FACT caught in the act of manipulating the nucleosome

Abstract

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone known as ‘facilitates chromatin transcription’ (FACT1) (consisting of subunits SPT16 and SSRP1) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication and DNA repair2. However, the mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-electron-microscopic structures of human FACT in complex with partially assembled subnucleosomes, with supporting biochemical and hydrogen–deuterium exchange data. We find that FACT is engaged in extensive interactions with nucleosomal DNA and all histone variants. The large DNA-binding surface on FACT appears to be protected by the carboxy-terminal domains of both of its subunits, and this inhibition is released by interaction with H2A–H2B, allowing FACT–H2A–H2B to dock onto a complex containing DNA and histones H3 and H4 (ref. 3). SPT16 binds nucleosomal DNA and tethers H2A–H2B through its carboxy-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A–H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating removal of the H2A–H2B dimer, stabilizing intermediate subnucleosomal states and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.

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Fig. 1: FACT forms two complexes with a partially assembled nucleosome.
Fig. 2: FACT makes extensive contacts with tetrasome DNA.
Fig. 3: SPT16 interactions with the subnucleosome.
Fig. 4: SSRP1 can assume two different positions on the subnucleosome, depending on the presence or absence of a second H2A–H2B dimer.

Data availability

Raw HDX-MS data are available in Supplementary Tables 2 and 3 for Figs. 2b–d, 3b, c. Atomic coordinates and cryo-EM maps have been deposited in the PDB and Electron Microscopy Data Bank (EMDB) under accession codes EMDB-20840, PDB 6UPK and EMDB-20841, PDB 6UPL. Raw cryoEM images have been deposited in the Electron Microscopy Public Image Archive (EMPIAR-10333).

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Acknowledgements

This work was supported by the Howard Hughes Medical Institute (Y.L., K.Z. and K.L.), by start-up funds (S.D.) and by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) (GM133751 to S.D.). Screening of the initial cryo-EM condition was performed at the CU Boulder Electron Microscopy Service, with help from C. Page and G. P. Morgan. Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy, located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247) and the NIH NIGMS (GM103310), with additional support from Agouron Institute (F00316) and the NIH (OD019994). We thank D. Reinberg for assistance in initial stages of the project.

Author information

Affiliations

Authors

Contributions

Y.L. cloned and expressed all FACT constructs with the help of K.Z. K.Z. and Y.L. optimized sample and grid preparation and performed extensive screening. H.W. and Z.Z. (with B.C. and C.S.P.) optimized the conditions for Chameleon/Spotiton vitrification. H.W. collected the data. Y.Z.T., H.W. and K.Z. analysed the electron-microscopy data and obtained the electron-density maps, with input from B.C. and C.S.P. K.Z. modelled and refined the structures. K.Z., Y.L. and K.L. interpreted electron-microscopy structures and drafted the manuscript. Y.L. designed and performed all biochemical assays, with input from K.Z. and K.L. N.Z. (with input from Y.L.) designed HDX experiments, with the help of S.D. and K.L. N.Z. and S.D. performed HDX experiments and analysed HDX data. Y.L., K.Z., S.D. and K.L. wrote the manuscript and prepared the figures.

Corresponding author

Correspondence to Karolin Luger.

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Competing interests

B.C. and S.C.P. have an intellectual property licensing arrangement with TTP Labtech Ltd for the Chameleon sample vitrification instrument.

Additional information

Peer review information Nature thanks Tim Formosa, Karl-Peter Hopfner, Hitoshi Kurumizaka and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Analysis of electron-microscopic data.

a, Representative micrograph of particles containing 79 bp of 601 DNA prebound to a (H3–H4)2 tetramer (‘tetrasome’), with FACT prebound to H2A–H2B. The inset is a lower-magnification view showing the holy substrate. The red X marks the location of this micrograph in the low-magnification map. The particles in the final picking are circled in green. This experiment was repeated with six independent grids in two Krios sessions, with similar results. b, Two-dimensional class averages generated from the dataset obtained from CryoSPARC 2.0. c, Flowchart showing the data-processing strategy.

Extended Data Fig. 2 Validation of electron-microscopic data.

a, Local-resolution map and FSC curve for class 1 (complex 1). Local resolution was calculated in CryoSPARC 2.0 and 3DFSC was calculated using the 3DFSC server. b, Local-resolution map and FSC curve for class 2 (complex 2). Local resolution was calculated as in a.

Extended Data Fig. 3 Electron density and models.

a, Density maps for class 1 and 2 particles in three orthogonal orientations. The subunits and domains are colour-coded as indicated. b, Representative views of models fit into electron-density maps for FACT–subnucleosome complex 1, DNA, H2A–H2B, (H3–H4)2, SPT16 middle domain, SSRP1 MD and SPT16–SSRP1 dimerization domains.

Extended Data Fig. 4 Coverage maps from FACT HDX experiments.

a, b, SPT16 and SSRP1 coverage maps from HDX experiments. Each rectangle represents one peptide, repeatably monitored through all time points and samples. Significant peptides (P < 0.01) (Fig. 2b) are coloured and listed at the bottom of each panel. Secondary-structure elements from the structures of the published domains that were used for fitting are also shown. c, Deuterium uptake plot for SPT16 residues 1016–1030, showing FACT (black) and FACT with subnucleosomes (red). Each data point is the average of 3 replicates with error bars of ± 2 s.d.

Extended Data Fig. 5 Changes in deuterium uptake mapped onto complex 1.

