Chromatin Assembly Using Drosophila Systems

Dmitry V. Fyodorov1, Mark E. Levenstein1

1 University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.7
DOI:  10.1002/0471142727.mb2107s58
Online Posting Date:  May, 2002
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Abstract

To successfully study chromatin structure and activity in vitro, it is essential to have a chromatin assembly system that will prepare extended nucleosome arrays with highly defined protein content that resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. The Drosophila ATPā€dependent chromatin assembly system described in this unit meets these requirements. The end product of the reaction described here has highly periodic extended arrays with physiologic spacing and positioning of the nucleosomes.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of the Drosophila S‐190 Chromatin Assembly Extract
  • Basic Protocol 2: Purification of Core Histones from the Drosophila Embryos
  • Basic Protocol 3: Chromatin Assembly with the S‐190 Extract
  • Basic Protocol 4: Expression and Purification of the Recombinant Drosophila ACF
  • Basic Protocol 5: Expression and Purification of the Recombinant Drosophila NAP‐1
  • Alternate Protocol 1: Expression and Purification of the Recombinant Drosophila NAP‐1 (NTA Superflow Resin)
  • Basic Protocol 6: Chromatin Assembly with Purified Recombinant Drosophila Factors
  • Alternate Protocol 2: Titration of the Ratio of Core Histones to DNA in the Recombinant Chromatin Assembly Reaction
  • Support Protocol 1: Expression and Purification of the Core Catalytic Domain of the Drosophila Topoisomerase I
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of the Drosophila S‐190 Chromatin Assembly Extract

  Materials
  • 0‐ to 6‐hr Drosophila embryos
  • Bleach wash: 50% (v/v) household bleach in distilled H 2O
  • Embryo wash: 0.7% (w/v) NaCl/0.4% (v/v) Triton X‐100, room temperature and 4°C
  • Saline wash: 0.7% (w/v) NaCl, 4°C
  • recipeBuffer R (see recipe), 4°C
  • 1 M MgCl 2 ( appendix 22)
  • Liquid N 2
  • Fine nylon mesh (e.g., Sefar America 03‐80/37)
  • 800‐ml glass beaker
  • Glass rod
  • Vacuum aspirator
  • 40 ml Wheaton Dounce homogenizer with “A” and “B” pestles
  • 17 × 100–mm polypropylene tubes (e.g., Falcon 2059)
  • Sorvall Superspeed centrifuge with SS‐34 rotor (or equivalent)
  • 50‐ml conical tubes
  • 10‐ml plastic syringes and 18.5‐G needles
  • Beckman ultracentrifuge with SW 41 and SW 55 rotors
  • Thin‐walled 14 × 89–mm (for SW 41 rotor) and 13 × 51–mm (for SW 55 rotor) Beckman ultracentrifuge tubes

Basic Protocol 2: Purification of Core Histones from the Drosophila Embryos

  Materials
  • 0‐ to 12‐hr Drosophila embryos
  • Bleach wash: 50% (v/v) household bleach in distilled H 2O
  • Embryo wash: 0.7% (w/v) NaCl/0.04% (v/v) Triton X‐100
  • recipeBuffer B (see recipe)
  • recipeBuffer A (see recipe)
  • 1 M NaOH
  • 0.1 M CaCl 2
  • recipe200 U/ml micrococcal nuclease stock solution (see recipe)
  • 10 mM and 500 mM EDTA
  • 10% (w/v) SDS
  • 5 M NaCl ( appendix 22)
  • 24:1 chloroform/isoamyl alcohol
  • recipeLinear 5% to 30% sucrose gradients (see recipe)
  • T 50E 4 buffer: 50 mM Tris⋅Cl, pH 7.9 ( appendix 22)/4 mM EDTA
  • Hydroxylapatite resin (e.g., BioGel HT gel; Bio‐Rad)
  • recipeHA chromatography buffer (see recipe) with 0 M, 0.35 M, and 2.5 M NaCl
  • recipeCore histone storage buffer (see recipe)
  • BCA assay kit (Pierce)
  • Fine nylon mesh (e.g., Sefar America 03‐80/37)
  • Weigh boats
  • Yamato LH‐21 homogenizer (alternatively, Potter‐Elvehjem homogenizer)
  • 500‐ml centrifuge bottles for Sorvall GSA rotor (or equivalent)
  • Mira cloth (Calbiochem‐Novabiochem)
  • Sorvall Superspeed centrifuge with GSA and SS‐34 rotors or equivalents
  • Beckman ultracentrifuge with SW‐28 rotor and appropriate tubes
  • 12,000 to 15,000 and 3,500 MWCO dialysis tubing
  • FPLC apparatus
  • Additional reagents and equipment for extraction of DNA (unit 2.1), agarose gel electrophoresis (unit 2.5), SDS‐PAGE (unit 10.2), Coomassie blue staining and destaining of proteins in gels (unit 10.6), and dialysis ( appendix 3C)

