5-hydroxymethylcytosine represses the activity of enhancers in embryonic stem cells: a new epigenetic signature for gene regulation

BMC Genomics

Kyoung-Jae Won¹  Klaus H Kaestner¹  Hee-Woong Lim¹  Rinho Kim¹  Inchan Choi¹ 

[1] Department of Genetics, Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania, 3400 Civic Center Blvd, 19104 Philadelphia, PA, USA

关键词: Enhancer;    mESC;    eRNA;    PolII;    GROseq;    5hmC;   

Others  :  1216279
DOI  :  10.1186/1471-2164-15-670

 received in 2014-05-19, accepted in 2014-07-31,  发布年份 2014

【 摘 要 】

Background

Recent mapping of 5-hydroxymethylcytosine (5hmC) provides a genome-wide view of the distribution of this important chromatin mark. However, the role of 5hmC in specific regulatory regions is not clear, especially at enhancers.

Results

We found a group of distal transcription factor binding sites highly enriched for 5-hdroxymethylcytosine (5hmC), but lacking any known activating histone marks and being depleted for nascent transcripts, suggesting a repressive role for 5hmC in mouse embryonic stem cells (mESCs). 5-formylcytosine (5fC), which is known to mark poised enhancers where H3K4me1 is enriched, is also observed at these sites. Furthermore, the 5hmC levels were inversely correlated with RNA polymerase II (PolII) occupancy in mESCs as well as in fully differentiated adipocytes. Interestingly, activating H3K4me1/2 histone marks were enriched at these sites when the associated genes become activated following lineage specification. These putative enhancers were shown to be functional in embryonic stem cells when unmethylated. Together, these data suggest that 5hmC suppresses the activity of this group of enhancers, which we termed “silenced enhancers”.

Conclusions

Our findings indicate that 5hmC has a repressive role at specific proximal and distal regulatory regions in mESCs, and suggest that 5hmC is a new epigenetic mark for silenced enhancers.