Deuterium-uptake plots are included for example peptides that show a significant change (P < 0.01) (Fig. 2c, d) when comparing FACT (black) with FACT plus subnucleosomes (red). Each data point is the average of 3 replicates, with error bars of ± 2 s.d. The subnucleosome is shown in wheat. For FACT, regions with no detectable change are in white; regions with a change are coloured according to their domain location (Fig. 1a); and regions with no peptide coverage are in grey.

Extended Data Fig. 6 Effects of FACT deletion mutants on DNA binding.

a, Full-length (FL) FACT and FACT CTD deletions (SPT16ΔC–SSRP1, SPT16–SSRP1ΔC or SPT16ΔC–SSRP1ΔC) were purified from Sf9 cells and visualized on 4–12% SDS–PAGE, stained with Blazin blue. This particular gel was run only once, although proteins were purified and analysed by SDS–PAGE four times. b, As for Fig. 2e, except that full-length FACT was pre-incubated with an equimolar amount of H2A–H2B dimer at 400 nM. The pre-incubation of wild-type FACT with H2A–H2B dimer has a similar effect on DNA binding as FACT ΔCTDs. Visualized by Atto N (647). The experiment was done twice with similar results.

Extended Data Fig. 7 Coverage maps from H2A–H2B HDX experiments.

a, b, H2A and H2B coverage maps from HDX experiments. Each rectangle shows one peptide repeatably monitored through all time points and samples. Significant peptides (P < 0.01) (Fig. 3b) are coloured and listed. Secondary-structure elements are shown. c, Deuterium-uptake plots for example peptides that show a significant change (P < 0.01) (Fig. 3c) when comparing H2A–H2B with FACT (black) and H2A–H2B with FACT plus tetrasomes (red). Each data point is the average of 3 replicates with error bars of ± 2 s.d. FACT, H3–H4 and DNA are wheat. For H2A–H2B, regions with no detectable change are white; regions with a change are in yellow for H2A and red for H2B; and regions with no peptide coverage are in grey.

Extended Data Fig. 8 Interactions between H3–H4 and the SPT16 middle domain are incompatible with FACT interactions made with the subnucleosome.

a, Domain structure of SPT16, and cartoon showing the overall architecture. b, Interactions between H3–H4 and the SPT16 MD (PDB code 4Z2M) are incompatible with FACT interactions made with the subnucleosome. These structures are for complex 1, which shows only the SPT16 MD. Two sites of interaction with the subnucleosome are outlined (interface 1, near the H2A-docking domain and nearby DNA; interface 2, near the H4 N-tail and DNA). c, The presence of DNA and the H2A–H2B dimer completely occludes interactions between the SPT16 MD and the (H3–H4)2 tetramer in the 4Z2M structure (clashes 1 and 2, indicated by red arrows).

Extended Data Fig. 9 Model-to-map fitting for the class 2 map of the SSRP1 MD.

a, The class 1 model of the SSRP1 MD (red ribbon) does not fit into the class 2 map (grey). The contour level of the map is 0.153. Left, the entire map. The orange oval highlights how the class 1 model overlaps with the density assigned to H2A–H2B. Right, close-up of the fit between the class 1 model and class 2 density for the SSRP1 MD. Arrows highlight problematic fits; a substantial part of the PH2 domain is outside of the density, and other parts of the density are empty. The correlation between model 1 (from class 1) and map 2 for the entire SSRP1 MD is 0.84 (calculated using UCSF Chimera). b, The class 2 model of the SSRP1 MD (purple ribbon) fits into the class 2 density map (grey). The contour level of the map is 0.153. Left, the entire map; no clashes are identified. Right, close-up of the fit between the model and density for the SSRP1 MD. Arrows indicate the same parts of the model or map as in a. The correlation between model 2 (from class 2) and map 2 for the entire SSRP1 MD is 0.93 (calculated using UCSF Chimera). c, Local-resolution heat map for the class 2 map. The resolution of the highlighted region (the PH2 domain from the SSRP1 MD at interface 2) is between 9 Å and 10 Å. The contour level is 0.177. d, Relative movement of the highlighted region (the PH2 domain of the SSRP1 MD at interface 2) between class 1 and 2 models. The distances or movements of the same amino acids in the two models (dotted ovals in c, d) are indicated.

Extended Data Table 1 Summary of cryoEM data collection and refinement

Supplementary information

Supplementary Figure

This file contains the uncropped gels used in Fig. 1b, Fig. 2e and Extended Data Fig. 6a,b

Reporting Summary

Supplementary Table 1 |Fluorescence polarization raw data (four replicates)

Supplementary Table 2 | HDX summary of details and statistics of HDX experiments with FACT, FACT with H2A-H2B, and FACT with sub-nucleosomes

Supplementary Table 3 | HDX raw data and statistics of HDX uptake for all peptides overtime in all conditions

Supplementary Video 1 | Overview of FACT - sub-nucleosome structures

Supplementary Video 2 | Morphing between complex 1 and complex 2

The two structures were superimposed by aligning the tetrasome. Morphing was accomplished by linear interpolation in Chimera26

Supplementary Video 3 | A hypothetical model of how SPT16 (MD and CTD) promotes the disassociation of histones from DNA while maintaining the integrity of the sub-nucleosome

Only MD and CTD of SPT16 are shown, the other visible parts of FACT were omitted for clarity. The removal of the DNA from the histones is ‘fictional’; the transition after DNA peeling was obtained by linear interpolation between the structures in this study and PDB code: 4z2m

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Liu, Y., Zhou, K., Zhang, N. et al. FACT caught in the act of manipulating the nucleosome. Nature 577, 426–431 (2020). https://doi.org/10.1038/s41586-019-1820-0

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