Basic Protocol 3: Chromatin Assembly with the S‐190 Extract

  Materials
  • recipeBuffer R (see recipe)
  • S‐190 extract (see protocol 1)
  • Core histones (see protocol 2)
  • recipeATP mix (see recipe)
  • 0.1 M MgCl 2
  • DNA template (2.5 to 25 kbp circular or linearized plasmid, or λ DNA)
  • 0.1 M CaCl 2
  • recipe200 U/ml micrococcal nuclease stock solution (see recipe)
  • 0.5 M EDTA
  • 10 mg/ml RNase A
  • recipeGlycogen stop buffer (see recipe)
  • 2.5 mg/ml proteinase K
  • 50:49:1 (v/v/v) phenol/chloroform/isoamyl alcohol, equilibrated with 10 mM Tris⋅Cl, pH 8.0 (see unit 2.1 for equilibration technique)
  • 2.5 M ammonium acetate
  • 70% and 100% ethanol
  • 1.2% agarose gel (unit 2.5)
  • TBE buffer ( appendix 22)
  • 123 bp DNA ladder (Life Technologies)
  • 27° and 37°C water baths
  • Additional reagents and equipment for extraction of DNA (unit 2.1) and agarose gel electrophoresis (unit 2.5)

Basic Protocol 4: Expression and Purification of the Recombinant Drosophila ACF

  Materials
  • High titer Acf1‐FLAG and ISWI baculovirus stocks (Orbigen; also see units 16.9 & 16.10)
  • Late log phase Sf9 cells cultured in suspension (>2 × 106 cells/ml; also see units 16.9 & 16.10)
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • recipeLysis buffer F (see recipe)
  • 1:1 (v/v) slurry of FLAG‐M2 resin (Sigma‐Aldrich), equilibrated in recipelysis buffer F
  • recipeDilution buffer F (see recipe)
  • recipeWash buffer F (see recipe)
  • recipeElution buffer F (see recipe)
  • Liquid nitrogen
  • Bovine serum albumin (BSA) standard, 2 mg/ml (Pierce, Cat. No. 23209)
  • Clinical centrifuge with swinging‐bucket rotor, 4°C
  • 250‐ml conical centrifuge bottles appropriate for clinical centrifuge, or 50‐ml conical tubes
  • 14‐ and 50‐ml disposable conical centrifuge tubes
  • 15‐ml Wheaton dounce homogenizer, “A” pestle
  • Sorvall Superspeed centrifuge with SS‐34 rotor (or equivalent)
  • 15‐ml capped polypropylene tubes
  • Siliconized 1.5‐ml polypropylene tubes (e.g., ISC BioExpress, Cat. # C‐3302‐1)
  • Additional reagents and equipment for baculovirus culture (units 16.9 & 16.10), SDS‐PAGE (unit 10.2), and staining of gels (unit 10.6)

Basic Protocol 5: Expression and Purification of the Recombinant Drosophila NAP‐1

