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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.


SWR1 binding to nucleosome core particle with gaps on both sides of dyad.Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles.DOI:http://dx.doi.org/10.7554/eLife.06845.010
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fig3s1: SWR1 binding to nucleosome core particle with gaps on both sides of dyad.Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles.DOI:http://dx.doi.org/10.7554/eLife.06845.010

Mentions: Furthermore, we examined how gaps in nucleosomal DNA interfere with histone replacement by SWR1. Systematic introduction of two-nucleotide gaps on one DNA strand of a nucleosome showed that a single gap at −17, −18 nt from the nucleosome dyad blocked the second round of H2A.Z replacement (Figure 3A). Further scanning identified a 6 nt region (−17 to −22 nt from the dyad), whose integrity is required for histone replacement (Figure 3B). This gap-sensitive region overlaps with the hydroxyl radical footprint of linker-oriented SWR1 at nucleosome position SHL2. Introduction of gaps on both sides of the nucleosome dyad caused a complete failure of H2A.Z replacement by SWR1 (Figure 3C). Taken together, our findings indicate that close contact between SWR1 and nucleosomal DNA around SHL2 is critical for enzyme activation. This activation likely occurs post-recruitment, as SWR1 binding is not adversely affected on the gap-containing nucleosome substrate (Figure 3—figure supplement 1).10.7554/eLife.06845.009Figure 3.DNA gaps block SWR1 activity when positioned 17–22 bp on either side from dyad.


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 binding to nucleosome core particle with gaps on both sides of dyad.Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles.DOI:http://dx.doi.org/10.7554/eLife.06845.010
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508883&req=5

fig3s1: SWR1 binding to nucleosome core particle with gaps on both sides of dyad.Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles.DOI:http://dx.doi.org/10.7554/eLife.06845.010
Mentions: Furthermore, we examined how gaps in nucleosomal DNA interfere with histone replacement by SWR1. Systematic introduction of two-nucleotide gaps on one DNA strand of a nucleosome showed that a single gap at −17, −18 nt from the nucleosome dyad blocked the second round of H2A.Z replacement (Figure 3A). Further scanning identified a 6 nt region (−17 to −22 nt from the dyad), whose integrity is required for histone replacement (Figure 3B). This gap-sensitive region overlaps with the hydroxyl radical footprint of linker-oriented SWR1 at nucleosome position SHL2. Introduction of gaps on both sides of the nucleosome dyad caused a complete failure of H2A.Z replacement by SWR1 (Figure 3C). Taken together, our findings indicate that close contact between SWR1 and nucleosomal DNA around SHL2 is critical for enzyme activation. This activation likely occurs post-recruitment, as SWR1 binding is not adversely affected on the gap-containing nucleosome substrate (Figure 3—figure supplement 1).10.7554/eLife.06845.009Figure 3.DNA gaps block SWR1 activity when positioned 17–22 bp on either side from dyad.

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.