxygenase Ten-Eleven Translocation (TET) 2, which is frequently mutated in myeloid malignancies. The siRNA-mediated knockdown of this enzyme in primary monocytes prevented active DNA demethylation, suggesting that TET2 is essential for the proper execution of this process in human monocytes.
The work described here provides definite evidence that TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine initiates targeted, active DNA demethylation in a mature postmitotic myeloid cell type.
KeywordsEpigenetics active DNA demethylation differentiation
DNA methylation is a frequent epigenetic modification that restricts the activity of regulatory elements, including cell type-specific gene promoters and enhancers. In mammals, methylated cytosines (5mC) mainly occur in the context of CpG dinucleotides and the targeted setting and erasure of the methylation mark is crucial for the silencing of repetitive and potentially harmful elements and for the proper execution of essential regulatory programs including embryonic development, X-chromosome inactivation, parental imprinting as well as cellular differentiation [1, 2]. While the process of cytosine methylation, which is catalyzed by a group of DNA methyl-transferases (DNMTs) is well characterized, the exact mechanisms facilitating the erasure of DNA methylation in mammals remain less clear and the proposed existence of active enzymatic demethylation processes has been a matter of controversy over the last decades .
Recent pioneering work has identified the family of Ten-Eleven-Translocation proteins (TET1-3) that catalyze the conversion of 5mC to 5-hydroxy-methylcytosine (5hmC) in mammalian cells , and has prompted speculations that these enzymes are involved in DNA demethylation processes [5, 6]. On the one hand, 5hmC could interfere with maintenance methylation and induce a passive demethylation process. On the other hand, TET enzymes may also initiate active demethylation processes through repair-associated mechanisms .
Global DNA demethylation is observed during early embryonal development in particular in zygotes and primordial germ cells and 5hmC has been detected in both pathways [8, 9]. The initial massive erasure of 5mC in primordial germ cells, however, appears to be a TET-independent, passive process that is likely controlled by the downregulation of UHRF1, which facilitates the recruitment of the maintenance DNA-methyltransferase DNMT1 to nascent hemimethylated DNA at the replication fork . In the zygote, however, TET3 mediated conversion of 5mC to 5hmC is essential for the reprogramming of the zygotic paternal DNA after fertilization [11–13]. 5hmC is then gradually replaced by unmethylated cytosines during preimplantation development, suggesting that the erasure of 5hmC in zygotes is also a DNA replication-dependent passive process .
Another member of this family (TET2) directly affects myelopoiesis and diverse myeloid malignancies (including myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloproliferative neoplasms, and acute myeloid leukemia) are frequently associated with mutations in this gene [14–16]. Targeted disruption or knockdown of TET2 results in reduced levels of 5hmC and affects self-renewal and differentiation of hematopoietic stem cells [17–20]. However, the exact mechanisms that contribute to disease pathology are currently unknown.
Here we provide direct evidence that the TET2-dependent conversion of 5mC to 5hmC is required for active DNA demethylation in primary human monocytes. Similar processes are likely to occur in other myeloid (progenitor) cells and the reduced ability to erase DNA methylation at critical regulatory sites in cases with TET2 loss-of-function mutations may therefore contribute to disease pathology.
Results and discussion
To study the effect of siRNA knockdowns, DNA methylation levels were analyzed at 27 h or 42 h using mass spectrometry of bisulfite treated DNA. Results are shown as heatmaps for the entire regions (Figure 4D) and bar charts for selected individual CpGs (Figure 4E). Interestingly, the methylation pattern derived from bisulfite treated DNA after knockdown of MBD4 or TDG was undistinguishable from control siRNA treatment (Figure 4D and 4E). This indicates that neither of these two enzymes actively converts 5hmC, which is in line with previous observations . Tet proteins were recently shown to metabolize 5hmC to 5-formylcytosine (5fC) and 5caC , which are both converted into uracil during bisulfite treatment [26, 29]. Because we cannot distinguish 5fC/5caC from unmethylated cytosine residues after bisulfite treatment, we are currently not able to address whether MBD4 or TDG are active to 'complete' the demethylation process in primary monocytes that is initiated by TET2 mediated processing of 5mC into 5hmC and further into 5fC/5caC. Since human blood monocytes are (in contrast to monocyte-derived macrophages or dendritic cells) severely impaired in base and DNA double-strand break repair , the exchange of the two 5mC derivatives may be delayed. To test whether 5fC/5caC accumulate as the end product of the demethylation process at the CCL13 promoter, we analyzed the restriction efficiency of MspI (which is inhibited by the presence of 5caC or 5fC ) at the site covering one of the demethylated CpGs. While MspI can also be inhibited by methylation of the outer C (5mCCGG) and thus may not allow the quantification of 5caC or 5fC, the fact that DNA from iDC (as well as from all knockdown experiments) could be efficiently cut with this restriction enzyme (Figure S4 in Additional File 5) suggests that the demethylation process is completed (5mC→5C) in iDC. MBD4 or TDG knockdown did not lead to a decrease in restriction efficiency, indicating that 5caC or 5fC do not accumulate at these sites in transfected monocytes . It is thus still unclear whether TDG, MBD4, or another enzyme initiates the last steps of the active demethylation process. Notably, a recent mass spectrometry study systematically identified readers in embryonic stem cells, neuronal progenitor cells, and brain for all known 5C derivates . This study identified a number of additional DNA glycosylases (Neil1, Neil3), as well as helicases (Hells, Harp, Recql, and its homolog Bloom) binding specifically to hmC suggesting that this derivate may already attract DNA-repair enzymes and perhaps initiate DNA demethylation in differentiating monocytes.