  Materials
  • Late‐log‐phase Sf9 cells cultured in suspension (>2 × 106 cells/ml; also see units 16.9 & 16.10)
  • High‐titer His‐NAP‐1 baculovirus stock (Orbigen; also see units 16.9 & 16.10)
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • recipeLysis buffer H (see recipe)
  • recipeWash buffer H (see recipe)
  • Elution buffer H: recipewash buffer H (see recipe) containing 480 mM imidazole
  • recipeHEGD buffer containing 0.1 M NaCl (see recipe)
  • recipeNAP‐1 purification buffer (see recipe) containing 0.0, 0.1, and 1.0 M NaCl
  • Ni‐NTA agarose resin (Qiagen)
  • Bovine serum albumin (BSA) standard, 2 mg/ml (Pierce, Cat. No. 23209)
  • Source 15Q resin (Amersham Pharmacia Biotech)
  • 20% (v/v) ethanol
  • 8% and 15% SDS‐PAGE gels (unit 10.2)
  • Liquid nitrogen
  • Clinical centrifuge with swinging‐bucket rotor, 4°C
  • 250‐ml conical centrifuge bottles appropriate for clinical centrifuge
  • 40‐ml Wheaton Dounce homogenizer with “A” pestle
  • Sorvall Superspeed centrifuge with SS34 rotor (or equivalent)
  • 15‐ and 50‐ml conical tubes
  • End‐over‐end rotator
  • 12,000 to 15,000 MWCO dialysis tubing
  • HR‐5 or HR‐10 FPLC column (Amersham Pharmacia Biotech)
  • FPLC apparatus
  • Siliconized 1.5 ml polypropylene tubes (e.g., ISC BioExpress, Cat. # C‐3302‐1)
  • Additional reagents and equipment for baculovirus culture and infection (units 16.9 16.11), dialysis ( 3.NaN), and SDS‐PAGE (unit 10.2)

Alternate Protocol 1: Expression and Purification of the Recombinant Drosophila NAP‐1 (NTA Superflow Resin)

  • NTA Superflow resin (Qiagen)
  • recipeSuperflow chromatography buffer (see recipe) containing 20 mM and 500 mM imidazole
  • C‐10/10 or C‐10/20 FPLC column (Amersham Pharmacia Biotech)

Basic Protocol 6: Chromatin Assembly with Purified Recombinant Drosophila Factors

  Materials
  • recipeHEG buffer (see recipe)
  • 300 mM KCl (store in aliquots of 0.1 to 1 ml at −20°C)
  • recipePvOH/PEG solution (see recipe)
  • 2 mg/ml BSA solution (store in aliquots of 0.1 to 1 ml at −20°C)
  • 0.5 to 4.0 mg/ml recombinant NAP‐1 (see protocol 5)
  • 0.3 to 2.0 mg/ml purified Drosophila core histones, 0.3 to 2.0 mg/ml (see protocol 2)
  • 0.5 M ATP (store in aliquots of 0.1 to 1 ml at −20°C)
  • recipe0.5 M creatine phosphate (see recipe)
  • recipe5 mg/ml creatine kinase (see recipe)
  • 100 mM MgCl 2 (store in aliquots of 0.1 to 1 ml at −20°C)
  • 0.3 to 2.0 mg/ml plasmid DNA, double CsCl‐purified (unit 1.7), in TE buffer ( appendix 22)
  • recipe10× topoisomerase I buffer (see recipe)
  • Recombinant topoisomerase I working solution (see protocol 9)
  • 0.002 to 0.2 mg/ml recombinant ACF (see protocol 4)
  • ACF dilution buffer: recipewash buffer F (see recipe) containing 0.4 mg/ml recombinant human insulin (Roche)
  • recipe200 U/ml micrococcal nuclease stock solution (see recipe)
  • recipeBuffer R (see recipe)
  • 10 mM CaCl 2 (store in aliquots of 0.1 to 1 ml at −20°C)
  • 0.5 M EDTA
  • 10 mg/ml RNase A
  • recipeGlycogen stop buffer (see recipe)
  • 2.5 mg/ml proteinase K
  • 50:49:1 (v/v/v) phenol/chloroform/isoamyl alcohol, equilibrated with 10 mM Tris⋅Cl, pH 8.0 (see unit 2.1 for equilibration technique)
  • 2.5 M ammonium acetate
  • 100% ethanol
  • Siliconized 1.5 ml polypropylene tubes (e.g., ISC BioExpress, Cat. # C‐3302‐1)
  • 27° and 30°C water baths
  • Additional reagents and equipment for extraction of DNA (unit 2.1) and agarose gel electrophoresis (unit 2.5)

Alternate Protocol 2: Titration of the Ratio of Core Histones to DNA in the Recombinant Chromatin Assembly Reaction

