NOMe-seq

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Overview of NOMe-seq Figure 1 NOMe-seq overview final 1.jpg
Overview of NOMe-seq

Nucleosome Occupancy and Methylome Sequencing (NOMe-seq) is a genomics technique used to simultaneously detect nucleosome positioning and DNA methylation... [1] This method is an extension of bisulfite sequencing, which is the gold standard for determining DNA methylation. [2] NOMe-seq relies on the methyltransferase M.CviPl, which methylates cytosines in GpC dinucleotides unbound by nucleosomes or other proteins, creating a nucleosome footprint. The mammalian genome naturally contains DNA methylation, but only at CpG sites, so GpC methylation can be differentiated from genomic methylation after bisulfite sequencing. This allows simultaneous analysis of the nucleosome footprint and endogenous methylation on the same DNA molecules. [1] In addition to nucleosome foot-printing, NOMe-seq can determine locations bound by transcription factors. Nucleosomes are bound by 147 base pairs of DNA [3] whereas transcription factors or other proteins will only bind a region of approximately 10-80 base pairs. Following treatment with M.CviPl, nucleosome and transcription factor sites can be differentiated based on the size of the unmethylated GpC region. [4]

Contents

Nucleosome occupancy determines DNA accessibility, which provides insight into regulatory regions of the genome. Important regulatory elements within a cell (such as promoters, enhancers, silencers, etc.), are located in open or accessible regions to allow binding of transcription factors or other regulatory molecules. [3] NOMe-seq can therefore be used to elucidate regulatory information. Alternative DNA accessibility techniques include MNase-seq, [5] DNase-seq, [6] FAIRE-seq, [7] and their successor ATAC-seq. [8] NOMe-seq has the additional benefit of providing DNA methylation status, which also plays a crucial role in the regulation of genomic activity. Interestingly, increased DNA methylation is associated with transcriptional silencing whereas accessible DNA unbound by nucleosomes is generally associated with transcriptional activation. In this sense, NOMe-seq consists of two independent methylation analyses that are functionally oppositional. [9]

History

The M.CviPl methyltransferase was first described in 1998, where the gene was cloned from Chorella virus NYs-1. [10] After its discovery, the methyltransferase was used for nucleosome foot-printing as early as 2004, [11] but NOMe-seq was not officially described until 2012. [12] M.CviPl was not the only methyltransferase used for nucleosome foot-printing; Methylase-sensitive Single Promoter Analysis (M-SPA) was described in 2005 using the CpG methyltransferase M.Sssi. [13] [14] M.CviPl techniques quickly overtook M-SPA as GpC specificity is preferable to CpG specificity, with GpC dinucleotides having a broader distribution throughout the genome and no endogenous methylation. [14] The NOMe-seq assay was subsequently developed, with the earliest mention being in 2011 [15] and an in depth description published in 2012. [12] The technique has since been adapted for single cell technologies, with single cell NOMe-seq (scNOMe-seq) described in 2017 [16] and NOMe-seq using nanopore sequencing (nanoNOMe) described in 2020. [17] These adaptations have allowed high resolution analyses that can compare and contrast DNA accessibility between single cells. [16]

Methods

Components
[9]
Workflow
[1] [9]
NOMe-seq experimental and analytical workflow New Figure.jpg
NOMe-seq experimental and analytical workflow

Use

Advantages

Limitations

Other Applications & Complementary Methods

scNOMe-seq

scNOMe-seq is a method that was adapted from NOMe-seq to be used in single cells studies. This has been found to produce similar results as NOMe-seq when using bulk samples of human cell cultures. Single cell analyses have many benefits in cases where gene expression can vary between cells. For example, to further develop cancer treatments, it would be useful to understand the differences that arise between individual cells using the scNOMe-seq method. [16]

nanoNOMe

nanoNOMe is a method that was adapted from NOMe-seq that uses nanopore sequencing instead of bisulfite sequencing. Nanopore sequencing is a long read sequencing method that also detects DNA methylation, providing additional insight into longe range patterns on individual molecules. [17]

NOMePlot

NOMePlot is a bioinformatic tool that was developed for datasets derived by NOMe-seq. This tool easily obtains single molecule locus-specific information in genome-wide datasets from bulk cell populations and has been validated using mouse embryonic stem cells. [20]

See also

Related Research Articles

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

Methylated DNA immunoprecipitation is a large-scale purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. et al. in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type.

FAIRE-Seq is a method in molecular biology used for determining the sequences of DNA regions in the genome associated with regulatory activity. The technique was developed in the laboratory of Jason D. Lieb at the University of North Carolina, Chapel Hill. In contrast to DNase-Seq, the FAIRE-Seq protocol doesn't require the permeabilization of cells or isolation of nuclei, and can analyse any cell type. In a study of seven diverse human cell types, DNase-seq and FAIRE-seq produced strong cross-validation, with each cell type having 1-2% of the human genome as open chromatin.

ATAC-seq is a technique used in molecular biology to assess genome-wide chromatin accessibility. In 2013, the technique was first described as an alternative advanced method for MNase-seq, FAIRE-Seq and DNase-Seq. ATAC-seq is a faster analysis of the epigenome than DNase-seq or MNase-seq.

H3K4me3 is an epigenetic modification to the DNA packaging protein Histone H3 that indicates tri-methylation at the 4th lysine residue of the histone H3 protein and is often involved in the regulation of gene expression. The name denotes the addition of three methyl groups (trimethylation) to the lysine 4 on the histone H3 protein.

<span class="mw-page-title-main">Single cell epigenomics</span> Study of epigenomics in individual cells by single cell sequencing

Single cell epigenomics is the study of epigenomics in individual cells by single cell sequencing. Since 2013, methods have been created including whole-genome single-cell bisulfite sequencing to measure DNA methylation, whole-genome ChIP-sequencing to measure histone modifications, whole-genome ATAC-seq to measure chromatin accessibility and chromosome conformation capture.

H3K9me3 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the tri-methylation at the 9th lysine residue of the histone H3 protein and is often associated with heterochromatin.

H3K4me1 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the mono-methylation at the 4th lysine residue of the histone H3 protein and often associated with gene enhancers.

H3K36me3 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the tri-methylation at the 36th lysine residue of the histone H3 protein and often associated with gene bodies.

H3K79me2 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the di-methylation at the 79th lysine residue of the histone H3 protein. H3K79me2 is detected in the transcribed regions of active genes.

H4K20me is an epigenetic modification to the DNA packaging protein Histone H4. It is a mark that indicates the mono-methylation at the 20th lysine residue of the histone H4 protein. This mark can be di- and tri-methylated. It is critical for genome integrity including DNA damage repair, DNA replication and chromatin compaction.

H3K14ac is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the acetylation at the 14th lysine residue of the histone H3 protein.

H3K36me2 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the di-methylation at the 36th lysine residue of the histone H3 protein.

H3K36me is an epigenetic modification to the DNA packaging protein Histone H3, specifically, the mono-methylation at the 36th lysine residue of the histone H3 protein.

H3R42me is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the mono-methylation at the 42nd arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.

H3R17me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 17th arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.

H3R26me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 26th arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.

H3R8me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 8th arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.

H3R2me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 2nd arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.

H4R3me2 is an epigenetic modification to the DNA packaging protein histone H4. It is a mark that indicates the di-methylation at the 3rd arginine residue of the histone H4 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.

