Simultaneous Single‐Molecule Mapping of Protein‐DNA Interactions and DNA Methylation by MAPit

Carolina E. Pardo1, Russell P. Darst1, Nancy H. Nabilsi1, Amber L. Delmas1, Michael P. Kladde1

1 Department of Biochemistry and Molecular Biology and UF Shands Cancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology, University of Florida College of Medicine, Gainesville, Florida
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.22
DOI:  10.1002/0471142727.mb2122s95
Online Posting Date:  July, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Sites of protein binding to DNA are inferred from footprints or spans of protection against a probing reagent. In most protocols, sites of accessibility to a probe are detected by mapping breaks in DNA strands. As discussed in this unit, such methods obscure molecular heterogeneity by averaging cuts at a given site over all DNA strands in a sample population. The DNA methyltransferase accessibility protocol for individual templates (MAPit), an alternative method described in this unit, localizes protein‐DNA interactions by probing with cytosine‐modifying DNA methyltransferases followed by bisulfite sequencing. Sequencing individual DNA products after amplification of bisulfite‐converted sequences permits assignment of the methylation status of every enzyme target site along a single DNA strand. Use of the GC‐methylating enzyme M.CviPI allows simultaneous mapping of chromatin accessibility and endogenous CpG methylation. MAPit is therefore the only footprinting method that can detect subpopulations of molecules with distinct patterns of protein binding or chromatin architecture and correlate them directly with the occurrence of endogenous methylation. Additional advantages of MAPit methylation footprinting as well as considerations for experimental design and potential sources of error are discussed. Curr. Protoc. Mol. Biol. 95:21.22.1‐21.22.18. © 2011 by John Wiley & Sons, Inc.

Keywords: chromatin; nucleosomes; DNA methylation; DNA methyltransferases; footprinting; single‐molecule analysis

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Probing Mammalian Nuclear Chromatin with DNMTs
  • Support Protocol 1: Verification of Methylation of DNA by M.CviPI
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Probing Mammalian Nuclear Chromatin with DNMTs

  Materials
  • Trypsin‐EDTA solution (see recipe), 37°C
  • Mammalian cell lines cultured under appropriate experimental conditions in tissue culture plates or flasks
  • Cell growth medium (store up to 4 months at 4°C), 37°C
  • Phosphate buffered saline (PBS; appendix 22), ice cold
  • 0.4% (w/v) trypan blue solution (store indefinitely at room temperature)
  • 80 U/µl M.CviPI fused to maltose binding protein (MBP, New England Biolabs) or fused to glutathione‐S‐transferase (GST, Zymo Research); store in 20‐µl aliquots up to 1 year at −20°C in non‐frost‐free freezer; see recipe for dilutions)
  • DNMT dilution buffer (see recipe), ice cold
  • DNMT storage buffer (see recipe), ice cold
  • Cell resuspension buffer (see recipe), ice cold
  • Cell lysis buffer (see recipe), ice cold
  • Methylation buffer (see recipe)
  • Methylation stop buffer (see recipe), room temperature
  • 20 mg/ml proteinase K (store up to 4 months at −20°C in non‐frost‐free freezer)
  • Phenol/chloroform solution (see recipe)
  • 10.0 M ammonium acetate, pH 8.0 ( appendix 22)
  • Absolute and 70% (v/v) ethanol (see recipe; store indefinitely at room temperature)
  • 0.1× TE buffer (see recipe)
  • Refrigerated microcentrifuge
  • Hemacytometer or automated cell‐counting device
  • Light microscope
  • 1.7‐ml microcentrifuge tubes
  • 37° and 50°C water baths
  • Additional reagents and equipment for bisulfite sequencing (unit 7.9)
