Chromatin Assembly Using Drosophila Systems

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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 ATP?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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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 H₂ O
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 MgCl₂ ( appendix 22 )
Liquid N₂

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 H₂ O
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 CaCl₂
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 )
T₅₀ E₄ 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 MgCl₂
DNA template (2.5 to 25 kbp circular or linearized plasmid, or λ DNA)
0.1 M CaCl₂
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 MgCl₂ (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 CaCl₂ (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

Figure 21.7.1 Chromatin assembly by the S‐190 extract. Lanes 1, 2—complete system, as described in ; lanes 3, 4—no S‐190 extract; lanes 5, 6—no core histones (note residual assembly activity with the endogenous histones of the extract); lanes 7, 8—no ATP; lanes 9, 10—no DNA template.

View Image

Figure 21.7.2 Chromatin assembly in the recombinant system. The reactions were performed as described in . The core histone to DNA ratios were initially estimated as: lanes 1, 2—0.8:1; lanes 3, 4—0.9:1; lanes 5, 6—1.0:1; lanes 7, 8—1.1:1; lanes 9, 10—1.2:1.

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Videos

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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Chromatin Assembly Using Drosophila Systems

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited