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Multiple aspects of ATP-dependent nucleosome translocation by RSC and Mi-2 are directed by the underlying DNA sequence.

van Vugt JJ, de Jager M, Murawska M, Brehm A, van Noort J, Logie C - PLoS ONE (2009)

Bottom Line: Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps.Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA.This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, NCMLS, Radboud University, Nijmegen, The Netherlands. j.vanvugt@ncmls.ru.nl;

ABSTRACT

Background: Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted.

Methodology/principal findings: We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps.

Conclusions/significance: Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.

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RSC titration and time course on centrally positioned nucleosomes.A) Native 4% acrylamide gel with 0–10 nM RSC titration on 7.7 nM nucleosomes with a 204 and a 309 bp arm for 1 hour with or without 1 mM ATP. B) Native 4% acrylamide gel with 1 mM ATP and 5 nM RSC on 7.7 nM nucleosomes with a 204 and 309 bp arm at time points indicated in minutes. C) Lineweaver-Burk plot from the RSC titration.
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pone-0006345-g004: RSC titration and time course on centrally positioned nucleosomes.A) Native 4% acrylamide gel with 0–10 nM RSC titration on 7.7 nM nucleosomes with a 204 and a 309 bp arm for 1 hour with or without 1 mM ATP. B) Native 4% acrylamide gel with 1 mM ATP and 5 nM RSC on 7.7 nM nucleosomes with a 204 and 309 bp arm at time points indicated in minutes. C) Lineweaver-Burk plot from the RSC titration.

Mentions: To observe the RSC induced nucleosome repositioning with a higher resolution and better statistics than with AFM, we performed time course and enzyme titration experiments using native PAGE. With this technique the band shift relative to naked DNA increases with the distance of the nucleosome from the DNA ends (Figure 4). Nucleosomes were reconstituted on radioactively end-labelled DNA templates harbouring a 601 element with DNA arms of 205 and 309 bp. Consistent with our AFM results, reconstitution yielded between 85–90% of the DNA template with a nucleosome at the 601 sequence and on average 10% bare DNA. Titration of RSC resulted in an increased amount of end positioned nucleosomes with increasing RSC concentration (Figure 4A). The fraction of bare DNA remained constant, reproducing the data obtained by AFM and showing that histone octamers were not displaced from the DNA under these reaction conditions. Time course experiments revealed that detectable levels of end-nucleosomes were obtained after 10 minutes using 5 nM RSC (Figure 4B), and after 30 minutes when 1 nM RSC was used (data not shown). Because the initial nucleosome was positioned slightly off centre, we could also resolve how fast nucleosomes were mobilized from the starting positing, resulting in bands above and below the starting position (Figure 4A and B). Consistent with the AFM data, only little radioactive signal was detected between the bands corresponding to the starting and the end positioned nucleosome at any RSC concentration or time point. Together, these results indicate that nucleosomes are moderately trapped in the 601 element, resulting in a larger band width surrounding the initial nucleosome, but are efficiently translocated over larger distances once they escape this region.


Multiple aspects of ATP-dependent nucleosome translocation by RSC and Mi-2 are directed by the underlying DNA sequence.

van Vugt JJ, de Jager M, Murawska M, Brehm A, van Noort J, Logie C - PLoS ONE (2009)

RSC titration and time course on centrally positioned nucleosomes.A) Native 4% acrylamide gel with 0–10 nM RSC titration on 7.7 nM nucleosomes with a 204 and a 309 bp arm for 1 hour with or without 1 mM ATP. B) Native 4% acrylamide gel with 1 mM ATP and 5 nM RSC on 7.7 nM nucleosomes with a 204 and 309 bp arm at time points indicated in minutes. C) Lineweaver-Burk plot from the RSC titration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2710519&req=5

pone-0006345-g004: RSC titration and time course on centrally positioned nucleosomes.A) Native 4% acrylamide gel with 0–10 nM RSC titration on 7.7 nM nucleosomes with a 204 and a 309 bp arm for 1 hour with or without 1 mM ATP. B) Native 4% acrylamide gel with 1 mM ATP and 5 nM RSC on 7.7 nM nucleosomes with a 204 and 309 bp arm at time points indicated in minutes. C) Lineweaver-Burk plot from the RSC titration.
Mentions: To observe the RSC induced nucleosome repositioning with a higher resolution and better statistics than with AFM, we performed time course and enzyme titration experiments using native PAGE. With this technique the band shift relative to naked DNA increases with the distance of the nucleosome from the DNA ends (Figure 4). Nucleosomes were reconstituted on radioactively end-labelled DNA templates harbouring a 601 element with DNA arms of 205 and 309 bp. Consistent with our AFM results, reconstitution yielded between 85–90% of the DNA template with a nucleosome at the 601 sequence and on average 10% bare DNA. Titration of RSC resulted in an increased amount of end positioned nucleosomes with increasing RSC concentration (Figure 4A). The fraction of bare DNA remained constant, reproducing the data obtained by AFM and showing that histone octamers were not displaced from the DNA under these reaction conditions. Time course experiments revealed that detectable levels of end-nucleosomes were obtained after 10 minutes using 5 nM RSC (Figure 4B), and after 30 minutes when 1 nM RSC was used (data not shown). Because the initial nucleosome was positioned slightly off centre, we could also resolve how fast nucleosomes were mobilized from the starting positing, resulting in bands above and below the starting position (Figure 4A and B). Consistent with the AFM data, only little radioactive signal was detected between the bands corresponding to the starting and the end positioned nucleosome at any RSC concentration or time point. Together, these results indicate that nucleosomes are moderately trapped in the 601 element, resulting in a larger band width surrounding the initial nucleosome, but are efficiently translocated over larger distances once they escape this region.

Bottom Line: Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps.Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA.This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, NCMLS, Radboud University, Nijmegen, The Netherlands. j.vanvugt@ncmls.ru.nl;

ABSTRACT

Background: Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted.

Methodology/principal findings: We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps.

Conclusions/significance: Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.

Show MeSH