Assembly of Nucleosomal Templates by Salt Dialysis

Kyu‐Min Lee1, Geeta Narlikar1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.6
DOI:  10.1002/0471142727.mb2106s54
Online Posting Date:  May, 2001
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Abstract

Because it is convenient to assemble nucleosomal templates through salt dialysis, large amounts of chromatin complexes can be made easily and in a short amount of time. This unit includes instructions for the various salt dialysis schemes (step versus gradient), which are accompanied by protocols for purification of core histones from bacteria and preparation of DNA for the nucleosomal arrays. Electrophoretic procedures to analyze the reconstituted complexes are also included.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Assembly of Nucleosomal Templates by Step Salt Dialysis
  • Basic Protocol 2: Assembly of High Concentration of Mononucleosomes by Gradient Salt Dialysis
  • Basic Protocol 3: Assembly of Nucleosomal Arrays by Gradient Salt Dialysis
  • Support Protocol 1: Purification of Recombinant Core Histones from Bacteria
  • Support Protocol 2: Preparation of Single 5′ End‐Labeled DNA for Mononucleosome Assembly
  • Support Protocol 3: Preparation of DNA for Assembly of High Concentration Unlabeled Mononucleosomes
  • Support Protocol 4: Preparation of DNA for Nucleosomal Arrays
  • Support Protocol 5: Analysis of Reconstituted Complexes by Agarose Gel Electrophoresis
  • Support Protocol 6: Analysis of Reconstituted Complexes by Polyacrylamide Gel Electrophoresis
  • Support Protocol 7: EcoRI Digestion to Determine Extent of G5E4 Array Assembly
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assembly of Nucleosomal Templates by Step Salt Dialysis

  Materials
  • ∼1 to 2 mg/ml sonicated calf thymus DNA (∼0.5 to 1 kb; Sigma)
  • Radioactively labeled DNA (see protocol 5)
  • Purified native core histone protein fractions ( protocol 4)
  • 5 M NaCl ( appendix 22)
  • TE buffer, pH 8.0 ( appendix 22)
  • TE buffer, pH 8.0 ( appendix 22), containing 1.2, 1.0, 0.8, and 0.6 M NaCl
  • 6000 to 8000 MWCO dialysis tubing (Spectrapor; boil in Milli‐Q water for 5 min and store at 4°C)
  • Additional reagents and equipment for dialysis ( appendix 3C) and purification of nucleosomes by glycerol gradient (unit 21.5)

Basic Protocol 2: Assembly of High Concentration of Mononucleosomes by Gradient Salt Dialysis

  Materials
  • Unlabeled DNA fragment at high concentration (>2 mg/ml; protocol 6)
  • 1 M Tris⋅Cl, pH 7.7 ( appendix 22)
  • 1 M dithiothreitol (DTT)
  • 0.5 M disodium EDTA, pH 8.0 ( appendix 22)
  • 2.5 M KCl
  • 0.5 M benzamidine
  • Core histones (purified from HeLa nuclear pellets; unit 21.5)
  • recipeHigh‐salt buffer (see recipe)
  • recipeLow‐salt Buffer (see recipe)
  • recipeZero‐salt buffer (see recipe)
  • 10% and 30% glycerol gradient buffer (see recipe in unit 21.5)
  • Rabbit pump (Rainin) with tubes
  • 6000 to 8000 MWCO dialysis tubing (Spectrapor, 6.4 mm diameter; boil in Milli‐Q water for 5 min and store at 4°C)
  • Conductivity meter calibrated for KCl concentration
  • Additional reagents and equipment for analysis of reconstituted complexes by agarose gel electrophoresis (see protocol 8) or polyacrylamide gel electrophoresis (see protocol 9)

Basic Protocol 3: Assembly of Nucleosomal Arrays by Gradient Salt Dialysis

  Materials
  • Unlabeled G5E4 DNA (Dr. Jerry Workman; )
  • End‐labeled G5E4 DNA (see protocol 5 for labeling)
  • Bovine serum albumin (BSA)
  • recipeHigh‐salt buffer (see recipe) with 2 M NaCl in place of KCl
  • recipeLow‐salt buffer (see recipe) with 250 mM NaCl in place of KCl
  • Additional reagents and equipment for assembly of mononucleosomes by gradient salt dialysis (see protocol 2) and EcoRI digestion to determine extent of array assembly (see protocol 10)

Support Protocol 1: Purification of Recombinant Core Histones from Bacteria

  Materials
  • Bacteria overexpressing core histones (BL21; Novagen; Luger et al., )
  • Luria broth (LB), sterile
  • 100× (0.2 M) isopropyl D‐thiogalactopyranoside (IPTG) stock
  • TE buffer, pH 8.0 ( appendix 22) containing 1 mM PMSF (add from saturated PMSF stock in 95% ethanol)
  • 10 mg/ml lysozyme solution
  • Triton X‐100 detergent
  • recipeDenaturing solution (see recipe)
  • 5 M NaCl ( appendix 22)
  • 50% (v/v) slurry of Bio‐Rex 50‐100 mesh chromatography resin (Bio‐Rad)
  • TE buffer, pH 8.0, containing 0.6, 1.0, and 2.0 M NaCl and 1 mM PMSF (add from saturated PMSF stock in 95% ethanol)
  • Oak Ridge centrifuge tubes
  • Sonicator
  • Beckman centrifuge with JA‐20 rotor (or equivalent)
  • 6000 to 8000 MWCO dialysis tubing (Spectrapor; boil in Milli‐Q water for 5 min and store at 4°C)
  • 10‐ml disposable plastic chromatography column (Bio‐Rad)
  • 15‐ and 50‐ml conical centrifuge tubes
  • Additional reagents and equipment for dialysis ( appendix 3C)

Support Protocol 2: Preparation of Single 5′ End‐Labeled DNA for Mononucleosome Assembly

  Materials
  • Plasmid DNA containing sequence of interest, with two convenient restriction sites that generate a ∼150‐bp fragment
  • Restriction endonuclease suitable for linearizing the plasmid, and corresponding endonuclease buffer
  • 3 M sodium acetate ( appendix 22)
  • 95% and 70% ethanol, –20°C
  • Alkaline phosphatase (Boehringer Mannheim) and alkaline phosphatase buffer
  • 10% (w/v) SDS
  • TE buffer, pH 8.0 ( appendix 22)
  • 10,000 U/ml T4 polynucleotide kinase (Promega) and 10× T4 polynucleotide kinase buffer
  • 10 µCi/µl [γ ‐32P]dATP (6000 Ci/mmol; NEN Life Science Products)
  • 2.5 M ammonium acetate
  • Restriction endonuclease appropriate for liberating the end‐labeled fragment of interest, and corresponding endonuclease buffer
  • 6% nondenaturing polyacrylamide gel
  • Pestle homogenizer fitting microcentrifuge tubes (VWR)
  • SpinX microcentrifuge tubes (Costar)
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA (unit 2.1), polyacrylamide gel electrophoresis of DNA (unit 2.7), and autoradiography ( appendix 3A)

Support Protocol 3: Preparation of DNA for Assembly of High Concentration Unlabeled Mononucleosomes

  Materials
  • p2085‐G5E4 plasmid (Dr. Jerry Workman; ) or other 12S array plasmid
  • 40 U/µl Asp718 and ClaI restriction endonucleases and buffer B (all from Boehringer‐Mannheim)
  • DdeI restriction endonclease and buffer 3 (both from New England Biolabs)
  • 65°C water bath
  • Additional reagents and equipment for phenol/chloroform extraction and ethanol precipitation of DNA (unit 2.1), purification of restriction fragments (unit 2.6), and labeling with Klenow fragment (unit 3.5)

Support Protocol 4: Preparation of DNA for Nucleosomal Arrays

  Materials
  • Agarose (Research Genetics)
  • recipe5× TBE buffer (see recipe)
  • Labeled or unlabeled nucleosomes (see Basic Protocols protocol 11 and protocol 22)
  • 50% (v/v) glycerol
  • recipeDNA loading dye (see recipe)
  • 0.5 µg/ml ethidium bromide in 0.5× TBE buffer (see recipe for recipe5× TBE buffer)
  • Whatman filter paper
  • X‐ray film (Kodak)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and autoradiography ( appendix 3A)