NOTE: Reagents should be prepared in sterile disposable labware. Use only distilled water in all steps and solutions. Nuclei isolation and methylation buffers should be freshly prepared on the day of the experiment. DTT, PMSF, and SAM should be added to solutions immediately before use to avoid oxidation or hydrolysis. M.CviPI activity is strongly dependent on fresh addition of DTT.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 21.22.1 MAPit overview for mapping m5CG and chromatin accessibility in mammalian nuclei. Nuclei are isolated from cultured cells of interest grown under desired experimental conditions. Isolated nuclei are then probed with M.CviPI, a DNA methyltransferase that methylates cytosines at accessible GC sites, i.e., nucleosome‐free or unbound by non‐histone proteins. After purification of DNA, cytosines will either be unmethylated (unfilled symbols), modified at CG by endogenous DNMTs (m5CG; black‐filled circles), or modified at GC by M.CviPI probe (G‐m5C; red‐filled inverted triangles). Purified DNA is then subjected to chemical conversion by bisulfite ion in which unmethylated C in denatured DNA is deaminated to U, whereas methylated C is not. During subsequent PCR of bisulfite‐treated DNA, U is converted through replicative complementarity to T and m5C to C. Sequencing after cloning individual DNA molecules from the bulk PCR product provides a single‐molecule readout of the methylation status at C in each CG and GC site. This output is representative of the combined endogenous methylation and chromatin accessibility at a single locus within the original nuclei. Sequence alignment and visual representation of methylation status are efficiently obtained computationally, e.g., with MethylViewer (Pardo et al., ). In the shown view in the bottom panel, each horizontal line indicates a single sequenced DNA molecule decorated with symbols representing the methylation status of each CG, GC, and overlapping CGC as defined (key at bottom right).
  •   FigureFigure 21.22.2 MAPit analysis of the TSS region of human SIM2 in MCF10A cells. Nuclei (106) were probed with 10 U of wild‐type M.CviPI for 30 min at 37°C. SIM2 is expressed in MCF‐10A cells. Each horizontal line represents 524 bp of chromatin from a single cell. Circles represent CG sites and triangles represent GC sites. Black filled circles and red filled triangles, represent m5CG and G‐m5C, respectively. GCG sites are represented by both gray triangles and circles. GCG site methylation cannot rigorously be discriminated as being placed by endogenous or exogenous DNMT, but this can often be inferred from context (see Anticipated Results for discussion). Blue highlighted areas represent 147 bp of contiguous M.CviPI DNA footprint. Note that about half of the alleles have relatively high levels of endogenous methylation (black filled circles). Based on molecules from cells not treated with M.CviPI, it can be inferred that gray GCG sites in these densely methylated MCF‐10A alleles were likely methylated by endogenous DNMTs. The other half of the molecules is almost free of endogenous methylation but shows an accessible, nucleosome‐length region high in M.CviPI methylation (red triangles) highlighted in red. No other technique can determine this bipartite pattern of chromosome structure. The high accessibility to M.CviPI is probably due to histone depletion near the TSS. In contrast, this putative histone‐free region is flanked by protected spans of median length ∼150 bp. Numbers at the right of each molecule depiction indicate the percentage of C conversion to T in non‐CG and non‐GC sequences. Nucleotides that failed to convert or reverted to a C during PCR amplification are indicated by vertical blue tick marks.

Videos

Literature Cited

   Bestor, T.H. 2000. The DNA methyltransferases of mammals. Hum. Mol. Genet. 9:2395‐2402.
   Bestor, T.H. and Bourc'his, D. 2004. Transposon silencing and imprint establishment in mammalian germ cells. Cold Spring Harb. Symp. Quant. Biol. 69:381‐387.
   Bird, A. 2002. DNA methylation patterns and epigenetic memory. Genes Dev. 16:6‐21.
   Boeger, H., Griesenbeck, J., Strattan, J.S., and Kornberg, R.D. 2004. Removal of promoter nucleosomes by disassembly rather than sliding in vivo. Mol. Cell 14:667‐673.
   Clapier, C.R. and Cairns, B.R. 2009. The biology of chromatin remodeling complexes. Ann. Rev. Biochem. 78:273‐304.
   Clark, S.J., Harrison, J., Paul, C.L., and Frommer, M. 1994. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22:2990‐2997.
   Cokus, S.J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C.D., Pradhan, S., Nelson, S.F., Pellegrini, M., and Jacobsen, S.E. 2008. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452:215‐219.
   Deaton, A.M. and Bird, A. 2011. CpG islands and the regulation of transcription. Genes Dev. 25:1010‐1022.
