Abstract
5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals1,2,3,4,5. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC)1,2,4,5,6,7. 5hmC and TET proteins have been implicated in stem cell biology and cancer1,4,5,8,9, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS5. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the ‘poised’ chromatin signature found at developmentally-regulated genes in ES cells.
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Acknowledgements
We thank B. Ren for assistance in next generation sequencing using the Illumina platform. We thank M. Guttman for making his RNASeq data set available to us. W.A.P. is supported by a predoctoral graduate research fellowship from the National Science Foundation, and Y.H. by a postdoctoral fellowship from the Leukemia and Lymphoma Society. R.L. is supported by a California Institute for Regenerative Medicine Training Grant. This study was supported by the National Institute of Health grants RC1 DA028422, R01 AI44432 and 1 R01 HD065812-01A1 and a grant from the California Institute of Regenerative Medicine (to A.R.), a pilot grant from Harvard Catalyst, The Harvard Clinical and Translational Science Center (NIH Grant 1 UL1 RR 025758-02) and NIH K08 HL089150 (to S.A.), and a grant from the Mary. K. Chapman Foundation (to J.R.E.).
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Authors and Affiliations
Contributions
W.A.P., Y.B. and S.A. devised the GLIB method. W.A.P., S.A., H.R.H. and E.M.M. optimized the GLIB method. Y.H. generated the anti-CMS antiserum, and Y.H. and W.A.P. optimized the anti-CMS pull-down. W.A.P. and Y.H. grew ES cells. W.A.P. prepared GLIB samples for sequencing, Y.H. prepared CMS samples, H.R.H. performed MeDIPs. Helicos sequencing and mapping was performed by P.K. and P.M.M., Illumina sequencing and mapping was performed by R.L. and J.R.E., and U.J.P. was responsible for bioinformatic analysis. M.K. performed the anti-5hmC dot blot. W.A.P. and M.T. performed anti-5hmC pull-downs. H.R.H. and S.M. performed and optimized in vitro tests of Tet substrate specificity. W.A.P., S.A. and A.R. wrote the manuscript. S.A. and A.R. coordinated research.
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P.K. and P.M.M. are employees of Helicos Biosciences.
Supplementary information
nature10102-s1.pdf Supplementary Information
This file contains Supplementary Figures 1-5 with legends, Supplementary Methods, additional references and Supplementary Tables 1-3 (PDF 1197 kb)
Supplementary Table 4. GLIB Peak Annotation.
This table shows the location of every GLIB HERG and the genomic features it is enclosed within (or in the case of exons, transcription start sites, or enhancers, touches). (XLS 22370 kb)
Supplementary Table 5. GLIB Peak Locations.
This table shows the location of each GLIB HERG in the Mus Musculus (mm9) genome. (TXT 5055 kb)
Supplementary Table 6. CMS Peak Annotation.
This table shows the location of every CMS HERG and the genomic features it is enclosed within (or in the case of exons, transcription start sites, or enhancers, touches). (XLS 23176 kb)
Supplementary Table 7. CMS Peak Locations.
This table shows the location of each CMS HERG in the Mus Musculus (mm9) genome. (TXT 4601 kb)
Supplementary Table 8. 5mC Peak Annotation.
This table shows the location of every MERG and the genomic features it is enclosed within (or in the case of exons, transcription start sites, or enhancers, touches). (XLS 21412 kb)
Supplementary Table 9. 5mC Peak Locations.
This table shows the location of each MERG in the Mus Musculus (mm9) genome. (TXT 2648 kb)
Supplementary Table 9. Visualization of hoxb locus
This file shows reads from the GLIB (reads.glib.hmc) and anti-CMS (reads.cms.hmc) precipitations. Also shown are the reads from the –BGT control (reads.glib.bg), the bisulphite treated input (reads.cms.bg), and the HERGs from each method (peaks.glib and peaks.cms). (TXT 3101 kb)
Supplementary Table 11. List of hydroxymethylated TSS genes (by GLIB).
This table lists every gene that overlaps with a HERG (as determined by GLIB) at or immediately prior to the TSS (-800bp to +200bp). Featured is the RefSeq output, the promoter CpG class and histone methylation state22, the expresson decile in ES cells21, the presence or absence of polycomb features at the promoter23, change in expression upon differentiation to embryoid bodies20, and upregulation or downregulation in response to Tet1 depletion8. (XLS 4771 kb)
Supplementary Table 12. List of hydroxymethylated TSS genes (by anti-CMS).
This table lists every gene that overlaps with a HERG (as determined by anti-CMS precipitation) at or immediately prior to the TSS (-800bp to +200bp). Featured is the RefSeq output, the promoter CpG class and histone methylation state22, the expresson decile in ES cells21, the presence or absence of polycomb features at the promoter23, change in expression upon differentiation to embryoid bodies20, and upregulation or downregulation in response to Tet1 depletion8. (XLS 4291 kb)
Supplementary Table 13. List of methylated TSS genes (by MeDIP).
This table lists every gene that overlaps with a MERG at or immediately prior to the TSS (-800bp to +200bp). Featured is the RefSeq output, the promoter CpG class and histone methylation state22, the expresson decile in ES cells21, the presence or absence of polycomb features at the promoter23, the change in expression upon differentiation to embryoid bodies20, and upregulation or downregulation in response to Tet1 depletion8. (XLS 644 kb)
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Pastor, W., Pape, U., Huang, Y. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011). https://doi.org/10.1038/nature10102
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DOI: https://doi.org/10.1038/nature10102
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