The TET2-siRNA treatment, however, resulted in significantly different methylation patterns: the local loss of DNA methylation at the two loci showing rapid 5mC erasure (CCL13 and USP20) was significantly delayed in cells with reduced TET2 expression (Figure 4D and 4E), while control regions were unaffected and the late demethylation targets did not show any signs of methylation loss at these early time points. We also analyzed local 5hmC levels using hMeDIP (Figure S5A in Additional File 5), as well as glycosylation-sensitive restriction (Figure S5B in Additional File 5) and detected a significant reduction of 5hmC at demethylated regions only in TET2-siRNA-treated monocytes. These results clearly establish that differentiating monocytes require TET2 to initiate the active demethylation process.
As shown previously, DNA demethylation in primary monocytes is characterized by the parallel appearance of activating histone marks, such as mono- and dimethylation of H3K4 or acetylation of histones H3 and H4 , which are typical features of enhancers. This is also in line with the recent observation of dynamic deposition of 5hmC at differentiation-associated enhancers in other cellular systems . The histone modifications likely follow the recruitment of DNA-binding factors that direct histone methyl- and/or acetyl-transferases to these sites . The local appearance of 5hmC suggests that the modified histones or the same factors responsible for the modification of histones may also recruit the 5-methylcytosine dioxygenase TET2 to initiate DNA demethylation of newly activated and/or remodeled sites.
Our data unequivocally show that the TET2-mediated conversion of 5mC to 5hmC is an essential intermediate in targeted, locus-specific active demethylation processes that are observed during the differentiation of non-dividing human monocytes. This function of TET2 may also be essential for the differentiation of earlier myeloid progenitor stages, as a significant proportion of myeloid dysplasia are characterized by loss-of-function mutations of TET2.
Materials and methods
Collection of blood cells from healthy donors was performed in compliance with the Helsinki Declaration. All donors signed an informed consent. The leukapheresis procedure, the subsequent purification of peripheral blood monocytes by density gradient centrifugation over Ficoll/Hypaque as well as the counter current centrifugal elutriation were approved by the local ethical committee (reference number 92-1782 and 09/066c). The generation of monocyte-derived dendritic cells and macrophages has been described previously .
Genomic DNA was prepared using the DNeasy Blood and Tissue Kit from Qiagen (Hilden, Germany).
Mass spectrometry analysis of bisulfite-converted DNA
Sodium bisulfite conversion and quantitative analysis of DNA methylation using MALDI-TOF mass spectrometry (MassARRAY Compact MALDI-TOF, Sequenom, San Diego, CA, USA) was performed as described [23, 33]. Primers for amplicon generation were described  or are listed in Table S1 in Additional File 1.
hMeDIP & MeDIP
Enrichment of 5- methylcytosine (5mC) or 5-hydroxy-methylcytosine (5hmC) was analyzed by immunoprecipitation using 5-methylcytidine and 5-hydroxy-methylcytidine antibodies (Diagenode and Active Motif, respectively) essentially as described for 5mC in Mohn et al. . Enriched DNA was purified with the PCR purification kit from Qiagen and quantified on a Realplex Mastercycler EP (Eppendorf, Hamburg, Germany) using the Quantifast SYBR Green PCR Kit (Qiagen) as indicated by the manufacturer. Primer sequences were previously described or are listed in Table S1 in Additional File 1.
