Base-Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome

The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance. The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance. Genome-wide Tet-assisted bisulfite sequencing for single-base detection of 5hmC Single-base resolution maps of 5hmC versus 5mC in human and mouse ESCs 5hmC is enriched at distal-regulatory elements and promoters with low CpG content 5hmC deposition is asymmetric and strand biased toward G-rich sequences 5-methylcytosine (5mC) in mammalian genomic DNA is essential for normal development and impacts a variety of biological functions. In 2009, 5-hydroxymethylcytosine (5hmC) was discovered as another relatively abundant form of cytosine modification in embryonic stem cells (ESCs) and Purkinje neurons (Kriaucionis and Heintz, 2009Kriaucionis S. Heintz N. 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Jacobsen S.E. 5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells.Genome Biol. 2011; 12: R54Crossref PubMed Scopus (346) Google Scholar, Szulwach et al., 2011aSzulwach K.E. Li X. Li Y. Song C.X. Han J.W. Kim S. Namburi S. Hermetz K. Kim J.J. Rudd M.K. et al.Integrating 5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells.PLoS Genet. 2011; 7: e1002154Crossref PubMed Scopus (219) Google Scholar, Williams et al., 2011Williams K. Christensen J. Pedersen M.T. Johansen J.V. Cloos P.A. Rappsilber J. Helin K. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity.Nature. 2011; 473: 343-348Crossref PubMed Scopus (800) Google Scholar, Wu et al., 2011Wu H. D'Alessio A.C. Ito S. Wang Z. Cui K. Zhao K. Sun Y.E. Zhang Y. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.Genes Dev. 2011; 25: 679-684Crossref PubMed Scopus (452) Google Scholar, Xu et al., 2011Xu Y. Wu F. Tan L. Kong L. Xiong L. Deng J. Barbera A.J. Zheng L. Zhang H. Huang S. et al.Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells.Mol. Cell. 2011; 42: 451-464Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). However, in all cases, the resolution of these maps was restricted by the size of the immunoprecipitated or chemically captured DNA, which varied from several hundred to over a thousand bases. The study of 5mC has been facilitated by the development of whole-genome bisulfite sequencing methods that can resolve the genomic location of methylcytosine at single-base resolution (Cokus et al., 2008Cokus S.J. Feng S. Zhang X. Chen Z. Merriman B. Haudenschild C.D. Pradhan S. Nelson S.F. Pellegrini M. Jacobsen S.E. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.Nature. 2008; 452: 215-219Crossref PubMed Scopus (1717) Google Scholar, Lister et al., 2008Lister R. O'Malley R.C. Tonti-Filippini J. Gregory B.D. Berry C.C. Millar A.H. Ecker J.R. Highly integrated single-base resolution maps of the epigenome in Arabidopsis.Cell. 2008; 133: 523-536Abstract Full Text Full Text PDF PubMed Scopus (1856) Google Scholar, Lister et al., 2009Lister, R., Pelizzola, M., Dowen, R.H., Hawkins, R.D., Hon, G., Tonti-Filippini, J., Nery, J.R., Lee, L., Ye, Z., Ngo, Q.M., et al. (2009). Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322.Google Scholar). However, current bisulfite sequencing methods cannot distinguish between 5mC and 5hmC (Huang et al., 2010Huang Y. Pastor W.A. Shen Y. Tahiliani M. Liu D.R. Rao A. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.PLoS ONE. 2010; 5: e8888Crossref PubMed Scopus (567) Google Scholar, Jin et al., 2010Jin S.G. Kadam S. Pfeifer G.P. Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine.Nucleic Acids Res. 2010; 38: e125Crossref PubMed Scopus (352) Google Scholar). Therefore, the genome-wide bisulfite sequencing maps generated in recent years may not accurately capture the true abundance of 5mC at each base in the genome. A more detailed understanding of the function of 5hmC as well as 5mC has, therefore, been hampered by the lack of a single-base resolution sequencing technology capable of detecting the relative abundance of 5hmC per cytosine. Here we present a Tet-assisted bisulfite sequencing (TAB-Seq) strategy, which provides a method for single-base resolution detection of 5hmC amenable to both genome-wide and loci-specific sequencing. Applying this method, we have generated genome-wide, single-base resolution maps of 5hmC in ESCs. Distinct classes of functional elements exhibit variable abundance of 5hmC, with promoter-distal regulatory elements harboring the highest levels of 5hmC. High levels of 5hmC and reciprocally low levels of 5mC can be found near binding sites of transcription factors. In contrast to 5mC, 5hmC sites display strand asymmetry and sequence bias. Finally, the base-resolution maps of 5hmC provide more accurate estimates of both 5hmC and 5mC levels at each modified cytosine than previous whole-genome bisulfite sequencing approaches. Our results support a dynamic DNA methylation process at distal-regulatory elements and suggest that different mechanisms of DNA modification may be involved at distinct classes of functional sequences in the genome. Traditional bisulfite sequencing cannot discriminate 5mC from 5hmC because both resist deamination by bisulfite treatment (Huang et al., 2010Huang Y. Pastor W.A. Shen Y. Tahiliani M. Liu D.R. Rao A. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.PLoS ONE. 2010; 5: e8888Crossref PubMed Scopus (567) Google Scholar, Jin et al., 2010Jin S.G. Kadam S. Pfeifer G.P. Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine.Nucleic Acids Res. 2010; 38: e125Crossref PubMed Scopus (352) Google Scholar). We have recently found that TET proteins not only oxidize 5mC to 5hmC but also further oxidize 5hmC to 5caC, and that 5caC exhibits behavior similar to that of unmodified cytosine after bisulfite treatment (He et al., 2011He Y.F. Li B.Z. Li Z. Liu P. Wang Y. Tang Q. Ding J. Jia Y. Chen Z. Li L. et al.Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.Science. 2011; 333: 1303-1307Crossref PubMed Scopus (2014) Google Scholar, Ito et al., 2011Ito S. Shen L. Dai Q. Wu S.C. Collins L.B. Swenberg J.A. He C. Zhang Y. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.Science. 2011; 333: 1300-1303Crossref PubMed Scopus (2463) Google Scholar). This deamination difference between 5caC and 5mC/5hmC under standard bisulfite conditions inspired us to explore TAB-Seq. In this approach, we use β-glucosyltransferase (βGT) to introduce a glucose onto 5hmC, generating β-glucosyl-5-hydroxymethylcytosine (5gmC) to protect 5hmC from further TET oxidation. After blocking of 5hmC, all 5mC is converted to 5caC by oxidation with an excess of recombinant Tet1 protein. Bisulfite treatment of the resulting DNA then converts all C and 5caC (derived from 5mC) to uracil or 5caU, respectively, whereas the original 5hmC bases remain protected as 5gmC. Thus, subsequent sequencing will reveal 5hmC as C, which, when combined with traditional bisulfite sequencing results, will provide an accurate assessment of abundance of this modification at each cytosine (Figure 1A ). We first confirmed that 5gmC is read as C in traditional bisulfite sequencing (data not shown). We cloned and expressed the catalytic domain of mouse Tet1 (mTet1) (Figure S1A available online), as previously reported (Ito et al., 2010Ito S. D'Alessio A.C. Taranova O.V. Hong K. Sowers L.C. Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.Nature. 2010; 466: 1129-1133Crossref PubMed Scopus (1925) Google Scholar). We tested a double-stranded DNA (dsDNA) with site-specifically incorporated 5mC or 5hmC modification (Figure 1B). Application of our method with Sanger sequencing of the PCR-amplified products showed that the original 5mC was completely converted into T after treatment, indicating efficient oxidation of 5mC to 5caC by mTet1 (Figure 1B). However, the original 5hmC was sequenced as C, confirming that the protected 5gmC is resistant to deamination under bisulfite treatment (Figure 1B). The products of each step were confirmed by MALDI-TOF/TOF using a shorter model duplex DNA (Figure 1C). Full conversion of 5mC in the context of genomic DNA was also confirmed by