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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 particles containing H2A or H2A.Z histone.EMSA shows SWR1 binding to Alexa 647-labeled H2A- and H2A.Z-nucleosome core particles (1 nM). Free and bound complexes are resolved on 1.3% agarose gel. Bottom: binding curves for H2A- and H2A.Z-nucleosome core particles.DOI:http://dx.doi.org/10.7554/eLife.06845.004
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fig1s1: SWR1 binding to nucleosome core particles containing H2A or H2A.Z histone.EMSA shows SWR1 binding to Alexa 647-labeled H2A- and H2A.Z-nucleosome core particles (1 nM). Free and bound complexes are resolved on 1.3% agarose gel. Bottom: binding curves for H2A- and H2A.Z-nucleosome core particles.DOI:http://dx.doi.org/10.7554/eLife.06845.004

Mentions: To identify elements of histone H2A on the canonical nucleosome that activate SWR1, we constructed hybrids in which H2A segments were systematically interchanged with segments of histone H2A.Z. Hybrid nucleosome substrates were reconstituted and analyzed by a SWR1-mediated histone H2A.Z replacement assay. Interchanging the M6 and α-C regions of H2A with H2A.Z on the nucleosome had only a small effect on SWR1 activity as measured by nucleosomal incorporation of H2A.Z-3xFlag, which generates a native gel mobility upshift (Figure 1A). Strikingly, an additional interchange of the M4 domain of nucleosomal H2A with H2A.Z caused a ∼90% decrease in H2A.Z replacement by SWR1, and extension of the interchange to M2 and M3 domains further reduced SWR1's activity (Figure 1A). Interchanging the M4 domain alone caused a large reduction in activity of SWR1, and activity was also reduced by interchange of M5 and M3A domains individually, whereas M2 and M3B domain interchanges had minimal effects (Figure 1B,C). Thus, residues contained entirely within the H2A histone-fold motif (the α1 helix, α2 helix, and loop 2) contribute to activation of SWR1. Underlying mechanism(s) could include improved enzyme binding, as the non-activating H2A.Z-nucleosome shows slightly decreased affinity for SWR1 (Figure 1—figure supplement 1), but activation at a post-recruitment step is required, because neither ATPase stimulation nor histone replacement occurs under saturating conditions ([Luk et al., 2010] and data not shown). Interchange of the M4 region of H2A for H2A.Z (substitution of five residues) in yeast causes lethality (Figure 1—figure supplement 2A). This indicates that the N-terminal portion of the H2A α2 helix provides an essential function apart from regulating SWR1 activity, which itself is not essential for viability. Glycine 47 is the only surface accessible H2A-specific residue in the M4 region. Strains bearing single or double amino acid substitutions to corresponding H2A.Z residues-G47K and P49A are viable. The single P49A substitution showed no reduction (even an increase) of H2A.Z levels at gene promoters by ChIP-PCR. However, the G47K interchange in the M4 region shows reduced H2A.Z incorporation (average 63% of WT), as does the double-substitution G47K, P49A (average 54% of WT), suggesting that G47 facilitates activation of SWR1 in vivo (Figure 1—figure supplement 2B).10.7554/eLife.06845.003Figure 1.H2A histone regions in the canonical nucleosome that activate H2A.Z replacement by SWR1.


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 particles containing H2A or H2A.Z histone.EMSA shows SWR1 binding to Alexa 647-labeled H2A- and H2A.Z-nucleosome core particles (1 nM). Free and bound complexes are resolved on 1.3% agarose gel. Bottom: binding curves for H2A- and H2A.Z-nucleosome core particles.DOI:http://dx.doi.org/10.7554/eLife.06845.004
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fig1s1: SWR1 binding to nucleosome core particles containing H2A or H2A.Z histone.EMSA shows SWR1 binding to Alexa 647-labeled H2A- and H2A.Z-nucleosome core particles (1 nM). Free and bound complexes are resolved on 1.3% agarose gel. Bottom: binding curves for H2A- and H2A.Z-nucleosome core particles.DOI:http://dx.doi.org/10.7554/eLife.06845.004
Mentions: To identify elements of histone H2A on the canonical nucleosome that activate SWR1, we constructed hybrids in which H2A segments were systematically interchanged with segments of histone H2A.Z. Hybrid nucleosome substrates were reconstituted and analyzed by a SWR1-mediated histone H2A.Z replacement assay. Interchanging the M6 and α-C regions of H2A with H2A.Z on the nucleosome had only a small effect on SWR1 activity as measured by nucleosomal incorporation of H2A.Z-3xFlag, which generates a native gel mobility upshift (Figure 1A). Strikingly, an additional interchange of the M4 domain of nucleosomal H2A with H2A.Z caused a ∼90% decrease in H2A.Z replacement by SWR1, and extension of the interchange to M2 and M3 domains further reduced SWR1's activity (Figure 1A). Interchanging the M4 domain alone caused a large reduction in activity of SWR1, and activity was also reduced by interchange of M5 and M3A domains individually, whereas M2 and M3B domain interchanges had minimal effects (Figure 1B,C). Thus, residues contained entirely within the H2A histone-fold motif (the α1 helix, α2 helix, and loop 2) contribute to activation of SWR1. Underlying mechanism(s) could include improved enzyme binding, as the non-activating H2A.Z-nucleosome shows slightly decreased affinity for SWR1 (Figure 1—figure supplement 1), but activation at a post-recruitment step is required, because neither ATPase stimulation nor histone replacement occurs under saturating conditions ([Luk et al., 2010] and data not shown). Interchange of the M4 region of H2A for H2A.Z (substitution of five residues) in yeast causes lethality (Figure 1—figure supplement 2A). This indicates that the N-terminal portion of the H2A α2 helix provides an essential function apart from regulating SWR1 activity, which itself is not essential for viability. Glycine 47 is the only surface accessible H2A-specific residue in the M4 region. Strains bearing single or double amino acid substitutions to corresponding H2A.Z residues-G47K and P49A are viable. The single P49A substitution showed no reduction (even an increase) of H2A.Z levels at gene promoters by ChIP-PCR. However, the G47K interchange in the M4 region shows reduced H2A.Z incorporation (average 63% of WT), as does the double-substitution G47K, P49A (average 54% of WT), suggesting that G47 facilitates activation of SWR1 in vivo (Figure 1—figure supplement 2B).10.7554/eLife.06845.003Figure 1.H2A histone regions in the canonical nucleosome that activate H2A.Z replacement by SWR1.

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.