   Dechassa, M.L., Sabri, A., Pondugula, S., Kassabov, S.R., Chatterjee, N., Kladde, M.P., and Bartholomew, B. 2010. SWI/SNF has intrinsic nucleosome disassembly activity that is dependent on adjacent nucleosomes. Mol. Cell 38:590‐602.
   Dong, L., Wang, W., Wang, F., Stoner, M., Reed, J.C., Harigai, M., Samudio, I., Kladde, M.P., Vyhlidal, C., and Safe, S. 1999. Mechanisms of transcriptional activation of bcl‐2 gene expression by 17β‐estradiol in breast cancer cells. J. Biol. Chem. 274:32099‐32107.
   Duan, R., Porter, W., Samudio, I., Vyhlidal, C., Kladde, M., and Safe, S. 1999. Transcriptional activation of c‐fos protooncogene by 17β‐estradiol: Mechanism of aryl hydrocarbon receptor‐mediated inhibition. Mol. Endocrinol. 13:1511‐1521.
   Fatemi, M., Pao, M.M., Jeong, S., Gal‐Yam, E.N., Egger, G., Weisenberger, D.J., and Jones, P.A. 2005. Footprinting of mammalian promoters: Use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level. Nucleic Acids Res. 33:e176.
   Fehér, Z., Kiss, A., and Venetianer, P. 1983. Expression of a bacterial modification methylase gene in yeast. Nature 302:266‐268.
   Feinberg, A.P., Ohlsson, R., and Henikoff, S. 2006. The epigenetic progenitor origin of human cancer. Nat. Rev. Genet. 7:21‐33.
   Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., and Paul, C.L. 1992. A genomic sequencing protocol that yields a positive display of 5‐methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. U.S.A. 89:1827‐1831.
   Gal‐Yam, E.N., Jeong, S., Tanay, A., Egger, G., Lee, A.S., and Jones, P.A. 2006. Constitutive nucleosome depletion and ordered factor assembly at the GRP78 promoter revealed by single molecule footprinting. PLoS Genet. 2:e160.
   Goll, M.G., and Bestor, T.H. 2005. Eukaryotic cytosine methyltransferases. Ann. Rev. Biochem. 74:481‐514.
   Gottschling, D.E. 1992. Telomere‐proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo. Proc. Natl. Acad. Sci. U.S.A. 89:4062‐4065.
   Grandjean, V., Yaman, R., Cuzin, F., and Rassoulzadegan, M. 2007. Inheritance of an epigenetic mark: The CpG DNA methyltransferase 1 is required for de novo establishment of a complex pattern of non‐CpG methylation. PLoS One 2:e1136.
   Gruenbaum, Y., Stein, R., Cedar, H., and Razin, A. 1981. Methylation of CpG sequences in eukaryotic DNA. FEBS Lett. 124:67‐71.
   Hawkins, R.D., Hon, G.C., Lee, L.K., Ngo, Q., Lister, R., Pelizzola, M., Edsall, L.E., Kuan, S., Luu, Y., Klugman, S., Antosiewicz‐Bourget, J., Ye, Z., Espinoza, C., Agarwahl, S., Shen, L., Ruotti, V., Wang, W., Stewart, R., Thomson, J.A., Ecker, J.R., and Ren, B. 2010. Distinct epigenomic landscapes of pluripotent and lineage‐committed human cells. Cell Stem Cell 6:479‐491.
   Hayatsu, H. 1976. Bisulfite modification of nucleic acids and their constituents. Prog. Nucleic Acids Res. 16:75‐124.
   Hayatsu, H., Shiraishi, M., and Negishi, K. 2008. Bisulfite modification for analysis of DNA methylation. Curr. Protoc. Nucleic Acid Chem. 33:6.10.1‐6.10.15.
   Henderson, I.R. and Jacobsen, S.E. 2007. Epigenetic inheritance in plants. Nature 447:418‐424.
   Hermann, A., Gowher, H., and Jeltsch, A. 2004. Biochemistry and biology of mammalian DNA methyltransferases. Cell. Mol. Life Sci. 61:2571‐2587.