conventional bisulfite, PCR, and both Sanger and semiconductor sequencing (Figures S1B and S1C). Additionally, application to genomic DNA confirmed conversion of 5mC to 5caC and protection of 5hmC, and that 5fC is undetectable by immunoblot on the final reaction products (Figure 1D). Thus, coupling βGT-mediated transfer of glucose to 5hmC with mTet1-catalyzed oxidation of 5mC to 5caC enables the distinction of 5hmC from both C and 5mC after sodium bisulfite treatment.Figure S1TAB-Seq of Specific Loci and 5mC Conversion Rate Test in the Context of Genomic DNA, Related to Figure 1Show full caption(A) Purified mTet1 catalytic domain used for oxidation of genomic DNA.(B) Sanger sequencing of M.SssI treated lambda DNA spiked into a genomic DNA background at 0.5% before (− mTet1) and after (+ mTet1) subjecting the DNA to TAB-Seq.(C) Semiconductor sequencing of M.SssI-treated lambda DNA spiked into a genomic DNA background at 0.5% before (− mTet1) and after (+ mTet1) subjecting the DNA to TAB-Seq. The left y axis shows the percentage of bases read as C, and the right y axis shows the depth of sequencing at each C position in the targeted amplicon, which is plotted on the x axis. For reference, a dotted line is plotted at 98% on the left y axis.(D) Several loci in mouse cerebellum were tested by both traditional bisulfite sequencing and TAB-Seq. Genuine 5hmC is read as C in both methods (left) whereas genuine 5mC is read as C in traditional bisulfite sequencing but display as T in TAB-Seq (right).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Purified mTet1 catalytic domain used for oxidation of genomic DNA. (B) Sanger sequencing of M.SssI treated lambda DNA spiked into a genomic DNA background at 0.5% before (− mTet1) and after (+ mTet1) subjecting the DNA to TAB-Seq. (C) Semiconductor sequencing of M.SssI-treated lambda DNA spiked into a genomic DNA background at 0.5% before (− mTet1) and after (+ mTet1) subjecting the DNA to TAB-Seq. The left y axis shows the percentage of bases read as C, and the right y axis shows the depth of sequencing at each C position in the targeted amplicon, which is plotted on the x axis. For reference, a dotted line is plotted at 98% on the left y axis. (D) Several loci in mouse cerebellum were tested by both traditional bisulfite sequencing and TAB-Seq. Genuine 5hmC is read as C in both methods (left) whereas genuine 5mC is read as C in traditional bisulfite sequencing but display as T in TAB-Seq (right). The ability to distinguish 5hmC at base resolution offers a significant opportunity to further parse DNA methylation/hydroxymethylation states at specific genomic loci. We applied traditional bisulfite sequencing and TAB-Seq to known 5hmC-enriched loci in mouse cerebellum that were identified previously (Song et al., 2011Song C.X. Szulwach K.E. Fu Y. Dai Q. Yi C. Li X. Li Y. Chen C.H. Zhang W. Jian X. et al.Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.Nat. Biotechnol. 2011; 29: 68-72Crossref PubMed Scopus (806) Google Scholar, Szulwach et al., 2011bSzulwach K.E. Li X. Li Y. Song C.X. Wu H. Dai Q. Irier H. Upadhyay A.K. Gearing M. Levey A.I. et al.5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.Nat. Neurosci. 2011; 14: 1607-1616Crossref PubMed Scopus (615) Google Scholar). Comparing the sequencing results, we were able to identify genuine 5hmC and 5mC sites (Figure S1D). We next applied TAB-Seq to genomic DNA from H1 hESCs and E14Tg2a mESCs and sequenced to an average depth of 26.5× and 17× per cytosine, respectively. Successful detection of 5hmC is governed by three key parameters: (1) efficient conversion of unmodified cytosine to uracil; (2) efficient conversion of 5mC to 5caU/U; and (3) efficient protection of 5hmC. To directly assess these conversion rates in the context of genomic DNA, sequenced samples were spiked in with fragments of lambda DNA amplified by PCR to contain three distinct domains having either unmodified cytosine, 5mC, or 5hmC. We observe low nonconversion rates for unmodified cytosine (0.38%) and 5mC (2.21%), contrasted to a high nonconversion rate of 5hmC (84.4%) (Figure S2B ). Further analysis indicates that this latter value is an underestimate of the true 5hmC protection rate in H1, which is close to 92.0% (Figures S2D and S2E). These data further confirm the capability of TAB-Seq for robust distinction of 5hmC from 5mC and unmodified cytosine in the context of genomic DNA. We next focused our analysis on the map of H1 hESCs. To confidently identify 5hmC-modified bases, we took advantage of the highly annotated H1 methylome generated with methylC-Seq, which identifies both 5mC as well as 5hmC. Accordingly, we restricted our search for 5hmC to the subset of methylated bases previously identified by methylC-Seq (Lister et al., 2009Lister R. Pelizzola M. Dowen R.H. Hawkins R.D. Hon G. Tonti-Filippini J. Nery J.R. Lee L. Ye Z. Ngo Q.M. et al.Human DNA methylomes at base resolution show widespread epigenomic differences.Nature. 2009; 462: 315-322Crossref PubMed Scopus (3352) Google Scholar). The probability that a cytosine can be confidently identified as 5hmC is governed by the sequencing depth at the cytosine and abundance of the modification (Figure S2C). Modeling this probabilistic event with a binomial distribution (Lister et al., 2009Lister R. Pelizzola M. Dowen R.H. Hawkins R.D. Hon G. Tonti-Filippini J. Nery J.R. Lee L. Ye Z. Ngo Q.M. et al.Human DNA methylomes at base resolution show widespread epigenomic differences.Nature. 2009; 462: 315-322Crossref PubMed Scopus (3352) Google Scholar) with N as the depth of sequencing at the cytosine and p as the 5mC nonconversion rate, we identified a total of 691,414 5hmCs with a false discovery rate of 5% (Figure S2F; see Extended Experimental Procedures). Given an average sequencing depth of 26.5, our assay can on average resolve 5hmC having an abundance of 20% or higher (Figure S2C). Genomic profiles of absolute 5hmC levels are comparable to a map previously generated with an affinity-based approach (Szulwach et al., 2011aSzulwach K.E. Li X. Li Y. Song C.X. Han J.W. Kim S. Namburi S. Hermetz K. Kim J.J. Rudd M.K. et al.Integrating 5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells.PLoS Genet. 2011; 7: e1002154Crossref PubMed Scopus (219) Google Scholar) (Figure 2A ). As sequenced fragments are equally distributed among the population of cells, TAB-Seq provides a steady-state glimpse of 5hmC in the entire population. This is in contrast to affinity-based approaches, which bias sequencing toward 5hmC-enriched DNA fragments. By TAB-Seq, identified 5hmCs are highly clustered, unlike 5mCs (Figure S3A ), and track well with peaks of 5hmC enrichment previously identified by affinity sequencing (Figure 2A). There are 7.6 times as many 5hmCs overlapping affinity-identified regions as expected by chance (Figure 2B, Z-score = 1,579). Furthermore, 81.5% of these 82,221 affinity-identified regions were recovered by at least one 5hmC. In contrast, only 35.6% of 5hmCs are recovered by affinity-based approaches, suggesting an increased sensitivity of TAB-Seq. Using semiconductor sequencing, we verified the presence/absence of 5hmC at 57 out of 59 individual cytosines (9 out of 11 hydroxymethylated CpGs, with depth ≥ 30) within regions that previously escaped detection by 5hmC affinity capture (Figure S2A), underscoring the sensitivity and specificity of our approach.Figure S3Related to Figure 3Show full caption(A) The distribution of pairwise distances between all 5hmCs identified in H1 (red), compared to the same number of randomly selected 5mCs (black).(B) The distribution of base-level phastCons conservation scores (Siepel et al., 2005Siepel, A., Bejerano, G., Pedersen, J.S., Hinrichs, A.S., Hou, M., Rosenbloom, K., Clawson, H., Spieth, J., Hillier, L.W., Richards, S., et al. (2005). Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050.Google Scholar) for several tiers of 5hmC abundance.(C) Total methylation level measured by methylC-Seq (left) and the 5hmC abundance measured by TAB-Seq (right) for DNase I hypersensitive elements ranked by sig