Support Protocol 5: Analysis of Reconstituted Complexes by Agarose Gel Electrophoresis

  Materials
  • 40% acrylamide 29:1 (acrylamide:bis) solution (see unit 2.7)
  • recipe5× TBE buffer (see recipe)
  • Labeled or unlabeled nucleosomes (see Basic Protocols protocol 11 and protocol 22)
  • Naked DNA template used to prepare nucleosomes, as control
  • 50% (v/v) glycerol
  • DNA loading dye
  • 0.5 µg/ml of ethidium bromide solution in 0.5× TBE buffer (see recipe for recipe5× TBE buffer)
  • X‐ray film (Kodak)
  • Additional reagents and equipment for polyacrylamide gel electrophoresis of DNA (unit 2.7)

Support Protocol 6: Analysis of Reconstituted Complexes by Polyacrylamide Gel Electrophoresis

  Materials
  • Nucleosomal array (see protocol 3)
  • EcoRI restriction endonuclease and corresponding buffer
  • 25% (v/v) glycerol/10 mM disodium EDTA, pH 8.0 (see appendix 22 for preparation of the EDTA)
  • Agarose (Research Genetics)
  • recipe5× TBE buffer with increased EDTA (see recipe)
  • 0.5 µg/ml ethidium bromide solution in 0.5× TBE buffer with regular EDTA concentration (see recipe for recipe5× TBE buffer)
  • Additional reagents and equipment for restriction endonuclease digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), and autoradiography ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Camerini‐Otero, R.D., Sollner‐Webb, B., and Felsenfeld, G. 1973. The organization of histones and DNA in chromatin: evidence for an arginine‐rich histone kernel. Cell 3:333‐347.
   Carruthers, L.M., Tse, C., Walker, K.P., and Hansen, J. 1999. Assembly of defined nucleosomal and chromatin arrays from pure components. Methods Enzymol. 304:19‐35.
   Hayes, J.J. and Lee, K.M. 1997. In vitro reconstitution and analysis of mononucleosomes containing defined DNAs and proteins. Methods 12:2‐9.
   Hayes, J.J., Tullius, T.D., and Wolffe, A.P. 1990. The structure of DNA in a nucleosome. Proc. Natl. Acad. Sci. U.S.A. 87:7405‐7409.
   Lee, K.M. and Hayes, J.J. 1997. The N‐terminal tail of histone H2A binds to two distinct sites within the nucleosome core. Proc. Natl. Acad. Sci. U.S.A. 94:8959‐8964.
   Lee, K.M., Chafin, D.R., and Hayes, J.J. 1999. Targeted cross‐linking and DNA cleavage within model chromatin complexes. Methods Enzymol. 304:231‐251.
   Luger, K., Rechsteiner, T.J., Flaus, A.J., Waye, M.M., and Richmond, T.J. 1997a. Characterization of nucleosome core particles containing histone protein made in bacteria. J. Mol. Biol. 272:301‐311.
   Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. 1997b. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389:251‐260.
   Luger, K., Rechsteiner, T.J., and Richmond, T.J. 1999. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304:3‐19.
   Neely, K.E., Hassan, A.H., Wallberg, A.E., Steger, D.J., Cairns, B.R., Wright, A.P. and Workman, J.L. 1999. Activation domain‐mediated targeting of the SWI/SNF complex to promoters stimulates transcription from nucleosome arrays. Mol. Cell. 4:649‐655.
   Rhodes, D. 1985. Structural analysis of a triple complex between the histone octamer, a Xenopus gene for 5S RNA and transcription factor IIIA. EMBO J. 4:3473‐3482.
   Schwarz, P.M. and Hansen, J.C. 1994. Formation and stability of higher order chromatin structures.contributions of the histone octamer. J. Biol. Chem. 269:16284‐16289.
   van Holde, K.E. 1989. Chromatin. Springer‐ Verlag, New York
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