   Holz‐Schietinger, C. and Reich, N.O. 2010. The inherent processivity of the human de novo methyltransferase 3A (DNMT3A) is enhanced by DNMT3L. J. Biol. Chem. 285:29091‐29100.
   Hoose, S.A. and Kladde, M.P. 2006. DNA methyltransferase probing of DNA‐protein interactions. Methods Mol. Biol. 338:225‐244.
   Huang, Y., Pastor, W.A., Shen, Y., Tahiliani, M., Liu, D.R., and Rao, A. 2010. The behaviour of 5‐hydroxymethylcytosine in bisulfite sequencing. PloS One 5:e8888.
   Ito, S., D'Alessio, A.C., Taranova, O.V., Hong, K., Sowers, L.C., and Zhang, Y. 2010. Role of Tet proteins in 5mC to 5hmC conversion, ES‐cell self‐renewal and inner cell mass specification. Nature 466:1129‐1133.
   Jaenisch, R. and Bird, A. 2003. Epigenetic regulation of gene expression: How the genome integrates intrinsic and environmental signals. Nat. Genet. 33:245‐254.
   Jessen, W.J., Dhasarathy, A., Hoose, S.A., Carvin, C.D., Risinger, A.L., and Kladde, M.P. 2004. Mapping chromatin structure in vivo using DNA methyltransferases. Methods 33:68‐80.
   Jessen, W.J., Hoose, S.A., Kilgore, J.A., and Kladde, M.P. 2006. Active PHO5 chromatin encompasses variable numbers of nucleosomes at individual promoters. Nat. Struct. Mol. Biol. 13:256‐263.
   Jiang, C. and Pugh, B.F. 2009. Nucleosome positioning and gene regulation: Advances through genomics. Nat. Rev. Genet. 10:161‐172.
   Jones, P.A. and Baylin, S.B. 2007. The epigenomics of cancer. Cell 128:683‐692.
   Kilgore, J.A., Hoose, S.A., Gustafson, T.L., Porter, W., and Kladde, M.P. 2007. Single‐molecule and population probing of chromatin structure using DNA methyltransferases. Methods 41:320‐332.
   Kladde, M.P. and Simpson, R.T. 1994. Positioned nucleosomes inhibit Dam methylation in vivo Proc. Natl. Acad. Sci. U.S.A. 91:1361‐1365.
   Kladde, M.P., Xu, M., and Simpson, R.T. 1996. Direct study of DNA‐protein interactions in repressed and active chromatin in living cells. EMBO J. 15:6290‐6300.
   Korber, P., Luckenbach, T., Blaschke, D., and Hörz, W. 2004. Evidence for histone eviction in trans upon induction of the yeast PHO5 promoter. Mol. Cell. Biol. 24:10965‐10974.
   Kouidou, S., Agidou, T., Kyrkou, A., Andreou, A., Katopodi, T., Georgiou, E., Krikelis, D., Dimitriadou, A., Spanos, P., Tsilikas, C., Destouni, H., and Tzimagiorgis, G. 2005. Non‐CpG cytosine methylation of p53 exon 5 in non‐small cell lung carcinoma. Lung Cancer 50:299‐307.
   Kouzarides, T. 2007. Chromatin modifications and their function. Cell 128:693‐705.
   Kriaucionis, S. and Heintz, N. 2009. The nuclear DNA base 5‐hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929‐930.
   Kwak, H.I., Gustafson, T., Metz, R.P., Laffin, B., Schedin, P., and Porter, W.W. 2007. Inhibition of breast cancer growth and invasion by single‐minded 2s. Carcinogenesis 28:259‐266.
   Lai, A.Y., Fatemi, M., Dhasarathy, A., Malone, C., Sobol, S.E., Geigerman, C., Jaye, D.L., Mav, D., Shah, R., Li, L., and Wade, P.A. 2010. DNA methylation prevents CTCF‐mediated silencing of the oncogene BCL6 in B cell lymphomas. J. Exp. Med. 207:1939‐1950.
   Längst, G. and Becker, P.B. 2004. Nucleosome remodeling: One mechanism, many phenomena? Biochim. Biophys. Acta 1677:58‐63.
   Latham, T., Gilbert, N., and Ramsahoye, B. 2008. DNA methylation in mouse embryonic stem cells and development. Cell Tissue Res. 331:31‐55.
   Laurent, L., Wong, E., Li, G., Huynh, T., Tsirigos, A., Ong, C.T., Low, H.M., Kin Sung, K.W., Rigoutsos, I., Loring, J., and Wei, C.L. 2010. Dynamic changes in the human methylome during differentiation. Genome Res. 20:320‐331.
   Li, B., Carey, M., and Workman, J.L. 2007. The role of chromatin during transcription. Cell 128:707‐719.
   Lister, R., Pelizzola, M., Dowen, R.H., Hawkins, R.D., Hon, G., Tonti‐Filippini, J., Nery, J.R., Lee, L., Ye, Z., Ngo, Q.M., Edsall, L., Antosiewicz‐Bourget, J., Stewart, R., Ruotti, V., Millar, A.H., Thomson, J.A., Ren, B., and Ecker, J.R. 2009. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462:315‐322.
   Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. 1997. Crystal structure of the nucleosome core particle at 2.8‐Å resolution. Nature 389:251‐260.
   Matsuo, K., Silke, J., Gramatikoff, K., and Schaffner, W. 1994. The CpG‐specific methylase SssI has topoisomerase activity in the presence of Mg2+. Nucleic Acids Res. 22:5354‐5359.
   McCabe, M.T., Brandes, J.C., and Vertino, P.M. 2009. Cancer DNA methylation: Molecular mechanisms and clinical implications. Clin. Cancer Res. 15:3927‐3937.
   Metz, R.P., Kwak, H.I., Gustafson, T., Laffin, B, and Porter, W.W. 2006. Differential transcriptional regulation by mouse single‐minded 2s. J. Biol. Chem. 281:10839‐10848.
   Mito, Y., Henikoff, J.G., and Henikoff, S. 2005. Genome‐scale profiling of histone H3.3 replacement patterns. Nat. Genet. 37:1090‐1097.
   Nabilsi, N.H., Broaddus, R.R., and Loose, D.S. 2009. DNA methylation inhibits p53‐mediated survivin repression. Oncogene 28:2046‐2050.
   Pardo, C., Hoose, S.A., Pondugula, S., and Kladde, M.P. 2009. DNA methyltransferase probing of chromatin structure within populations and on single molecules. Methods Mol. Biol. 523:41‐65.
   Pardo, C.E., Carr, I.M., Hoffman, C.J., Darst, R.P., Markham, A.F., Bonthron, D.T., and Kladde, M.P. 2010. MethylViewer: Computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects. Nucleic Acids Res. 39:e5.
   Pondugula, S. and Kladde, M.P. 2008. Single‐molecule analysis of chromatin: Changing the view of genomes one molecule at a time. J. Cell. Biochem. 105:330‐337.
   Pratt, K.I. and Hattman, S. 1981. Deoxyribonucleic acid methylation and chromatin organization in Tetrahymena thermophila. Mol. Cell. Biol. 1:600‐608.
   Pratt, K.I. and Hattman, S. 1983. Nucleosome phasing in Tetrahymena macronuclei. J. Protozool. 30:592‐598.
   Proffitt, J.H., Davie, J.R., Swinton, D., and Hattman, S. 1984. 5‐Methylcytosine is not detectable in Saccharomyces cerevisiae DNA. Mol. Cell. Biol. 4:985‐988.
   Ramsahoye, B.H., Biniszkiewicz, D., Lyko, F., Clark, V., Bird, A.P., and Jaenisch, R. 2000. Non‐CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc. Natl. Acad. Sci. USA 97:5237‐5242.
   Renbaum, P. and Razin, A. 1992. Mode of action of the Spiroplasma CpG methylase M.SssI. FEBS Lett. 313:243‐247.
   Renbaum, P., Abrahamove, D., Fainsod, A., Wilson, G., Rottem, S., and Razin, A. 1990. Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA from Spiroplasma sp strain MQ‐1 (M.SssI). Nucleic Acids Res. 18:1145‐1152.
   Richard‐Foy, H. and Hager, G.L. 1987. Sequence specific positioning of nucleosomes over the steroid inducible MMTV promoter. EMBO J. 6:2321‐2328.