References

  1. 1 2 3 4 Lay, Fides D.; Kelly, Theresa K.; Jones, Peter A. (2018), Tost, Jörg (ed.), "Nucleosome Occupancy and Methylome Sequencing (NOMe-seq)", DNA Methylation Protocols, Methods in Molecular Biology, New York, NY: Springer, vol. 1708, pp. 267–284, doi:10.1007/978-1-4939-7481-8_14, ISBN   978-1-4939-7481-8, PMID   29224149 , retrieved 2023-02-28
  2. ↑ Li, Yuanyuan; Tollefsbol, Trygve O. (2011). "DNA Methylation Detection: Bisulfite Genomic Sequencing Analysis". Epigenetics Protocols. Methods in Molecular Biology. Vol. 791. pp. 11–21. doi:10.1007/978-1-61779-316-5_2. ISBN   978-1-61779-315-8. ISSN   1064-3745. PMC   3233226 . PMID   21913068.
  3. 1 2 Tsompana, Maria; Buck, Michael J. (2014-11-20). "Chromatin accessibility: a window into the genome". Epigenetics & Chromatin. 7 (1): 33. doi: 10.1186/1756-8935-7-33 . ISSN   1756-8935. PMC   4253006 . PMID   25473421.
  4. ↑ "NOMe-Seq (Nucleosome Occupancy and Methylome Sequencing)". www.activemotif.com. Retrieved 2023-02-28.
  5. ↑ Johnson, Steven M.; Tan, Frederick J.; McCullough, Heather L.; Riordan, Daniel P.; Fire, Andrew Z. (December 2006). "Flexibility and constraint in the nucleosome core landscape of Caenorhabditis elegans chromatin". Genome Research. 16 (12): 1505–1516. doi:10.1101/gr.5560806. ISSN   1088-9051. PMC   1665634 . PMID   17038564.
  6. ↑ Boyle, Alan P.; Davis, Sean; Shulha, Hennady P.; Meltzer, Paul; Margulies, Elliott H.; Weng, Zhiping; Furey, Terrence S.; Crawford, Gregory E. (2008-01-25). "High-resolution mapping and characterization of open chromatin across the genome". Cell. 132 (2): 311–322. doi:10.1016/j.cell.2007.12.014. ISSN   1097-4172. PMC   2669738 . PMID   18243105.
  7. ↑ Giresi, Paul G.; Kim, Jonghwan; McDaniell, Ryan M.; Iyer, Vishwanath R.; Lieb, Jason D. (2006-12-19). "FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin". Genome Research. 17 (6): 877–885. doi:10.1101/gr.5533506. ISSN   1088-9051. PMC   1891346 . PMID   17179217.
  8. ↑ Buenrostro, Jason D.; Wu, Beijing; Chang, Howard Y.; Greenleaf, William J. (January 2015). "ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide". Current Protocols in Molecular Biology. 109 (1): 21.29.1–21.29.9. doi:10.1002/0471142727.mb2129s109. ISSN   1934-3639. PMC   4374986 . PMID   25559105.
  9. 1 2 3 4 Rhie, Suhn Kyong; Schreiner, Shannon; Farnham, Peggy J. (2018). "Defining Regulatory Elements in the Human Genome Using Nucleosome Occupancy and Methylome Sequencing (NOMe-Seq)". CPG Islands. Methods in Molecular Biology. Vol. 1766. pp. 209–229. doi:10.1007/978-1-4939-7768-0_12. ISBN   978-1-4939-7767-3. ISSN   1940-6029. PMC   6019634 . PMID   29605855.
  10. ↑ Xu, M.; Kladde, M. P.; Van Etten, J. L.; Simpson, R. T. (1998-09-01). "Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC". Nucleic Acids Research. 26 (17): 3961–3966. doi:10.1093/nar/26.17.3961. ISSN   0305-1048. PMC   147793 . PMID   9705505.
  11. ↑ Jessen, Walter J; Dhasarathy, Archana; Hoose, Scott A; Carvin, Christopher D; Risinger, April L; Kladde, Michael P (2004-05-01). "Mapping chromatin structure in vivo using DNA methyltransferases". Methods. In vitro analysis of chromatin structure in model systems. 33 (1): 68–80. doi:10.1016/j.ymeth.2003.10.025. ISSN   1046-2023. PMID   15039089.