【 授权许可】

   
2014 Choi et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Williams K, Christensen J, Pedersen MT, Johansen JV, Cloos PA, Rappsilber J, Helin K: TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 2011, 473(7347):343-348.
• [2]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(5929):930-935.
• [3]Yu M, Hon GC, Szulwach KE, Song CX, Zhang L, Kim A, Li X, Dai Q, Shen Y, Park B, Min JH, Jin P, Ren B, He C: Base-resolution analysis of 5-hydroxymethylcytosine in the Mammalian genome. Cell 2012, 149(6):1368-1380.
• [4]Kriaucionis S, Heintz N: The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 2009, 324(5929):929-930.
• [5]Song CX, Szulwach KE, Fu Y, Dai Q, Yi C, Li X, Li Y, Chen CH, Zhang W, Jian X, Wang J, Zhang L, Looney TJ, Zhang B, Godley LA, Hicks LM, Lahn BT, Jin P, He C: Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol 2011, 29(1):68-72.
• [6]Mellen M, Ayata P, Dewell S, Kriaucionis S, Heintz N: MeCP2 Binds to 5hmC Enriched within Active Genes and Accessible Chromatin in the Nervous System. Cell 2012, 151(7):1417-1430.
• [7]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, Métivier R, Eeckhoute J, Salbert G: Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers. Nucleic Acids Res 2012, 40(17):8255-8265.
• [8]Matarese F: Carrillo-de Santa Pau E, Stunnenberg HG: 5-Hydroxymethylcytosine: a new kid on the epigenetic block? Mol Syst Biol 2011, 7:562.
• [9]Szulwach KE, Li X, Li Y, Song CX, Han JW, Kim S, Namburi S, Hermetz K, Kim JJ, Rudd MK, Yoon YS, Ren B, He C, Jin P: Integrating 5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells. PLoS Genet 2011, 7(6):e1002154.
• [10]Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, Marques CJ, Andrews S, Reik W: Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 2011, 473(7347):398-402.
• [11]Stroud H, Feng S, Morey Kinney S, Pradhan S, Jacobsen SE: 5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol 2011, 12(6):R54.
• [12]Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, 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(7):679-684.
• [13]Xu Y, Wu F, Tan L, Kong L, Xiong L, Deng J, Barbera AJ, Zheng L, Zhang H, Huang S, Min J, Nicholson T, Chen T, Xu G, Shi Y, Zhang K, Shi YG: Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol Cell 2011, 42(4):451-464.
• [14]Pastor WA, Pape UJ, Huang Y, Henderson HR, Lister R, Ko M, McLoughlin EM, Brudno Y, Mahapatra S, Kapranov P, Tahiliani M, Daley GQ, Liu XS, Ecker JR, Milos PM, Agarwal S, Rao A: Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 2011, 473(7347):394-397.
• [15]Zhang Y, Wong CH, Birnbaum RY, Li G, Favaro R, Ngan CY, Lim J, Tai E, Poh HM, Wong E, Mulawadi FH, Sung WK, Nicolis S, Ahituv N, Ruan Y, Wei CL: Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations. Nature 2013, 504(7479):306-310.
• [16]Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH: Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 2008, 133(6):1106-1117.
• [17]Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, Lee W, Mendenhall E, O'Donovan A, Presser A, Russ C, Xie X, Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander ES, Bernstein BE: Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 2007, 448(7153):553-560.
• [18]Min IM, Waterfall JJ, Core LJ, Munroe RJ, Schimenti J, Lis JT: Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev 2011, 25(7):742-754.
• [19]Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, Cabili MN, Jaenisch R, Mikkelsen TS, Jacks T, Hacohen N, Bernstein BE, Kellis M, Regev A, Rinn JL, Lander ES: Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 2009, 458(7235):223-227.
• [20]Core LJ, Waterfall JJ, Lis JT: Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 2008, 322(5909):1845-1848.
• [21]Wang D, Garcia-Bassets I, Benner C, Li W, Su X, Zhou Y, Qiu J, Liu W, Kaikkonen MU, Ohgi KA, Glass CK, Rosenfeld MG, Fu XD: Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 2011, 474(7351):390-394.
• [22]Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, Barrera LO, Van Calcar S, Qu C, Ching KA, Wang W, Weng Z, Green RD, Crawford GE, Ren B: Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet 2007, 39(3):311-318.
• [23]Rada-Iglesias A, Bajpai R, Swigut T, Brugmann SA, Flynn RA, Wysocka J: A unique chromatin signature uncovers early developmental enhancers in humans. Nature 2011, 470(7333):279-283.
• [24]Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, Hanna J, Lodato MA, Frampton GM, Sharp PA, Boyer LA, Young RA, Jaenisch R: Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc Natl Acad Sci USA 2010, 107(50):21931-21936.
