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. 2015 Jun 5;4:e06249. doi: 10.7554/eLife.06249

Figure 4. SWI/SNF remodeling evicts a bound Gal4DBD from its DNA template.

Nucleosomes were remodeled by 1.5 nM SWI/SNF with 1 mM ATP for 10 min with or without Gal4DBD. Shaded regions indicate locations of Gal4 binding sequence and 601NPE. (A) Distributions of the locations of the nucleosome and bound Gal4DBD before remodeling (upper plot), after remodeling without Gal4DBD (middle plot), and after remodeling with Gal4DBD (lower plot). (B) Representative traces in the case of before remodeling (top plot; N = 55) and after remodeling (middle and bottom plots; N = 50). The middle plot shows an example trace where a nucleosome was remodeled to the opposite side of Gal4DBD relative to its original position; while the bottom plot shows an example trace where a nucleosome was remodeled to the same side of Gal4DBD relative to its original position. Gray traces were taken from the corresponding naked DNA.

DOI: http://dx.doi.org/10.7554/eLife.06249.015

Figure 4—source data 1. SWI/SNF is unable to evict a bound Gal4DBD in the absence of a nucleosome or in the absence of ATP.
To determine whether SWI/SNF with ATP alone is able to displace a bound Gal4DBD in the absence of a nucleosome, we carried out experiments on a DNA template preloaded with Gal4DBD but without a nucleosome in 1.5 nM SWI/SNF with 1 mM ATP for 10 min. DNA molecules were subsequently unzipped to determine the presence of Gal4DBD. The fraction of templates containing a bound Gal4DBD remained the same before and after the remodeling reaction, indicating that SWI/SNF with ATP alone is not able to displace a bound Gal4DBD. To rule out the possibility that Gal4DBD disruption was due to binding of SWI/SNF to DNA or the nucleosome and not due to nucleosome remodeling, we carried out a control experiment on a DNA template containing a bound Gal4DBD and a nucleosome by incubating the sample with 1.5 nM SWI/SNF for 10 min in the absence of ATP. We subsequently unzipped the DNA template to determine if Gal4DBD was still bound. The fraction of templates containing a bound Gal4DBD was comparable to that of a template without a nucleosome and without SWI/SNF and ATP added, indicating that in the absence of ATP, SWI/SNF is unable to evict a bound Gal4DBD even in the presence of a nucleosome adjacent to a bound Gal4DBD.
DOI: http://dx.doi.org/10.7554/eLife.06249.016
elife06249s002.docx (12.3KB, docx)
DOI: 10.7554/eLife.06249.016

Figure 4.

Figure 4—figure supplement 1. Distributions of the locations of SWI/SNF remodeled nucleosomes as determined by unzipping from both directions.

Figure 4—figure supplement 1.

To determine whether the distributions of the locations of remodeled nucleosomes were similar for measurements made by unzipping the DNA in one direction vs in the other direction, we unzipped multiple DNA molecules, each containing either an unremodeled or a remodeled nucleosome, from both directions. Nucleosome remodeling was carried out in 1 nM SWI/SNF with 1 mM ATP for 5 min. Our data show similar distributions for data obtained in both directions.
DOI: http://dx.doi.org/10.7554/eLife.06249.017
Figure 4—figure supplement 2. Nucleosome remodeling by SWI/SNF on a template with the Gal4 binding site separated from the 601NPE by 24 bp.

Figure 4—figure supplement 2.

To test whether SWI/SNF is able to evict Gal4DBD via nucleosome remodeling when a bound Gal4DBD is located farther away from a nucleosome, we used a template where the Gal4 binding site was separated from the 601NPS by 24 bp and carried out unzipping experiments under identical conditions as those shown in Figure 4. Out of all traces where the nucleosome was repositioned to the opposite side of the Gal4 binding site by SWI/SNF (N = 13), we did not detect any Gal4DBD binding signature on the template, indicating eviction of Gal4DBD. Shown are example traces, with arrows indicating the unzipping directions.
DOI: http://dx.doi.org/10.7554/eLife.06249.018