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H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast.

Ranjan A, Wang F, Mizuguchi G, Wei D, Huang Y, Wu C - Elife (2015)

Bottom Line: We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity.Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site.Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

ABSTRACT
The histone variant H2A.Z is a universal mark of gene promoters, enhancers, and regulatory elements in eukaryotic chromatin. The chromatin remodeler SWR1 mediates site-specific incorporation of H2A.Z by a multi-step histone replacement reaction, evicting histone H2A-H2B from the canonical nucleosome and depositing the H2A.Z-H2B dimer. Binding of both substrates, the canonical nucleosome and the H2A.Z-H2B dimer, is essential for activation of SWR1. We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity. Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site. Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.

No MeSH data available.


Related in: MedlinePlus

SWR1 mediates histone exchange without net change of nucleosome position.(A) Left: EMSA (6% native PAGE) shows INO80-mediated nucleosome sliding. An asymmetrically positioned 601 nucleosome with a 43 bp and 0 bp DNA linker was used for the sliding assay. Right: SWR1-mediated incorporation of H2A.Z-H2B dimer (without 3FLAG epitope tag). Incorporation of H2A.Z in nucleosome was confirmed by immunoblotting with anti-H2A.Z antibody. (B) Hydroxyl radical footprinting strategy. A canonical nucleosome with 60 bp and 0 bp linker DNA and fluorescence end-label (bottom strand) was used as substrate for histone replacement, followed by hydroxyl radical treatment and separation by 6% native PAGE. (C) Recovered DNA from gel slices containing AA, AZ, and ZZ states was analyzed on DNA sequencing gels. (D) Intensity plots for AA, AZ, and ZZ nucleosomes.DOI:http://dx.doi.org/10.7554/eLife.06845.012
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fig4: SWR1 mediates histone exchange without net change of nucleosome position.(A) Left: EMSA (6% native PAGE) shows INO80-mediated nucleosome sliding. An asymmetrically positioned 601 nucleosome with a 43 bp and 0 bp DNA linker was used for the sliding assay. Right: SWR1-mediated incorporation of H2A.Z-H2B dimer (without 3FLAG epitope tag). Incorporation of H2A.Z in nucleosome was confirmed by immunoblotting with anti-H2A.Z antibody. (B) Hydroxyl radical footprinting strategy. A canonical nucleosome with 60 bp and 0 bp linker DNA and fluorescence end-label (bottom strand) was used as substrate for histone replacement, followed by hydroxyl radical treatment and separation by 6% native PAGE. (C) Recovered DNA from gel slices containing AA, AZ, and ZZ states was analyzed on DNA sequencing gels. (D) Intensity plots for AA, AZ, and ZZ nucleosomes.DOI:http://dx.doi.org/10.7554/eLife.06845.012

Mentions: To date, all chromatin remodelers examined are able to mobilize positioned nucleosome in vitro, including the strongly positioned 601 nucleosome (Lowary and Widom, 1998; Becker and Workman, 2013; Bartholomew, 2014). SWR1 quantitatively evicts both H2A-H2B dimers on this nucleosome, replacing them with H2A.Z-H2B, but whether nucleosome positioning was also altered was unknown. To examine this question, we used a 601 nucleosome bearing a 60 bp linker on one side, and a native PAGE mobility assay, which separates nucleosomes on the basis of different linker lengths and spatial orientation (Hamiche et al., 1999). We found no substantial mobility shift indicative of a repositioned nucleosome after incorporation of (untagged) H2A.Z-H2B (Figure 4A). Similar results were obtained for a center-positioned 601 nucleosome (data not shown). By contrast, the INO80 remodeler mobilized the nucleosome from the end- to center-position, as shown by gel mobility shift (Figure 4A) (Shen et al., 2000; Udugama et al., 2011). For a more discerning technique, we mapped the precise position of AA, AZ, and ZZ nucleosomes after histone H2A.Z replacement by hydroxyl radical footprinting (Figure 4B). Strikingly, at single nucleotide resolution, there was no net change of the 601 nucleosome position after it underwent one or two rounds of histone H2A.Z replacement (Figure 4C).10.7554/eLife.06845.012Figure 4.SWR1 mediates histone exchange without net change of nucleosome position.