Glycosylation of 5hmC
Site-specific detection of 5hmC by glycosylation was done using the Quest 5-hmC detection kit (Zymo Research) following the manufacturer's instructions with modifications. After the glycosylation step (prolonged to 3 h), samples were cleaned using the DNA clean and concentrator kit (Zymo) and subsequently digested with 30 U MspI (NEB), or 30 U HpaII (NEB) at 37°C overnight. The fraction of glycosylated and therefore protected MspI sites as well as the fraction of 5mC- and 5hmC-sensitive sites (determined using HpaII restriction) at specific gene loci were quantified by qPCR using primers described in  or primers listed in Table S1 in Additional File 1.
Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse transcribed using Superscript II MMLV-RT (Promega, Mannheim, Germany). Real-time PCR was performed on a Realplex Mastercycler EP (Eppendorf, Hamburg, Germany) as described above. Primer sequences are listed in Table S1 in Additional File 1.
Transfection of primary human monocytes
Peripheral blood monocytes were transfected using the Human Monocyte Nucleofector Kit from Lonza (Cologne, Germany). In brief, 6×106 cells were resuspended in 100 μL Nucleofector solution (Lonza) with 600 nM TET2-, MBD4-, TDG-, or control-siRNA (all from Thermo Scientific Dharmacon) and electroporated using the Nucleofector I device. Cells were cultured as described without the addition of antibiotics. Expression of targeted genes as well as DNA methylation was measured after 27 h or 42 h in culture.
To follow knockdown efficiency on protein level, cells were harvested 27 h and 42 h after transfection, washed with PBS and lysed in 2x SDS-Lysis Buffer (20% Glycerin, 125mM Tris pH 6.8, 4% SDS, 10% 2-Mercaptoethanol, 0.02% Bromophenolblue). Lysates were boiled (95°C, 10 min) and 1.5 × 105 to 5 × 105 cells per lane separated on 8% or 10% polyacrylamide gels (Biometra Minigel Gelelectrophoresis device). Proteins were transferred to nitrocellulose membranes (Live Technologies, 0.45 μM pore size) using the Biometra Fastblot semi-dry blotter or the Biorad Mini Transblot Cell wet system according to the protein size. After 1 h of blocking in TBS-T with 5% dry milk at room temperature the membranes were incubated with either Anti-TET2 (1:2,000, a gift from O. Bernard), Anti-TDG (1:10,000, a gift from Primo Schär), Anti-MBD4 (1:2,000, from Diagenode) or Anti-actin (Sigma Aldrich) overnight at 4°C. Second antibody (Dako, Glostrup, Denmark) incubation was carried out at room temperature for 1 h. Flourescence signals were detected after exposure to ECL hyperfilm or using a fluorescence scanner (BioRad, Chemi Doc XRS+).
Quantification of MspI restriction efficiency
DNA from monocyte-derived dendritic cells (iDC, 100ng) was digested with MspI plus HhaI or with HhaI alone (20 U each, New England BioLabs) overnight (approximately 16 h) at 37°C. The efficiency of MspI cutting was measured by comparing the qPCR amplification of DNA fragments across the MspI site in MspI-digested and -undigested DNA samples.
immature dendritic cell
The authors thank Ireen Ritter, Lucia Schwarzfischer-Pfeilschifter, and Dagmar Glatz for excellent technical assistance. They are also grateful to Olivier Bernard for sharing the TET2 antiserum and to Primo Schär for providing his TDG antibody. This work was funded by a grant from the Deutsche Forschungsgemeinschaft to MR (RE1310/7).