  12. 1 2 Kelly, Theresa K.; Liu, Yaping; Lay, Fides D.; Liang, Gangning; Berman, Benjamin P.; Jones, Peter A. (December 2012). "Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules". Genome Research. 22 (12): 2497–2506. doi:10.1101/gr.143008.112. ISSN   1088-9051. PMC   3514679 . PMID   22960375.
  13. ↑ Fatemi, Mehrnaz; Pao, Martha M.; Jeong, Shinwu; Gal-Yam, Einav Nili; Egger, Gerda; Weisenberger, Daniel J.; Jones, Peter A. (2005-11-27). "Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level". Nucleic Acids Research. 33 (20): e176. doi:10.1093/nar/gni180. ISSN   1362-4962. PMC   1292996 . PMID   16314307.
  14. 1 2 Wolff, Erika M.; Byun, Hyang-Min; Han, Han F.; Sharma, Shikhar; Nichols, Peter W.; Siegmund, Kimberly D.; Yang, Allen S.; Jones, Peter A.; Liang, Gangning (2010-04-22). "Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer". PLOS Genetics. 6 (4): e1000917. doi: 10.1371/journal.pgen.1000917 . ISSN   1553-7404. PMC   2858672 . PMID   20421991.
  15. ↑ Han, Han; Cortez, Connie C.; Yang, Xiaojing; Nichols, Peter W.; Jones, Peter A.; Liang, Gangning (2011-11-15). "DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter". Human Molecular Genetics. 20 (22): 4299–4310. doi:10.1093/hmg/ddr356. ISSN   1460-2083. PMC   3196883 . PMID   21835883.
  16. 1 2 3 Pott, Sebastian (2017-06-27). Ren, Bing (ed.). "Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells". eLife. 6: e23203. doi: 10.7554/eLife.23203 . ISSN   2050-084X. PMC   5487215 . PMID   28653622.
  17. 1 2 Lee, Isac; Razaghi, Roham; Gilpatrick, Timothy; Molnar, Michael; Gershman, Ariel; Sadowski, Norah; Sedlazeck, Fritz J.; Hansen, Kasper D.; Simpson, Jared T.; Timp, Winston (December 2020). "Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing". Nature Methods. 17 (12): 1191–1199. doi:10.1038/s41592-020-01000-7. ISSN   1548-7105. PMC   7704922 . PMID   33230324.
  18. ↑ Ishii, Haruhiko; Kadonaga, James T.; Ren, Bing (2015-07-07). "MPE-seq, a new method for the genome-wide analysis of chromatin structure". Proceedings of the National Academy of Sciences of the United States of America. 112 (27): E3457–E3465. Bibcode:2015PNAS..112E3457I. doi: 10.1073/pnas.1424804112 . ISSN   0027-8424. PMC   4500276 . PMID   26080409.
  19. ↑ Nordström, Karl J. V.; Schmidt, Florian; Gasparoni, Nina; Salhab, Abdulrahman; Gasparoni, Gilles; Kattler, Kathrin; Müller, Fabian; Ebert, Peter; Costa, Ivan G.; DEEP consortium; Pfeifer, Nico; Lengauer, Thomas; Schulz, Marcel H.; Walter, Jörn (2019-11-18). "Unique and assay specific features of NOMe-, ATAC- and DNase I-seq data". Nucleic Acids Research. 47 (20): 10580–10596. doi:10.1093/nar/gkz799. ISSN   1362-4962. PMC   6847574 . PMID   31584093.
  20. ↑ Requena, Francisco; Asenjo, Helena G.; Barturen, Guillermo; Martorell-Marugán, Jordi; Carmona-Sáez, Pedro; Landeira, David (2019-05-31). "NOMePlot: analysis of DNA methylation and nucleosome occupancy at the single molecule". Scientific Reports. 9 (1): 8140. Bibcode:2019NatSR...9.8140R. doi:10.1038/s41598-019-44597-2. ISSN   2045-2322. PMC   6544651 . PMID   31148571.
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