  Materials
  • Competent BL21(DE3) bacteria (Novagen)
  • PET‐NDH6 plasmid (available on request from Dmitry Fyodorov; )
  • LB plates and medium (unit 1.1) containing 50 µg/ml kanamycin (add from 10 mg/ml kanamycin stock)
  • 100 mM IPTG
  • Liquid nitrogen
  • recipeLysis buffer T (see recipe)
  • Ni‐NTA resin (Qiagen)
  • Elution buffer T: recipelysis buffer T containing 0.5 M imidazole
  • recipeDialysis buffer T (see recipe)
  • recipeStorage buffer T (see recipe)
  • 8% SDS‐PAGE gel (unit 10.2)
  • Bovine serum albumin (BSA) standard, 2 mg/ml (Pierce, Cat. No. 23209)
  • 0.8% agarose gel (unit 2.5)
  • recipe10× topoisomerase I buffer (see recipe)
  • Sorvall Superspeed centrifuge with GSA and SS‐34 rotors (or equivalents)
  • Microtip sonicator (e.g., Branson Sonifier 450; VWR Scientific)
  • 10‐ml polypropylene chromatography column
  • 0.5 to 3.0 ml Slide‐A‐Lyzer, 10,000 MWCO (Pierce)
  • Additional reagents and equipment for transformation of bacteria (unit 1.8), growth of bacteria in solid (unit 1.3) and liquid (unit 1.2) media, SDS‐PAGE (unit 10.2), and agarose gel electrophoresis (unit 2.5)
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Figures

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Literature Cited

Literature Cited
   Banerjee, S. and Cantor, C.R. 1990. Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract. Mol. Cell. Biol. 10:2863‐2873.
   Becker, P.B. and Wu, C. 1992. Cell‐free system for assembly of transcriptionally repressed chromatin from Drosophila embryos. Mol. Cell. Biol. 12:2241‐2249.
   Bulger, M. and Kadonaga, J.T. 1994. Biochemical reconstitution of chromatin with physiological nucleosome spacing. Methods Mol. Genet. 5:241‐262.
   Bulger, M., Ito, T., Kamakaka, R.T., and Kadonaga, J.T. 1995. Assembly of regularly‐spaced nucleosome arrays by dCAF‐1 and a 56 kDa histone‐binding protein. Proc. Natl. Acad. Sci. U.S.A. 92:11726‐11730.
   Glikin, G.C., Ruberti, I., and Worcel, A. 1984. Chromatin assembly in Xenopus oocytes: In vitro studies. Cell 37:33‐41.
   Ito, T., Tyler, J.K., and Kadonaga, J.T. 1997a. Chromatin assembly factors: A dual function in nucleosome formation and mobilization?. Genes Cells 2:593‐600.
   Ito, T., Bulger, M., Pazin, M.J., Kobayashi, R., and Kadonaga, J.T. 1997b. ACF, an ISWI‐containing and ATP‐utilizing chromatin assembly and remodeling factor. Cell 90:145‐155.
   Ito, T., Levenstein, M.E., Fyodorov, D.V., Kutach, A.K., Kobayashi, R., and Kadonaga, J.T. 1999. ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP‐dependent catalysis of chromatin assembly. Genes & Dev. 13:1529‐1539.
   Levenstein, M.E. and Kadonaga, J.T. 2002. Biochemical analysis of chromatin containing recombinant Drosophila core histones Submitted.
   Nakagawa, T., Bulger, M., Muramatsu, M., and Ito, T. 2001. Multistep chromatin assembly on supercoiled plasmid DNA by nucleosome assembly protein‐1 and ATP‐utilizing chromatin assembly and remodeling factor. J. Biol. Chem. 276:27384‐27391.
   Shaiu, W.L. and Hsieh, T.S. 1998. Targeting to transcriptionally active loci by the hydrophilic N‐terminal domain of Drosophila DNA Topoisomerase I. Mol. Cell. Biol. 18:4358‐4367.
   Stillman, B. 1986. Chromatin assembly during SV40 DNA replication in vitro. Cell 45:555‐565.
   Tyler, J.K., Adams, C.R., Chen, S.‐R., Kobayashi, R., Kamakaka, R.T., and Kadonaga, J.T. 1999. The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402:555‐560.
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