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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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Stepwise nucleosome remodeling on off-centre nucleosomes.Native 4% acrylamide gel with 5 nM RSC or 10 nM Mi-2 on 11.5 nM nucleosomes with a 87 and a 422 bp arm for 1 hour and a 0–100 µM ATP titration (RSC) or 1 mM ATP (Mi-2) (Upper panel). The graph in the lower panel shows the relative signal intensity of two acrylamide gel lanes, one with RSC remodeled mononucleosomes (3 µM ATP) and one with Mi-2 remodeled mononucleosomes (1 mM ATP).
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pone-0006345-g005: Stepwise nucleosome remodeling on off-centre nucleosomes.Native 4% acrylamide gel with 5 nM RSC or 10 nM Mi-2 on 11.5 nM nucleosomes with a 87 and a 422 bp arm for 1 hour and a 0–100 µM ATP titration (RSC) or 1 mM ATP (Mi-2) (Upper panel). The graph in the lower panel shows the relative signal intensity of two acrylamide gel lanes, one with RSC remodeled mononucleosomes (3 µM ATP) and one with Mi-2 remodeled mononucleosomes (1 mM ATP).

Mentions: Careful inspection of the gels in Figure 4 revealed a subtle band pattern around the 601 positioning sequence that was not resolved by AFM. In the absence of ATP this band pattern was not observed, confirming that they are products of ATP-dependent remodeling (Figure 4A). However, the band distribution around the slightly off-centred nucleosome was too compressed to unambiguously quantify individual bands. Furthermore, its quasi-central position did not allow us to determine the direction of nucleosome translocation. We therefore produced a nucleosome with one short (87 bp) and one long (422 bp) DNA arm. Using this substrate a clear ladder of nucleosome positions could be resolved after remodeling (Figure 5). One of the bands that appeared after RSC remodeling, ran faster through the gel than the end-positioned nucleosomes that were a side product in this reconstitution, confirming that RSC pushes nucleosomes over the DNA end (Figure 5). Titration of ATP had no effect on the position of the bands, it only changed the number of bands of the nucleosome ladder, demonstrating ATP concentration independence of the catalyzed nucleosome ‘step size’.


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)

Stepwise nucleosome remodeling on off-centre nucleosomes.Native 4% acrylamide gel with 5 nM RSC or 10 nM Mi-2 on 11.5 nM nucleosomes with a 87 and a 422 bp arm for 1 hour and a 0–100 µM ATP titration (RSC) or 1 mM ATP (Mi-2) (Upper panel). The graph in the lower panel shows the relative signal intensity of two acrylamide gel lanes, one with RSC remodeled mononucleosomes (3 µM ATP) and one with Mi-2 remodeled mononucleosomes (1 mM ATP).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006345-g005: Stepwise nucleosome remodeling on off-centre nucleosomes.Native 4% acrylamide gel with 5 nM RSC or 10 nM Mi-2 on 11.5 nM nucleosomes with a 87 and a 422 bp arm for 1 hour and a 0–100 µM ATP titration (RSC) or 1 mM ATP (Mi-2) (Upper panel). The graph in the lower panel shows the relative signal intensity of two acrylamide gel lanes, one with RSC remodeled mononucleosomes (3 µM ATP) and one with Mi-2 remodeled mononucleosomes (1 mM ATP).
Mentions: Careful inspection of the gels in Figure 4 revealed a subtle band pattern around the 601 positioning sequence that was not resolved by AFM. In the absence of ATP this band pattern was not observed, confirming that they are products of ATP-dependent remodeling (Figure 4A). However, the band distribution around the slightly off-centred nucleosome was too compressed to unambiguously quantify individual bands. Furthermore, its quasi-central position did not allow us to determine the direction of nucleosome translocation. We therefore produced a nucleosome with one short (87 bp) and one long (422 bp) DNA arm. Using this substrate a clear ladder of nucleosome positions could be resolved after remodeling (Figure 5). One of the bands that appeared after RSC remodeling, ran faster through the gel than the end-positioned nucleosomes that were a side product in this reconstitution, confirming that RSC pushes nucleosomes over the DNA end (Figure 5). Titration of ATP had no effect on the position of the bands, it only changed the number of bands of the nucleosome ladder, demonstrating ATP concentration independence of the catalyzed nucleosome ‘step size’.

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