Krajewski WA et al. (2018), Effects of histone H2B ubiquitylation on the nu...

XB-ART-56241

Nucleic Acids Res 2018 Sep 06;4615:7631-7642. doi: 10.1093/nar/gky526.

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Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics.

Krajewski WA , Li J , Dou Y .

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DNA in nucleosomes has restricted nucleosome dynamics and is refractory to DNA-templated processes. Histone post-translational modifications play important roles in regulating DNA accessibility in nucleosomes. Whereas most histone modifications function either by mitigating the electrostatic shielding of histone tails or by recruiting 'reader' proteins, we show that ubiquitylation of H2B K34, which is located in a tight space protected by two coils of DNA superhelix, is able to directly influence the canonical nucleosome conformation via steric hindrances by ubiquitin groups. H2B K34 ubiquitylation significantly enhances nucleosome dynamics and promotes generation of hexasomes both with symmetrically or asymmetrically modified nucleosomes. Our results indicate a direct mechanism by which a histone modification regulates the chromatin structural states.

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Species referenced: Xenopus laevis
Genes referenced: h1-0 h2bc21 napsa scai tab3
GO keywords: chromatin [+]

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	Fig 1. Assembly of H2B K34ub and unmodified nucleosomes. (A) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. (B) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. (C) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. (D) Left panel: SDS-PAGE of recombinant linker histone H10. Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H10.
	Fig 2. Stability of H2B K34ub nucleosomes. (A) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table 1. The representative result from three repeats was presented. (B) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. (C) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. (D) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.
	Fig 3. Dynamics of H2B K34ub nucleosomes. (A) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. (B) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
	Fig 4. Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. (A) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. (B) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. (C) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).
	Fig 5. Stability of H2B K34ub- versus H2B K120ub-nucleosomes. (A) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. (B) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. (C) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.
	Fig 6. Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. (A) Nucleosomes assembled on 146 bp 5S DNA. (B) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. (C) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. (D and E) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.
	Figure 1. Assembly of H2B K34ub and unmodified nucleosomes. (A) Left panel: SDS-PAGE of H2B K34ub- (lane 1) and unmodified (lane 2) histone octamers. Right panel: native PAGE of modified nucleosomes assembled on 147 bp 601 DNA using increasing amounts histones. (B) SDS-PAGE of faster- (lane 1) and slower- (lane 2) migrating H2B K34ub assembly products extracted from the native gel in A. The densitometric tracing of the gel lanes is shown on the right. Quantification, by ImageJ, is normalized to that of the histone H4, which is arbitrarily set as 1. (C) Native PAGE for samples prepared by serial dilution of 2M NaCl mixtures of 147 bp 601 DNA and modified or unmodified histones to 1 M NaCl. (D) Left panel: SDS-PAGE of recombinant linker histone H10. Right panel: assembly of H2B K34ub modified and unmodified nucleosomes with increasing concentration of recombinant histone H10.
	Figure 2. Stability of H2B K34ub nucleosomes. (A) Native PAGE for nucleosome assembly with increasing amounts of unmodified and H2B K34ub histone octamers and their mixture at ratios indicated on top. Quantification was performed by Image J and relative abundance of each species was summarized in Table 1. The representative result from three repeats was presented. (B) H2B K34ub (top panel) or Unmodified (bottom panel) nucleosomes were assembled on nicked, positively or negatively supercoiled plasmids containing 12 copies of 177 bp 601 DNA. After assembly 601 nucleosomes were released with ScaI and resolved on native PAGE. (C) The nucleosome assembly containing histones of indicated ratios (top) were incubated with competitor DNA for 2 h at 26°C in a buffer containing 200 mm NaCl. (D) Nucleosomes assembled on 147 bp 601 DNA (as indicated on top) were incubated with or without CM-50 Sephadex.
	Figure 3. Dynamics of H2B K34ub nucleosomes. (A) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. (B) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
	Figure 4. Eviction of histone dimer in H2B K34ub nucleosome by histone dimer acceptors. (A) Left panel: SDS-PAGE of NAP1. Middle, right panels: unmodified and H2B K34ub nucleosomes assembled on a 147 or 177 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26 or 37°C in a buffer containing 100 mM NaCl. (B) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at 26/37°C in a buffer containing at 100 mM NaCl. (C) H2B K34ub nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA at 26/37°C for 2.5 h or at 26°C for 20 h (the ‘20 h’ and ‘2.5 h’ samples were resolved in different gels).
	Figure 5. Stability of H2B K34ub- versus H2B K120ub-nucleosomes. (A) Left panel: SDS-PAGE of H2B K120ub and K34ub histone octamers. Right panel: unmodified and H2B K120ub/ K34ub nucleosomes assembled on 147 bp 601 DNA. (B) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with competitor DNA for 2.5 h at indicated temperature/ionic conditions. (C) Nucleosomes assembled on 147 bp 601 DNA were post-assembly incubated with NAP1 for 2.5 h at 26/37°C in a buffer containing at 140 mM NaCl.
	Figure 6. Effects of underlying DNA sequence on stability of H2B K120ub and K34ub nucleosomes. (A) Nucleosomes assembled on 146 bp 5S DNA. (B) Assembled nucleosomes were incubated with competitor DNA for 2.5 h at 26°C in a buffer containing at 200 mM NaCl. (C) Nucleosomes incubated with competitor DNA for 2.5 h at indicated temperature/ ionic conditions. (D and E) Nucleosomes assembled with unmodified or H2B K34ub histones, or their 1/0.57 mixture, were post-assembly incubated for 2.5 h at 26 or 37°C with (D) competitor DNA in a buffer containing at 100 mM NaCl or (E) NAP1 in a buffer containing at 150 mM NaCl. Densitometry tracing of indicated gel lanes is shown at the bottom of gel images.

References [+] :

Belotserkovskaya, FACT facilitates transcription-dependent nucleosome alteration. 2003, Pubmed