Abstract
NOMe-seq (nucleosome occupancy and methylome sequencing) identifies nucleosome-depleted regions that correspond to promoters, enhancers, and insulators. The NOMe-seq method is based on the treatment of chromatin with the M.CviPI methyltransferase, which methylates GpC dinucleotides that are not protected by nucleosomes or other proteins that are tightly bound to the chromatin (GpCm does not occur in the human genome and therefore there is no endogenous background of GpCm). Following bisulfite treatment of the M.CviPI-methylated chromatin (which converts unmethylated Cs to Ts and thus allows the distinction of GpC from GpCm) and subsequent genomic sequencing, nucleosome-depleted regions can be ascertained on a genome-wide scale. The bisulfite treatment also allows the distinction of CpG from CmpG (most endogenous methylation occurs at CpG dinucleotides) and thus the endogenous methylation status of the genome can also be obtained in the same sequencing reaction. Importantly, open chromatin is expected to have high levels of GpCm but low levels of CmpG; thus, each of the two separate methylation analyses serve as independent (but opposite) measures which provide matching chromatin designations for each regulatory element.
NOMe-seq has advantages over ChIP-seq for identification of regulatory elements because it is not reliant upon knowing the exact modifications on the surrounding nucleosomes. Also, NOMe-seq has advantages over DHS (DNase hypersensitive site)-seq, FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)-seq, and ATAC (Assay for Transposase-Accessible Chromatin)-seq because it also gives positioning information for several nucleosomes on either side of each open regulatory element. Here, we provide a detailed protocol for NOMe-seq that begins with the isolation of chromatin, followed by methylation of GpCs with M.CviPI and treatment with bisulfite, and ending with the creation of next generation sequencing libraries. We also include sequencing QC analysis metrics and bioinformatics steps that can be used to identify nucleosome-depleted regions throughout the genome.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Consortium EP (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489(7414):57–74. https://doi.org/10.1038/nature11247
Rada-Iglesias A, Bajpai R, Swigut T, Brugmann SA, Flynn RA, Wysocka J (2011) A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470(7333):279–283. https://doi.org/10.1038/nature09692
RoadmapEpigenomicsConsortium (2015) Integrative analysis of 111 reference human epigenomes. Nature 19:317–330
Heintzman ND, Ren B (2009) Finding distal regulatory elements in the human genome. Curr Opin Genet Dev 19(6):541–549. https://doi.org/10.1016/j.gde.2009.09.006
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 (2007) Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet 39(3):311–318. https://doi.org/10.1038/ng1966
DesJarlais R, Tummino PJ (2016) Role of histone-modifying enzymes and their complexes in regulation of chromatin biology. Biochemistry 55(11):1584–1599. https://doi.org/10.1021/acs.biochem.5b01210
Allis CD, Jenuwein T (2016) The molecular hallmarks of epigenetic control. Nat Rev Genet 17(8):487–500. https://doi.org/10.1038/nrg.2016.59
Merkenschlager M, Nora EP (2016) CTCF and Cohesin in genome folding and transcriptional gene regulation. Annu Rev Genomics Hum Genet 17:17–43. https://doi.org/10.1146/annurev-genom-083115-022339
Kelly TK, Liu Y, Lay FD, Liang G, Berman BP, Jones PA (2012) Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res 22(12):2497–2506. https://doi.org/10.1101/gr.143008.112
Xu M, Kladde MP, Van Etten JL, Simpson RT (1998) Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC. Nucleic Acids Res 26(17):3961–3966
O'Geen H, Echipare L, Farnham PJ (2011) Using ChIP-Seq technology to generate high-resolution profiles of histone modifications. Methods Mol Biol 791:265–286. https://doi.org/10.1007/978-1-61779-316-5_20
Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, Sheffield NC, Stergachis AB, Wang H, Vernot B, Garg K, John S, Sandstrom R, Bates D, Boatman L, Canfield TK, Diegel M, Dunn D, Ebersol AK, Frum T, Giste E, Johnson AK, Johnson EM, Kutyavin T, Lajoie B, Lee BK, Lee K, London D, Lotakis D, Neph S, Neri F, Nguyen ED, Qu H, Reynolds AP, Roach V, Safi A, Sanchez ME, Sanyal A, Shafer A, Simon JM, Song L, Vong S, Weaver M, Yan Y, Zhang Z, Zhang Z, Lenhard B, Tewari M, Dorschner MO, Hansen RS, Navas PA, Stamatoyannopoulos G, Iyer VR, Lieb JD, Sunyaev SR, Akey JM, Sabo PJ, Kaul R, Furey TS, Dekker J, Crawford GE, Stamatoyannopoulos JA (2012) The accessible chromatin landscape of the human genome. Nature 489(7414):75–82. https://doi.org/10.1038/nature11232
Giresi PG, Kim J, McDaniell RM, Iyer VR, Lieb JD (2007) FAIRE (formaldehyde-assisted isolation of regulatory elements) isolates active regulatory elements from human chromatin. Genome Res 17(6):877–885. https://doi.org/10.1101/gr.5533506
Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ (2013) Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10(12):1213–1218. https://doi.org/10.1038/nmeth.2688
Buenrostro JD, Wu B, Chang HY, Greenleaf WJ (2015) ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol 109(21 29):21–29. https://doi.org/10.1002/0471142727.mb2129s109
Taberlay PC, Statham AL, Kelly TK, Clark SJ, Jones PA (2014) Reconfiguration of nucleosome-depleted regions at distal regulatory elements accompanies DNA methylation of enhancers and insulators in cancer. Genome Res 24(9):1421–1432. https://doi.org/10.1101/gr.163485.113
Statham AL, Taberlay PC, Kelly TK, Jones PA, Clark SJ (2015) Genome-wide nucleosome occupancy and DNA methylation profiling of four human cell lines. Genom Data 3:94–96. https://doi.org/10.1016/j.gdata.2014.11.012
Lay FD, Liu Y, Kelly TK, Witt H, Farnham PJ, Jones PA, Berman BP (2015) The role of DNA methylation in directing the functional organization of the cancer epigenome. Genome Res 25(4):467–477. https://doi.org/10.1101/gr.183368.114
Xi Y, Li W (2009) BSMAP: whole genome bisulfite sequence MAPping program. BMC Bioinformatics 10:232. https://doi.org/10.1186/1471-2105-10-232
Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for bisulfite-Seq applications. Bioinformatics 27(11):1571–1572. https://doi.org/10.1093/bioinformatics/btr167
Guo W, Fiziev P, Yan W, Cokus S, Sun X, Zhang MQ, Chen PY, Pellegrini M (2013) BS-Seeker2: a versatile aligning pipeline for bisulfite sequencing data. BMC Genomics 14:774. https://doi.org/10.1186/1471-2164-14-774
Chen PY, Cokus SJ, Pellegrini M (2010) BS Seeker: precise mapping for bisulfite sequencing. BMC Bioinformatics 11:203. https://doi.org/10.1186/1471-2105-11-203
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Liu Y, Siegmund KD, Laird PW, Berman BP (2012) Bis-SNP: combined DNA methylation and SNP calling for bisulfite-seq data. Genome Biol 13(7):R61. https://doi.org/10.1186/gb-2012-13-7-r61
Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24–26. https://doi.org/10.1038/nbt.1754
Nicol JW, Helt GA, Jr. Blanchard SG, Raja A, Loraine AE (2009) The integrated genome browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics 25(20):2730–2731. https://doi.org/10.1093/bioinformatics/btp472
Evgrafov OV, Wrobel BB, Kang X, Simpson G, Malaspina D, Knowles JA (2011) Olfactory neuroepithelium-derived neural progenitor cells as a model system for investigating the molecular mechanisms of neuropsychiatric disorders. Psychiatr Genet 21(5):217–228. https://doi.org/10.1097/YPG.0b013e328341a2f0
ThePsychEncodeConsortium, Akbarian S, Liu C, Knowles JA, Vaccarino FM, Farnham PJ, Crawford GE, Jaffe AE, Pinto D, Dracheva S, Geschwind DH, Mill J, Nairn AC, Abyzov A, Pochareddy S, Prabhakar S, Weissman S, Sullivan PF, State MW, Weng Z, Peters MA, White KP, Gerstein MB, Amiri A, Armoskus C, Ashley-Koch AE, Bae T, Beckel-Mitchener A, Berman BP, Coetzee GA, Coppola G, Francoeur N, Fromer M, Gao R, Grennan K, Herstein J, Kavanagh DH, Ivanov NA, Jiang Y, Kitchen RR, Kozlenkov A, Kundakovic M, Li M, Li Z, Liu S, Mangravite LM, Mattei E, Markenscoff-Papadimitriou E, Navarro FC, North N, Omberg L, Panchision D, Parikshak N, Poschmann J, Price AJ, Purcaro M, Reddy TE, Roussos P, Schreiner S, Scuderi S, Sebra R, Shibata M, Shieh AW, Skarica M, Sun W, Swarup V, Thomas A, Tsuji J, van Bakel H, Wang D, Wang Y, Wang K, Werling DM, Willsey AJ, Witt H, Won H, Wong CC, Wray GA, Wu EY, Xu X, Yao L, Senthil G, Lehner T, Sklar P, Sestan N (2015) The PsychENCODE project. Nat Neurosci 18(12):1707–1712. https://doi.org/10.1038/nn.4156
O'Geen H, Frietze S, Farnham PJ (2010) Using ChIP-seq technology to identify targets of zinc finger transcription factors. Methods Mol Biol 649:437–455. https://doi.org/10.1007/978-1-60761-753-2_27
Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol 9(9):R137. https://doi.org/10.1186/gb-2008-9-9-r137
O'Neill LP, Turner BM (2003) Immunoprecipitation of native chromatin: NChIP. Methods 31:76–82
Brind'Amour J, Liu S, Hudson M, Chen C, Karimi MM, Lorincz MC (2015) An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations. Nat Commun 6:6033. https://doi.org/10.1038/ncomms7033
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
1 Electronic Supplementary Material
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rhie, S.K., Schreiner, S., Farnham, P.J. (2018). Defining Regulatory Elements in the Human Genome Using Nucleosome Occupancy and Methylome Sequencing (NOMe-Seq). In: Vavouri, T., Peinado, M. (eds) CpG Islands. Methods in Molecular Biology, vol 1766. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7768-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7768-0_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7767-3
Online ISBN: 978-1-4939-7768-0
eBook Packages: Springer Protocols