Micrococcal Nuclease Analysis of Chromatin Structure

Ken Zaret1

1 Fox Chase Cancer Center, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.1
DOI:  10.1002/0471142727.mb2101s69
Online Posting Date:  February, 2005
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Abstract

This unit describes methodology for using micrococcal nuclease to investigate the presence of nucleosomes at a particular location in chromatin and to map the positions of nucleosomes at various levels of resolution. The approaches are readily adaptable to other probes of chromatin structure that cause DNA cleavage. Results obtained from such chromatin studies provide a structural view of the molecular environment of gene in their native context in cells.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Micrococcal Nuclease Digestion of Chromatin in Permeabilized Cells
  • Basic Protocol 2: Micrococcal Nuclease Digestion of Chromatin in Isolated Nuclei
  • Basic Protocol 3: Micrococcal Nuclease Digestion of Purified Genomic DNA
  • Support Protocol 1: Purification and Characterization of DNA from Chromatin Digestions
  • Support Protocol 2: Nuclease Cleavage Mapping Strategies
  • Support Protocol 3: Using a Modified LM‐PCR Procedure to Map Double‐Stranded MNase Cleavages At the Nucleotide Level of Resolution
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Micrococcal Nuclease Digestion of Chromatin in Permeabilized Cells

  Materials
  • Cells cultured on 100‐mm petri dishes or cells in suspension
  • Permeabilization solution 1, room temperature and 37°C (see recipe)
  • 1 mg/ml lysolecithin (Sigma) in permeabilization solution 1 (mix fresh before use)
  • Permeabilization solution 2 (see recipe), with and without MNase (see step below; mix fresh before use)
  • 2× TNESK solution (see recipe)
  • Lysis dilution buffer: 150 mM NaCl/5 mM EDTA
  • Phase‐contrast microscope
  • 12‐ml conical polypropylene tubes with caps (e.g., Sarstedt)
NOTE: All solutions, pipets, and pipet tips should be at room temperature so that the permeabilization reaction proceeds rapidly.

Basic Protocol 2: Micrococcal Nuclease Digestion of Chromatin in Isolated Nuclei

  Materials
  • Animal tissues (e.g., liver, kidney, and/or spleen) from sacrificed animal(s) or biopsy
  • Nuclear buffers A, B, and C (see recipe)
  • Calcium‐ and magnesium‐free (CMF) PBS (e.g., appendix 222)
  • 1 M NaOH
  • 2× TNESK solution (see recipe)
  • 0.1 M CaCl 2
  • Micrococcal nuclease (MNase) stock solution (see recipe)
  • Razor blades or scalpels
  • 100‐mm petri dishes on ice
  • 10‐ and/or 15‐ml tissue homogenizers with Teflon‐coated pestles
  • Tissue grinder motor and chuck
  • Phase‐contrast microscope
  • Cheesecloth
  • 15‐ and 30‐ml glass centrifuge tubes (e.g., Corex)
  • Refrigerated high‐speed centrifuge with fixed‐angle rotor
  • Ultracentrifuge with SW50.1 rotor and 1/2 × 2–in. Ultraclear tubes
  • Ultraviolet light spectrophotometer
NOTE: All solutions, test tubes, pipets, and pipet tips should be ice cold.

Basic Protocol 3: Micrococcal Nuclease Digestion of Purified Genomic DNA

  Materials
  • 0.5 to 1 mg purified genomic DNA (see protocol 4)
  • Nuclear buffer C (see recipe)
  • 0.5 M CaCl 2
  • Micrococcal nuclease (MNase)
  • 0.25 M EGTA
  • Chloroform
  • Heating block set to 68°C
  • Bucket of ice
  • Additional reagents and equipment for agarose minigel electrophoresis (unit 2.5)

Support Protocol 1: Purification and Characterization of DNA from Chromatin Digestions

  Materials
  • MNase‐digested cell or nuclear lysate (see Basic Protocols protocol 11, protocol 22, and protocol 33)
  • TE buffer, pH 7.9 ( appendix 222)
  • Neutralized phenol (see recipe)
  • Chloroform
  • Ether (for permeabilized cell preparations)
  • 5 mg/ml RNase A (for permeabilized cell preparations)
  • 3 M sodium acetate
  • 95% and 70% ethanol, room temperature
  • 10‐ or 12‐ml polypropylene tubes with tight caps (e.g., 12‐ml Sarstedt tubes)
  • Shaker or rocking device
  • 6‐in. Pasteur pipets
  • 30‐ml Corex tubes, silanized ( appendix 3B; for permeabilized cell preparations)
  • 6000‐ to 8000‐MWCO dialysis tubing (for permeabilized cell preparations)
  • 1‐ to 5‐µl glass capillary pipets
  • Glass capillary pipettor
  • Plastic wrap (e.g., Saran Wrap)
  • Ultraviolet light spectrophotometer
  • Additional reagents and equipment for dialysis ( appendix 3C) and agarose minigel electrophoresis (unit 2.5)

Support Protocol 2: Nuclease Cleavage Mapping Strategies

  Materials
  • DNA from MNase digest of chromatin (e.g., see Basic Protocols protocol 11 and protocol 22; see protocol 4 for purification procedure)
  • DNA from MNase digest of purified genomic DNA (e.g., see protocol 3)
  • 10 U/µl T4 polynucleotide kinase (e.g., New England Biolabs) and 10× buffer (supplied with enzyme)
  • 10 mM ATP
  • 0.5 M EDTA, pH 8 ( appendix 222)
  • 3 M sodium acetate
  • 95% and 70% ethanol, room temperature
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Figures

Videos

Literature Cited

Literature Cited
   Felsenfeld, G. 1992. Chromatin as an essential part of the transcriptional mechanism. Nature 355:219‐222.
   Hewish, D.R. and Burgoyne, L.A. 1973. Chromatin sub‐structure. The digestion of chromatin DNA at regularly space sites by a nuclear deoxyribonuclease. Biochem. Biophys. Res. Comm. 52:504‐510.
   Kornberg, R.D., LaPointe, J.W., and Lorch, Y. 1989. Preparation of nucleosomes and chromatin. Methods Enzymol. 170:3‐14.
   Liu, J.‐K., Bergman, Y., and Zaret, K.S. 1988. The mouse albumin promoter and a distal upstream site are simultaneously DNAaseI‐hypersensitive in liver chromatin and bind similar liver‐abundant factors in vitro. Genes Dev. 2:528‐541.
   McPherson, C.E., Shim, E.‐Y., Friedman, D.S., and Zaret, K.S. 1993. A tissue‐specific enhancer and bound transcription factors existing in a precisely positioned nucleosome array. Cell 75:387‐398.
   Pfeifer, G.P. and Riggs, A.D. 1991. Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation‐mediated PCR. Genes Dev. 5:1102‐1113.
   Shim, E.Y., Woodcock, C., and Zaret, K.S. 1998. Nucleosome positioning by the winged‐helix transcription factor HNF3. Genes Dev. 12:5‐10.
   van Holde, K.E. 1989. (pp. 497). Chromatin. Springer‐Verlag, New York.
   Wu, C. 1980. The 5′ ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature 286:854‐860.
   Zhang, L. and Gralla, J.D. 1990. In situ nucleoprotein structure involving origin‐proximal SV40 DNA control elements. Nucl. Acids Res. 18:1797‐1803.
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