• [25]Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, Nery JR, Lee L, Ye Z, Ngo QM, Edsall L, Antosiewicz-Bourget J, Stewart R, Ruotti V, Millar AH, Thomson JA, Ren B, Ecker JR: Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 2009, 462(7271):315-322.
• [26]Wu H, Zhang Y: Tet1 and 5-hydroxymethylation: a genome-wide view in mouse embryonic stem cells. Cell Cycle 2011, 10(15):2428-2436.
• [27]Tan L, Xiong L, Xu W, Wu F, Huang N, Xu Y, Kong L, Zheng L, Schwartz L, Shi Y, Shi YG: Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method. Nucleic Acids Res 2013, 41(7):e84.
• [28]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(6047):1300-1303.
• [29]Maiti A, Drohat AC: Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J Biol Chem 2011, 286(41):35334-35338.
• [30]Song CX, Szulwach KE, Dai Q, Fu Y, Mao SQ, Lin L, Street C, Li Y, Poidevin M, Wu H, Gao J, Liu P, Li L, Xu GL, Jin P, He C: Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 2013, 153(3):678-691.
• [31]Bernstein BE, Stamatoyannopoulos JA, Costello JF, Ren B, Milosavljevic A, Meissner A, Kellis M, Marra MA, Beaudet AL, Ecker JR, Farnham PJ, Hirst M, Lander ES, Mikkelsen TS, Thomson JA: The NIH Roadmap Epigenomics Mapping Consortium. Nat Biotechnol 2010, 28(10):1045-1048.
• [32]Sigova AA, Mullen AC, Molinie B, Gupta S, Orlando DA, Guenther MG, Almada AE, Lin C, Sharp PA, Giallourakis CC, Young RA: Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Proc Natl Acad Sci USA 2013, 110(8):2876-2881.
• [33]Nielsen R, Pedersen TA, Hagenbeek D, Moulos P, Siersbaek R, Megens E, Denissov S, Borgesen M, Francoijs KJ, Mandrup S, Stunnenberg HG: Genome-wide profiling of PPARgamma:RXR and RNA polymerase II occupancy reveals temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis. Genes Dev 2008, 22(21):2953-2967.
• [34]Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA: Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005, 122(6):947-956.
• [35]McLean CY, Bristor D, Hiller M, Clarke SL, Schaar BT, Lowe CB, Wenger AM, Bejerano G: GREAT improves functional interpretation of cis-regulatory regions. Nat Biotechnol 2010, 28(5):495-501.
• [36]Rezgaoui M, Hermey G, Riedel IB, Hampe W, Schaller HC, Hermans-Borgmeyer I: Identification of SorCS2, a novel member of the VPS10 domain containing receptor family, prominently expressed in the developing mouse brain. Mech Dev 2001, 100(2):335-338.
• [37]Yu P, Xiao S, Xin X, Song CX, Huang W, McDee D, Tanaka T, Wang T, He C, Zhong S: Spatiotemporal clustering of the epigenome reveals rules of dynamic gene regulation. Genome Res 2013, 23(2):352-364.
• [38]Huang Y, Chavez L, Chang X, Wang X, Pastor WA, Kang J, Zepeda-Martinez JA, Pape UJ, Jacobsen SE, Peters B, Rao A: Distinct roles of the methylcytosine oxidases Tet1 and Tet2 in mouse embryonic stem cells. Proc Natl Acad Sci USA 2014, 111(4):1361-1366.
• [39]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(6047):1303-1307.
• [40]Hahn MA, Qiu R, Wu X, Li AX, Zhang H, Wang J, Jui J, Jin SG, Jiang Y, Pfeifer GP, Lu Q: Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis. Cell Rep 2013, 3(2):291-300.
• [41]Robertson J, Robertson AB, Klungland A: The presence of 5-hydroxymethylcytosine at the gene promoter and not in the gene body negatively regulates gene expression. Biochem Biophys Res Commun 2011, 411(1):40-43.
• [42]Kim M, Park YK, Kang TW, Lee SH, Rhee YH, Park JL, Kim HJ, Lee D, Kim SY, Kim YS: Dynamic changes in DNA methylation and hydroxymethylation when hES cells undergo differentiation toward a neuronal lineage. Hum Mol Genet 2014, 23(3):657-667.
• [43]Guttman M, Garber M, Levin JZ, Donaghey J, Robinson J, Adiconis X, Fan L, Koziol MJ, Gnirke A, Nusbaum C, Rinn JL, Lander ES, Regev A: Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat Biotechnol 2010, 28(5):503-510.
• [44]Nikolaev LG, Akopov SB, Didych DA, Sverdlov ED: Vertebrate Protein CTCF and its Multiple Roles in a Large-Scale Regulation of Genome Activity. Curr Genomics 2009, 10(5):294-302.
• [45]Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A, Zhang X, Bernstein BE, Nusbaum C, Jaffe DB, Gnirke A, Jaenisch R, Lander ES: Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 2008, 454(7205):766-770.
• [46]Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5(7):621-628.
• [47]Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, Li J, Thiagarajan M, White JA, Quackenbush J: TM4 microarray software suite. Methods Enzymol 2006, 411:134-193.
• [48]Rahl PB, Lin CY, Seila AC, Flynn RA, McCuine S, Burge CB, Sharp PA, Young RA: c-Myc regulates transcriptional pause release. Cell 2010, 141(3):432-445.
• [49]Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC: Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 1993, 90(18):8424-8428.