Persistent site-specific remodeling of a nucleosome array by transient action of the SWI/SNF complex

Science

Volumn 273, Issue 5274, 1996, Pages 513-516

  Owen Hughes, T ^a   Utley, R T ^a   Cote J ^a   Peterson, C L ^b   Workman, J L ^a  

^a PENNSYLVANIA STATE UNIVERSITY   (United States)

^b UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE TRIPHOSPHATE; DEOXYRIBONUCLEASE I; DNA FRAGMENT; HISTONE; PROTEIN SUBUNIT; TRANSCRIPTION FACTOR;

ARTICLE; BINDING AFFINITY; CHROMATIN STRUCTURE; COMPLEX FORMATION; DNA FOOTPRINTING; GENETIC TRANSCRIPTION; NONHUMAN; NUCLEOSOME; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN DNA BINDING; PROTEIN PURIFICATION; SACCHAROMYCES CEREVISIAE; TRANSCRIPTION REGULATION;

EID: 0029826906     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5274.513     Document Type: Article

References (43)

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16. Plasmid pG5208-10 was constructed as follows: Five copies of the sea urchin 5S RNA nucleosome-positioning sequence were isolated from a partial Ava I digest of p5S207-12 (12) and subcloned into the Eco RV and Bgl II sites of pBENDΔ, a derivative of pBEND (J. Kim. C. Zwieb, C. Wu, S. Adhya, Gene 85, 15 (1989)] in which one copy of the polylinker had been deleted by Bam HI digestion, DNA containing five repeats of the Gal4 binding site was amplified by the polymerase chain reaction (PCR) from the plasmid pG5l160E4T [ Y. S. Lin, M. F. Carey, M. Ptashne, M. R. Green, Cell 54, 659 (1988)] with the primers 5′-CTAGCTAGCGTCGACGTC-GACGTCGACAAGCTTGCATGCCTGC-3′ and 5′-TCAACTAGTCGACGTCGACGTCGACAATCTTTT-TGTTGTCAAGCTG-3′. The PCR product was digested with Nhe I and Spe I and then subcloned into the Nhe I and Spe I sites of the pBEND derivative such that five nucleosome-positioning sequences flanked either side of the Gal4 binding sites.
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21. 2-terminal 94 amino acids containing DNA binding and dimerization domains), Gal4-AH, and Gal4-VP16 all functioned equally in the assays described here (T. Owen-Hughes and J. L. Workman, unpublished results). For analysis of the array construct, 1 fmol of probe DNA and 62.5 fmol of donor nucleosomes were included in each reaction. When Gal4-AH and SWI/ SNF were to be removed by competition, 1 μg of duplex Gal4 site-containing DNA (created by the annealing of the primers 5′-CTAGACCGGACGACAGTACTCCGACT-3′ and 5′-CTAGAGTCGGAGTACTGTCGTCCGGT-3′), 0.5 μg of HeLa nucleosomes, and 0.5 μg of calf thymus DNA (Sigma) were added to each binding reaction in 2 μl of CB [0.5 mM tris (pH 7.5), 0.5 mM EDTA, 1 M NaCl). Incubation was extended for 1 hour at 37°C. Competition was judged to be complete by the inability of Gal4-AH or SWI/SNF to induce a shift in the mobility of nucleosomes subsequent to this treatment (Fig. 3).
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31. The loss of histones from Gal4-site mononucleosomes was dependent on Gal4-AH, SWI/SNF, and ATP (Fig. 3). Although SWI/SNF participated in the disruption of these nucleosomes, leading to histone loss, it is likely that the evicted histones were transferred to the donor oligonucleosomes or DNA present in the reactions. Histones have been shown to transfer from factor-bound nucleosomes to both DNA and oligonucleosomes (9, 22, 23). 26. Histone loss (in trans or in cis) is likely to occur from many enhancers or promoters in vivo in response to factor binding and disruption by SWI/SNF or other nucleosome-remodeling activities. However, at other regulatory elements, the action of SWI/SNF may stabilize factor binding without actually evicting histones, leading to a persistently disrupted factor-bound nucleosome [E. H. Bresnick, M. Bustin, V. Marsaud, H. Richard-Foy, G. L. Hager Nucleic Acids Res. 20, 273 (1992); M. Truss, J. Bartsch, A. Schelbert, R. J. Hache, M. Beato, EMBO J. 14, 1737 (1995)].
32. The loss of histones from Gal4-site mononucleosomes was dependent on Gal4-AH, SWI/SNF, and ATP (Fig. 3). Although SWI/SNF participated in the disruption of these nucleosomes, leading to histone loss, it is likely that the evicted histones were transferred to the donor oligonucleosomes or DNA present in the reactions. Histones have been shown to transfer from factor-bound nucleosomes to both DNA and oligonucleosomes (9, 22, 23). 26. Histone loss (in trans or in cis) is likely to occur from many enhancers or promoters in vivo in response to factor binding and disruption by SWI/SNF or other nucleosome-remodeling activities. However, at other regulatory elements, the action of SWI/SNF may stabilize factor binding without actually evicting histones, leading to a persistently disrupted factor-bound nucleosome [E. H. Bresnick, M. Bustin, V. Marsaud, H. Richard-Foy, G. L. Hager Nucleic Acids Res. 20, 273 (1992); M. Truss, J. Bartsch, A. Schelbert, R. J. Hache, M. Beato, EMBO J. 14, 1737 (1995)].
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39. A single copy of the 5S nucleosome-positioning sequence was generated by digestion of p207-12 with Ava I and Sca I, and labeled at the Ava I ends with the Klenow fragment.
40. The nucleosome-length DNA fragment containing five Gal4 binding sites was excised from plasmid pGSH (18) with the enzymes Nhe I and Pst I, and end-labeled at the Nhe I site.
41. Binding reactions for the gel mobility-shift analysis were performed for 30 min at 30°C. SWI/SNF was then removed from the DNA by competition with 0.5 μg of HeLa oligonucleosomes and 0.5 μg of calf thymus DNA at 30°C for 30 min, enabling Gal4-AH bound complexes to be resolved by native polyacrylamide gel electrophoresis in 0.5× tris-borate-EDTA. Binding reactions shown in Fig. 2 all contained 25 ng of total nucleosomes and 0.2 ng of probe DNA.
42. The DNA sequence containing the five Gal4 sites was excised from plasmid pG5208-10 (11) by digestion with Sal I, end-labeled with the Klenow fragment, and reconstituted into nucleosome cores.
43. Supported by NIH grant GM47867 to J.L.W. T.O.-H. is the recipient of a long-term European Molecular Biology Organization fellowship, J.C. is a Canadian Medical Research Council postdoctoral fellow, and J.L.W. and C.L.P. are Leukemia Society scholars.