Detecting Epigenetic Changes: DNA Methylation

Limor Broday1, Max Costa1

1 New York University School of Medicine, New York, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.6
DOI:  10.1002/0471140856.tx0306s03
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit provides protocols for assessing the state of DNA methylation, including assays for determining the overall DNA methylation levels, measuring DNA methyltranseferase (MTase) activity, determining gene‐specific methylation and overall chromatin condensation patterns.

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

Table of Contents

  • Basic Protocol 1: Determining the Overall 5‐Methylcytosine Content in Genomic DNA
  • Basic Protocol 2: Measuring DNA: Methyltransferase Activity Assay
  • Basic Protocol 3: Measuring Gene‐Specific Methylation: Modification‐Sensitive Restriction Endonucleases Assay
  • Basic Protocol 4: Measuring Deoxyribonuclease I Sensitivity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Determining the Overall 5‐Methylcytosine Content in Genomic DNA

  Materials
  • Genomic DNA purified from treated and control cells
  • SssI methylase (New England Biolabs)
  • 60 to 85 Ci/mmol S‐adenosyl‐L‐[methyl‐3H]methionine (Amersham Pharmacia)
  • 15 mM nonradioactive S‐adenosylmethionine (SAM; prepare fresh)
  • SssI reaction buffer (see recipe or use as supplied by manufacturer)
  • 5% (w/v) trichloroacetic acid
  • 70% (v/v) ethanol
  • Scintillation cocktail
  • Millipore Ultrafree‐MC 100,000 NMWL filter unit (polysulfone membrane)
  • Scintillation vials
  • Liquid scintillation counter

Basic Protocol 2: Measuring DNA: Methyltransferase Activity Assay

  Materials
  • ∼1 × 107 treated and control cells to be tested
  • PBS ( appendix 2A)
  • Methyltransferase lysis buffer(see recipe)
  • Polydeoxyinosinic‐deoxycytidylic acid [poly (dI‐dC)]
  • 60 to 85 Ci/mmol S‐adenosyl‐L‐[methyl‐3H]methionine (Amerham Pharmacia)
  • Methyltransferase stop solution(see recipe)
  • 1:1 (v/v) phenol/chloroform
  • 100% ethanol, ice‐cold
  • 0.3 M NaOH
  • 5% (w/v) trichloroacetic acid
  • 70% ethanol
  • 0.5 M perchloric acid
  • Scintillation cocktail
  • Syringe and 25‐G needle
  • GF/C Whatman filter discs
  • Fitration unit (e.g., Millipore)
  • Liquid scintillation counter
  • Additional reagents and equipment for protein assay and phenyl/chloroform extraction/ethanol precipitation ( appendix 3A).

Basic Protocol 3: Measuring Gene‐Specific Methylation: Modification‐Sensitive Restriction Endonucleases Assay

  Materials
  • Genomic DNA from treated and control cells to be tested, from tissue culture cells or tissues
  • Methylation‐insensitive restriction enzyme (with recognition sites located within the borders of the tested DNA region) and buffer
  • Methylation‐sensitive restriction enzyme (such as the 4 cutters HpaII and HaeI, 6 cutters EagI and SacII, or 8 cutter NotI) and buffer
  • Appropriate PCR primers designed for the target region or appropriate probe for Southern blot hybridization analysis
  • Additional reagents and equipment for ethanol precipitation of DNA ( appendix 3A), PCR amplification ( appendix 3C) or Southern blot hybridization (Ausubel et al., ), and agarose gel electrophoresis (unit 2.2)

Basic Protocol 4: Measuring Deoxyribonuclease I Sensitivity

  Materials
  • Cell line to be examined, treated and control
  • DNase I buffer (see recipe)
  • DNase I
  • DNase I stop solution (see recipe)
  • 1:1 (v/v) phenol/chloroform
  • 100% ethanol, ice‐cold
  • 25° and 55°C water baths
  • Additional reagents and equipment for phenol/chloroform extraction and ethanol precipitation of DNA ( appendix 3A), Southern blot hybridization (Ausubel et al., ), and PCR ( appendix 3C)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Adams, R.L.P., Rinalsi, A., and Seivwright, C. 1991. Microassay for DNA methyltransferase. J. Biochem. Biophys. Methods 22:19‐22.
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 1992. Short Protocols in Molecular Biology, Second Edition John Wiley & Sons, New York.
   Bestor, T.H. 1998. Methylation meets acetylation. Nature 393:311‐312.
   Bird, A.P. and Southern, E.M. 1978. Use of restriction enzymes to study eukaryotic DNA methylation: The methylation pattern in ribosomal DNA from Xenopus laevis. J. Mol. Biol. 118:27‐47.
   Clark, S.J., Harrison, J., Paul, C.L., and Frommer, M. 1994. High sensitivity mapping of methylated cytosines. Nucl. Acids Res. 22:2990‐2997.
   Gonzalgo, M.L. and Jones, P.A. 1997. Mutagenic andepigenetic effects of DNA methylation. Mutat. Res. 386:107‐118.
   Lee, Y.W., Klein, C.B., Kargacin, B., Salnikow, K., Kitahara, J., Dowjat, K., Zhitkovich, A., Christie, N.T., and Costa, M. 1995. Carcinogenic nickel silences gene expression by chromatin condensation andDNA methylation: A new model for epigenetic carcinogens. Mol.Cell Biol. 15:2547‐2557.
   McGrew, M.J. and Rosenthal, N. 1993. Quantitation of genomic methylation using ligation‐mediated PCR. Biotech. 4:722‐729.
   Nan, X., Ng, H‐H., Johnson, C.A., Laherty, C.D., Turner, B.M., Eisenman, R.N., and Bird, A. 1998. Transcriptional repression by the methyl‐CpG‐binding protein MeCP2 involves a histone deacetylase complex. Nature 393:386‐389.
   Pfeifer, G.P., Steigerwald, S.D., Mueller, P.R., Wold, B., and Riggs, A.D. 1989. Genomic sequencing andmethylation analysis by ligation‐mediated PCR. Science 246:810‐813.
   Rein, T., Depamphilis, M.L., and Zorbas, H. 1998. Identifying 5‐methylcytosine andrelated modifications in DNA genomes. Nucl. Acids Res. 26:2255‐2264.
   Renbaum, P., Abrahamove, D., Fainsod, A., Wilson, G.G., Rottem, S., and Razin, A. 1990. Cloning, characterization, andexpression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1 (M. SssI). Nucl. Acids Res. 18:1145‐1152.
   Singer‐Sam, J., Le Bon, J.M., Tanguay, R.L., and Riggs, A.D. 1990. A quantitative Hpa II‐PCR assay to measure methylation of DNA from a small number of cells. Nucl. Acids Res. 18:687.
   Weintraub, H. 1985. High‐resolution mapping of S1 and DNaseI‐hypersensitive sites in chromatin. Mol. Cell Biol. 5:1538‐1539.
   Wu, J., Issa, J.P., Herman, J., Bassett, D.E., Jr., Nelkin, B.D., and Baylin, S.B. 1993. Expression of an exogenous eukaryotic DNA methyltransferase gene includes transformation of N1H3T3 cells. Proc. Natl. Acad.Sci. U.S.A. 90:8891‐8895.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library