   Robertson, K.D. 2001. DNA methylation, methyltransferases, and cancer. Oncogene 20:3139‐3155.
   Robertson, K.D. 2005. DNA methylation and human disease. Nat. Rev. Genet. 6:597‐610.
   Robertson, K.D. and Wolffe, A.P. 2000. DNA methylation in health and disease. Nat. Rev. Genet. 1:11‐19.
   Saha, A., Wittmeyer, J., and Cairns, B.R. 2006. Chromatin remodeling: The industrial revolution of DNA around histones. Nat. Rev. Mol. Cell. Biol. 7:437‐447.
   Samudio, I., Vyhlidal, C., Wang, F., Stoner, M., Chen, I., Kladde, M., Barhoumi, R., Burghardt, R., and Safe, S. 2001. Transcriptional activation of deoxyribonucleic acid polymerase α gene expression in MCF‐7 cells by 17β‐estradiol. Endocrinology 142:1000‐1008.
   Singh, J. and Klar, A.J.S. 1992. Active genes in yeast display enhanced in vivo accessibility to foreign DNA methylases: A novel in vivo probe for chromatin structure of yeast. Genes Dev. 6:186‐196.
   Tahiliani, M., Koh, K.P., Shen, Y., Pastor, W.A., Bandukwala, H., Brudno, Y., Agarwal, S., Iyer, L.M., Liu, D.R., Aravind, L., and Rao, A. 2009. Conversion of 5‐methylcytosine to 5‐hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930‐935.
   Vilkaitis, G. and Klimasauskas, S. 1999. Bisulfite sequencing protocol displays both 5‐methylcytosine and N4‐methylcytosine. Anal. Biochem. 271:116‐119.
   Vilkaitis, G., Suetake, I., Klimasauskas, S., and Tajima, S. 2005. Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase. J. Biol. Chem. 280:64‐72.
   Vyhlidal, C., Samudio, I., Kladde, M.P., and Safe, S. 2000. Transcriptional activation of transforming growth factor a by estradiol: Requirement for both a GC‐rich site and an estrogen response element half‐site. J. Mol. Endocrinol. 24:329‐338.
   Warnecke, P.M., Stirzaker, C., Song, J., Grunau, C., Melki, J.R., and Clark, S.J. 2002. Identification and resolution of artifacts in bisulfite sequencing. Methods 27:101‐107.
   Wu, H., Coskun, V., Tao, J.F., Xie, W., Ge, W.H., Yoshikawa, K., Li, E., Zhang, Y., and Sun, Y.E. 2010. Dnmt3a‐dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes. Science 329:444‐448.
   Xu, G.L. and Bestor, T.H. 1997. Cytosine methylation targeted to pre‐determined sequences. Nat. Genet. 17:376‐378.
   Xu, M., Kladde, M.P., Van Etten, J.L., and Simpson, R.T. 1998a. Cloning, characterization and expression of the gene coding for a cytosine‐5‐DNA methyltransferase recognizing GpC. Nucleic Acids Res. 26:3961‐3966.
   Xu, M., Simpson, R.T., and Kladde, M.P. 1998b. Gal4p‐mediated chromatin remodeling depends on binding site position in nucleosomes but does not require DNA replication. Mol. Cell. Biol. 18:1201‐1212.
   Zilberman, D., Gehring, M., Tran, R.K., Ballinger, T., and Henikoff, S. 2007. Genome‐wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nat. Genet. 39:61‐69.
Key References
  Kladde et al., 1996. See above.
  First demonstration of the utility of M.SssI for detection of nucleosome position and transcription factor binding.
  Fatemi et al., 2005. See above.
  First demonstrations of the use of C‐5 DNMTs in single‐molecule footprinting.
  Jessen et al., 2006. See above.
  First documented use of MAPit with M.CviPI, yielding simultaneous detection of chromatin accessibility and endogenous m5CG at the single‐molecule level.
  Kilgore et al., 2007. See above.
  Development of MethylViewer program for rapid analysis of MAPit datasets.
  Pardo et al., 2010. See above.
Internet Resources
  http://dna.leeds.ac.uk/methylviewer/
  Site for download of MethylViewer program and detailed usage instructions.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library