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Histone dynamics play a critical role in SNF2h-mediated nucleosome sliding

Nature Structural & Molecular Biology volume 28, pages 548–551 (2021)Cite this article

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Matters Arising to this article was published on 05 July 2021

The Original Article was published on 14 March 2019

arising from: L. Yan et al. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-019-0199-9 (2019)

To test whether the oxidation method may affect the results in the context of the H3(C110A) sCX2 octamer, we generated H3(C110A) sCX2 crosslinked nucleosomes using Cu-Phe or oxidized glutathione (GSSG), a gentler oxidizing agent that does not generate free radicals and that has previously been used to investigate nucleosome dynamics6. Oxidation with Cu-Phe went to near-completion as assessed by a non-reducing SDS–PAGE gel, whereas GSSG oxidation was less efficient (Extended Data Fig. 1a). We then reduced a portion of each H3(C110A) sCX2 crosslinked octamer by dialysis into buffer containing 100 mM DTT (Extended Data Fig. 1b). H3(C110A) sCX2 octamers showed no visible precipitates upon oxidation or after reduction and, like cysteine-free octamers, readily assembled into nucleosome core particles that were purified by glycerol gradient ultracentrifugation. H3(C110A) sCX2 nucleosomes assembled from octamers oxidized with Cu-Phe slowed SNF2h-driven sliding by approximately 40-fold compared with reduced controls under saturating concentrations of SNF2h, whereas nucleosomes assembled from octamers oxidized by GSSG slowed sliding by around 15-fold (Fig. 1b,c). This quantitative difference can be attributed to the different crosslinking efficiencies of the two oxidation protocols and cannot be explained by nucleosome disassembly (Supplementary Figs. 1, 2). Reduction of either type of oxidized octamer fully restored SNF2h-dependent sliding of nucleosomes assembled from their respective octamers (Fig. 1b,c). To further test whether the oxidation-dependent effect on SNF2h-dependent sCX2 nucleosome remodeling is due to the formation of a disulfide bond, we also tested remodeling on nucleosomes assembled using oxidized H3(C110A) octamers containing single cysteines from the sCX2 cysteine pair that were purified by ultracentrifugation (Extended Data Fig. 2a). In this side-by-side experiment, SNF2h remodeled Cu-Phe oxidized H3(C110A) sCX2 nucleosomes around 30-fold slower than untreated H3(C110A) nucleosomes in line with our previous observations, whereas single-cysteine-containing nucleosomes were remodeled only approximately twofold slower. Oxidation-dependent remodeling defects were also observed with sub-saturating concentrations of enzyme (Extended Data Fig. 2b). These results indicate that non-specific oxidative damage to the octamer cannot explain the remodeling defects.

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Fig. 1: Site-specific cysteine crosslinking, not general oxidative damage, inhibits remodeling regardless of oxidation method.

Data availability

All relevant data are included in the paper and its Supplementary Information. Uncropped images of gels and blots are provided as source data, and numerical values plotted in the graphs are available in Supplementary Table 1. Source data are provided with this paper.

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Acknowledgements

We thank K. Sinha for help generating the disulfide-crosslinked nucleosome; J. Tretyakova for preparing histones; S. Sanulli, J. Gross, J. Lobel and Y. Cheng for feedback in preparing this manuscript; and all members of the Narlikar laboratory for discussions. This work was supported by a grant from the NIH to G.J.N. (R35GM127020) and an NSF predoctoral and UCSF discovery fellowship to N.G.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA

    Nathan Gamarra & Geeta J. Narlikar

  2. TETRAD Graduate Program, University of California, San Francisco, San Francisco, CA, USA

    Nathan Gamarra

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  1. Nathan Gamarra
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  2. Geeta J. Narlikar
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Contributions

N.G. conceived and performed the experiments, performed data analysis and wrote and edited the original draft of the manuscript. G.J.N. helped to design experiments, contributed to the writing and editing of the manuscript and supervised the overall project.

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Correspondence to Geeta J. Narlikar.

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The authors declare no competing interests.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Preparation of crosslinked and reduced histone octamers.