- Bird A: DNA methylation patterns and epigenetic memory. Genes Dev. 2002, 16: 6-21. 10.1101/gad.947102.PubMedView ArticleGoogle Scholar
- Jaenisch R, Bird A: Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 2003, Suppl: 245-254.View ArticleGoogle Scholar
- Ooi SK, Bestor TH: The colorful history of active DNA demethylation. Cell. 2008, 133: 1145-1148. 10.1016/j.cell.2008.06.009.PubMedView ArticleGoogle Scholar
- Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A: Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009, 324: 930-935. 10.1126/science.1170116.PubMedPubMed CentralView ArticleGoogle Scholar
- Ito S, D'Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y: Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature. 2010, 466: 1129-1133. 10.1038/nature09303.PubMedPubMed CentralView ArticleGoogle Scholar
- Koh KP, Yabuuchi A, Rao S, Huang Y, Cunniff K, Nardone J, Laiho A, Tahiliani M, Sommer CA, Mostoslavsky G, Lahesmaa R, Orkin SH, Rodig SJ, Daley GQ, Rao A: Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell. 2011, 8: 200-213. 10.1016/j.stem.2011.01.008.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhu JK: Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet. 2009, 43: 143-166. 10.1146/annurev-genet-102108-134205.PubMedPubMed CentralView ArticleGoogle Scholar
- Iqbal K, Jin SG, Pfeifer GP, Szabo PE: Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc Natl Acad Sci USA. 2011, 108: 3642-3647. 10.1073/pnas.1014033108.PubMedPubMed CentralView ArticleGoogle Scholar
- Yamaguchi S, Hong K, Liu R, Inoue A, Shen L, Zhang K, Zhang Y: Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during germ cell reprogramming. Cell Res. 2013, 23: 329-339. 10.1038/cr.2013.22.PubMedPubMed CentralView ArticleGoogle Scholar
- Kagiwada S, Kurimoto K, Hirota T, Yamaji M, Saitou M: Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice. EMBO J. 2013, 32: 340-353.PubMedPubMed CentralView ArticleGoogle Scholar
- Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, Xie ZG, Shi L, He X, Jin SG, Iqbal K, Shi YG, Deng Z, Szabo PE, Pfeifer GP, Li J, Xu GL: The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature. 2011, 477: 606-610. 10.1038/nature10443.PubMedView ArticleGoogle Scholar
- Inoue A, Zhang Y: Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos. Science. 2011, 334: 194-10.1126/science.1212483.PubMedPubMed CentralView ArticleGoogle Scholar
- Wossidlo M, Nakamura T, Lepikhov K, Marques CJ, Zakhartchenko V, Boiani M, Arand J, Nakano T, Reik W, Walter J: 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nat Commun. 2011, 2: 241-PubMedView ArticleGoogle Scholar
- Delhommeau F, Dupont S, Della Valle V, James C, Trannoy S, Masse A, Kosmider O, Le Couedic JP, Robert F, Alberdi A, Lecluse Y, Plo I, Dreyfus FJ, Marzac C, Casadevall N, Lacombe C, Romana SP, Dessen P, Soulier J, Viguie F, Fontenay M, Vainchenker W, Bernard OA: Mutation in TET2 in myeloid cancers. N Engl J Med. 2009, 360: 2289-2301. 10.1056/NEJMoa0810069.PubMedView ArticleGoogle Scholar
- Langemeijer SM, Kuiper RP, Berends M, Knops R, Aslanyan MG, Massop M, Stevens-Linders E, van Hoogen P, van Kessel AG, Raymakers RA, Kamping EJ, Verhoef GE, Verburgh E, Hagemeijer A, Vandenberghe P, de Witte T, van der Reijden BA, Jansen JH: Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat Genet. 2009, 41: 838-842. 10.1038/ng.391.PubMedView ArticleGoogle Scholar
- Mullighan CG: TET2 mutations in myelodysplasia and myeloid malignancies. Nat Genet. 2009, 41: 766-767. 10.1038/ng0709-766.PubMedView ArticleGoogle Scholar
- Ko M, Bandukwala HS, An J, Lamperti ED, Thompson EC, Hastie R, Tsangaratou A, Rajewsky K, Koralov SB, Rao A: Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice. Proc Natl Acad Sci USA. 2011, 108: 14566-14571. 10.1073/pnas.1112317108.PubMedPubMed CentralView ArticleGoogle Scholar
- Ko M, Huang Y, Jankowska AM, Pape UJ, Tahiliani M, Bandukwala HS, An J, Lamperti ED, Koh KP, Ganetzky R, Liu XS, Aravind L, Agarwal S, Maciejewski JP, Rao A: Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature. 2010, 468: 839-843. 10.1038/nature09586.PubMedPubMed CentralView ArticleGoogle Scholar
- Moran-Crusio K, Reavie L, Shih A, Abdel-Wahab O, Ndiaye-Lobry D, Lobry C, Figueroa ME, Vasanthakumar A, Patel J, Zhao X, Perna F, Pandey S, Madzo J, Song C, Dai Q, He C, Ibrahim S, Beran M, Zavadil J, Nimer SD, Melnick A, Godley LA, Aifantis I, Levine RL: Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell. 2011, 20: 11-24. 10.1016/j.ccr.2011.06.001.PubMedPubMed CentralView ArticleGoogle Scholar