H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast.

Ranjan A, Wang F, Mizuguchi G, Wei D, Huang Y, Wu C - Elife (2015)

SWR1 mediates histone exchange without net change of nucleosome position.(A) Left: EMSA (6% native PAGE) shows INO80-mediated nucleosome sliding. An asymmetrically positioned 601 nucleosome with a 43 bp and 0 bp DNA linker was used for the sliding assay. Right: SWR1-mediated incorporation of H2A.Z-H2B dimer (without 3FLAG epitope tag). Incorporation of H2A.Z in nucleosome was confirmed by immunoblotting with anti-H2A.Z antibody. (B) Hydroxyl radical footprinting strategy. A canonical nucleosome with 60 bp and 0 bp linker DNA and fluorescence end-label (bottom strand) was used as substrate for histone replacement, followed by hydroxyl radical treatment and separation by 6% native PAGE. (C) Recovered DNA from gel slices containing AA, AZ, and ZZ states was analyzed on DNA sequencing gels. (D) Intensity plots for AA, AZ, and ZZ nucleosomes.DOI:http://dx.doi.org/10.7554/eLife.06845.012
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4508883&req=5

fig4: SWR1 mediates histone exchange without net change of nucleosome position.(A) Left: EMSA (6% native PAGE) shows INO80-mediated nucleosome sliding. An asymmetrically positioned 601 nucleosome with a 43 bp and 0 bp DNA linker was used for the sliding assay. Right: SWR1-mediated incorporation of H2A.Z-H2B dimer (without 3FLAG epitope tag). Incorporation of H2A.Z in nucleosome was confirmed by immunoblotting with anti-H2A.Z antibody. (B) Hydroxyl radical footprinting strategy. A canonical nucleosome with 60 bp and 0 bp linker DNA and fluorescence end-label (bottom strand) was used as substrate for histone replacement, followed by hydroxyl radical treatment and separation by 6% native PAGE. (C) Recovered DNA from gel slices containing AA, AZ, and ZZ states was analyzed on DNA sequencing gels. (D) Intensity plots for AA, AZ, and ZZ nucleosomes.DOI:http://dx.doi.org/10.7554/eLife.06845.012
Mentions: To date, all chromatin remodelers examined are able to mobilize positioned nucleosome in vitro, including the strongly positioned 601 nucleosome (Lowary and Widom, 1998; Becker and Workman, 2013; Bartholomew, 2014). SWR1 quantitatively evicts both H2A-H2B dimers on this nucleosome, replacing them with H2A.Z-H2B, but whether nucleosome positioning was also altered was unknown. To examine this question, we used a 601 nucleosome bearing a 60 bp linker on one side, and a native PAGE mobility assay, which separates nucleosomes on the basis of different linker lengths and spatial orientation (Hamiche et al., 1999). We found no substantial mobility shift indicative of a repositioned nucleosome after incorporation of (untagged) H2A.Z-H2B (Figure 4A). Similar results were obtained for a center-positioned 601 nucleosome (data not shown). By contrast, the INO80 remodeler mobilized the nucleosome from the end- to center-position, as shown by gel mobility shift (Figure 4A) (Shen et al., 2000; Udugama et al., 2011). For a more discerning technique, we mapped the precise position of AA, AZ, and ZZ nucleosomes after histone H2A.Z replacement by hydroxyl radical footprinting (Figure 4B). Strikingly, at single nucleotide resolution, there was no net change of the 601 nucleosome position after it underwent one or two rounds of histone H2A.Z replacement (Figure 4C).10.7554/eLife.06845.012Figure 4.SWR1 mediates histone exchange without net change of nucleosome position.

Bottom Line: We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity.Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site.Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

ABSTRACT
The histone variant H2A.Z is a universal mark of gene promoters, enhancers, and regulatory elements in eukaryotic chromatin. The chromatin remodeler SWR1 mediates site-specific incorporation of H2A.Z by a multi-step histone replacement reaction, evicting histone H2A-H2B from the canonical nucleosome and depositing the H2A.Z-H2B dimer. Binding of both substrates, the canonical nucleosome and the H2A.Z-H2B dimer, is essential for activation of SWR1. We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity. Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site. Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.

No MeSH data available.


Related in: MedlinePlus