SDS-PAGE gels of Xenopus laevis of histones used to prepare all nucleosomes in this study. (a) Wild type (WT) Histone H2A-H2B dimer as well as H3C110A histone octamer and H3C110A sCX2 (H3 L82C, H4 V81C) histone octamer oxidized using copper phenanthroline (CuPhe) or oxidized glutathione (GSSG). (b) The same samples used in (A) treated with 100 mM DTT in order to reduce the disulfide bond. Uncropped images are available as source data files. SDS-PAGE analysis was only performed once for all samples.

Source data

Extended Data Fig. 2 Remodeling of oxidized nucleosomes is slowed specifically due to disulfide bond formation and is robust to remodeling conditions.

(a) Left. Native gel remodeling assay with saturating SNF2h (1 μM), saturating ATP, and 15 nM cy3-nucleosomes as in Fig. 1. Middle. Quantification of the experiment at the left including a plot of all time points; and for ease of comparison a plot of the first 15 minutes of the reaction normalized to the best-fit parameters for Y0 and plateau. This experiment was performed 3 times with similar results. Right. Mean observed rate constants (kobs) from 3 independent experiments. Error bars reflect the standard error of the mean (SEM). (b) Left. Native gel remodeling assay with sub-saturating SNF2h (50 nM), saturating ATP, and 15 nM cy3-nucleosomes as in Fig. 1. Middle. Quantification of the experiment on the left. This experiment was performed 3 times with similar results. Right. Mean and SEM of the observed rate constants (kobs) from 3 independent experiments. The asterisk denotes that the rate constant for the oxidized reaction condition was too slow to reliably quantify with the time points taken. (C) Left. Native gel remodeling assay under the conditions of Yan et al. 2 using 50 nM SNF2h, saturating ATP, and 15 nM cy3-nucleosomes. Remodeling overall is substantially faster likely because of the different conditions used (higher temperature, lower salt concentration, the absence of .02% (v/v) NP40, and the presence of 0.1 mg/mL BSA). Middle. Quantification of the gel on the left along with the indicated observed rate constants. This experiment was performed once. Right. Time courses shown in the middle panel normalized to the best fit parameters for Y0 and Plateau of the exponential decay and zoomed in to the first 10 minutes of the reaction to better evaluate the fits. Uncropped images for panels A-C are available in Source Data and values obtained in quantifications are in Supplementary Table 1.

Source data

Extended Data Fig. 3 Disulfide reduction is impaired in the context of the nucleosome.

(a) Native gel remodeling assay with saturating SNF2h (1 µM), saturating ATP, and 15 nM cy3-nucleosomes as in Fig. 1. Nucleosomes containing the oxidized sCX2 bonds were generated by oxidizing the H3C110A sCX2 octamer using CuPhe, and then assembling nucleosomes. Treatment of these nucleosomes with excess DTT as in Yan et al. fails to reverse the remodeling defect. (b) Scheme for the samples run in C. Nucleosomes treated with DTT were either directly added to non-reducing SDS-PAGE loading buffer or quenched with 500 mM N-Ethyl Maleimide freshly dissolved in DMSO (final [DMSO] ≈ 10%(v/v)). Additionally, a condition where N-Ethyl Maleimide and DTT were added simultaneously is included to evaluate the efficacy of the quench. (c) SDS-PAGE of samples treated as in B. Samples with reducing agent quenched prior to running on the gel are near-completely oxidized. The experiments shown here were performed once.

Source data

Supplementary information

Supplementary Information

Supplementary Methods and Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Table 1

Numerical values for all plots in the manuscript.

Source data

Source Data Fig. 1

Uncropped native page gels.

Source Data Extended Data Fig. 1

Uncropped SDS–PAGE gels.

Source Data Extended Data Fig. 2

Uncropped native page gels.

Source Data Extended Data Fig. 3

Uncropped native page and SDS–PAGE gels.

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Gamarra, N., Narlikar, G.J. Histone dynamics play a critical role in SNF2h-mediated nucleosome sliding. Nat Struct Mol Biol 28, 548–551 (2021). https://doi.org/10.1038/s41594-021-00620-7

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