- Pronier E, Almire C, Mokrani H, Vasanthakumar A, Simon A, da Costa Reis Monte Mor B, Masse A, Le Couedic JP, Pendino F, Carbonne B, Larghero J, Ravanat JL, Casadevall N, Bernard OA, Droin N, Solary E, Godley LA, Vainchenker W, Plo I, Delhommeau F: Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors. Blood. 2011, 118: 2551-2555. 10.1182/blood-2010-12-324707.PubMedPubMed CentralView ArticleGoogle Scholar
- Gebhard C, Schwarzfischer L, Pham TH, Schilling E, Klug M, Andreesen R, Rehli M: Genome-wide profiling of CpG methylation identifies novel targets of aberrant hypermethylation in myeloid leukemia. Cancer Res. 2006, 66: 6118-6128. 10.1158/0008-5472.CAN-06-0376.PubMedView ArticleGoogle Scholar
- Schmidl C, Klug M, Boeld TJ, Andreesen R, Hoffmann P, Edinger M, Rehli M: Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity. Genome Res. 2009, 19: 1165-1174. 10.1101/gr.091470.109.PubMedPubMed CentralView ArticleGoogle Scholar
- Klug M, Heinz S, Gebhard C, Schwarzfischer L, Krause SW, Andreesen R, Rehli M: Active DNA demethylation in human postmitotic cells correlates with activating histone modifications, but not transcription levels. Genome Biol. 2010, 11: R63-10.1186/gb-2010-11-6-r63.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim MS, Kondo T, Takada I, Youn MY, Yamamoto Y, Takahashi S, Matsumoto T, Fujiyama S, Shirode Y, Yamaoka I, Kitagawa H, Takeyama K, Shibuya H, Ohtake F, Kato S: DNA demethylation in hormone-induced transcriptional derepression. Nature. 2009, 461: 1007-1012. 10.1038/nature08456.PubMedView ArticleGoogle Scholar
- Zhu B, Zheng Y, Angliker H, Schwarz S, Thiry S, Siegmann M, Jost JP: 5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence. Nucleic Acids Res. 2000, 28: 4157-4165. 10.1093/nar/28.21.4157.PubMedPubMed CentralView ArticleGoogle Scholar
- He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, Ding J, Jia Y, Chen Z, Li L, Sun Y, Li X, Dai Q, Song CX, Zhang K, He C, Xu GL: Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science. 2011, 333: 1303-1307. 10.1126/science.1210944.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang L, Lu X, Lu J, Liang H, Dai Q, Xu GL, Luo C, Jiang H, He C: Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA. Nat Chem Biol. 2012, 8: 328-330. 10.1038/nchembio.914.PubMedPubMed CentralView ArticleGoogle Scholar
- Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, He C, Zhang Y: Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011, 333: 1300-1303. 10.1126/science.1210597.PubMedPubMed CentralView ArticleGoogle Scholar
- Booth MJ, Branco MR, Ficz G, Oxley D, Krueger F, Reik W, Balasubramanian S: Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science. 2012, 336: 934-937. 10.1126/science.1220671.PubMedView ArticleGoogle Scholar
- Bauer M, Goldstein M, Christmann M, Becker H, Heylmann D, Kaina B: Human monocytes are severely impaired in base and DNA double-strand break repair that renders them vulnerable to oxidative stress. Proc Natl Acad Sci USA. 2011, 108: 21105-21110. 10.1073/pnas.1111919109.PubMedPubMed CentralView ArticleGoogle Scholar
- Spruijt CG, Gnerlich F, Smits AH, Pfaffeneder T, Jansen PW, Bauer C, Munzel M, Wagner M, Muller M, Khan F, Eberl HC, Mensinga A, Brinkman AB, Lephikov K, Muller U, Walter J, Boelens R, van Ingen H, Leonhardt H, Carell T, Vermeulen M: Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell. 2013, 152: 1146-1159. 10.1016/j.cell.2013.02.004.PubMedView ArticleGoogle Scholar
- Serandour AA, Avner S, Oger F, Bizot M, Percevault F, Lucchetti-Miganeh C, Palierne G, Gheeraert C, Barloy-Hubler F, Peron CL, Madigou T, Durand E, Froguel P, Staels B, Lefebvre P, Metivier R, Eeckhoute J, Salbert G: Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers. Nucleic Acids Res. 2012, 40: 8255-8265. 10.1093/nar/gks595.PubMedPubMed CentralView ArticleGoogle Scholar
- Gebhard C, Benner C, Ehrich M, Schwarzfischer L, Schilling E, Klug M, Dietmaier W, Thiede C, Holler E, Andreesen R, Rehli M: General transcription factor binding at CpG islands in normal cells correlates with resistance to de novo DNA methylation in cancer cells. Cancer Res. 2010, 70: 1398-1407. 10.1158/0008-5472.CAN-09-3406.PubMedView ArticleGoogle Scholar
- Mohn F, Weber M, Schubeler D, Roloff TC: Methylated DNA immunoprecipitation (MeDIP). Methods Mol Biol. 2009, 507: 55-64. 10.1007/978-1-59745-522-0_5.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.