Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Subnucleosome preference of human chromatin remodeller SMARCAD1

Nature volume 644, pages 818–826 (2025)Cite this article

Subjects

Abstract

Chromatin remodellers are pivotal in the regulation of nucleosome dynamics in cells, and they are important for chromatin packaging, transcription, replication and DNA repair1. Here we show that the human chromatin remodeller SMARCAD1 exhibits a substrate preference for subnucleosomal particles over the canonical nucleosome. Cryo-electron microscopy structures of SMARCAD1 bound to the nucleosome and hexasome provide mechanistic insights into the substrate selectivity. SMARCAD1 binds to the hexasome through multiple family-specific elements that are essential for the functions in vitro and in cells. The enzyme binds to the canonical nucleosome in an inactive conformation, which accounts for its diminished activity towards the nucleosome. Notably, the histone chaperone FACT complex acts synergistically with H2A–H2B to promote the activity of SMARCAD1 in nucleosome remodelling. Together, our findings reveal an avenue for chromatin regulation, whereby subnucleosomes are remodelled through an ATP-dependent process.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: SMARCAD1 shows preference for subnucleosomes over the canonical nucleosome in ATPase and chromatin-remodelling activities.
Fig. 2: Cryo-EM structure of SMARCAD1 bound to the hexasome.
Fig. 3: Hexasome binding mode of SMARCAD1 motor.
Fig. 4: Structures of SMARCAD1 bound to the nucleosome.
Fig. 5: Mechanism of action of SMARCAD1.

Similar content being viewed by others

Data availability

Coordinates and EM maps have been deposited in the EMData Resource and PDB under accession codes EMD-61298 (SMARCAD1-NCP, apo), EMD-61297 (SMARCAD1-NCP, ADP-BeFx), EMD-61296 (SMARCAD1-hexa, ADP-BeFx), with related PBD 9JAO and EMD-61295 (SMARCAD1 motor). Source data are provided with this paper.

References

  1. Clapier, C. R. & Cairns, B. R. The biology of chromatin remodeling complexes. Annu. Rev. Biochem. 78, 273–304 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251–260 (1997).

    Article  CAS  PubMed  Google Scholar 

  3. Smith, S. & Stillman, B. Stepwise assembly of chromatin during DNA-replication in vitro. EMBO J. 10, 971–980 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Luger, K., Dechassa, M. L. & Tremethick, D. J. New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat. Rev. Mol. Cell Biol. 13, 436–447 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Serra-Cardona, A. & Zhang, Z. Replication-coupled nucleosome assembly in the passage of epigenetic information and cell identity. Trends Biochem. Sci. 43, 136–148 (2018).

    Article  CAS  PubMed  Google Scholar 

  6. Liu, Y. et al. FACT caught in the act of manipulating the nucleosome. Nature 577, 426–431 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Li, N. N. et al. Parental histone transfer caught at the replication fork. Nature 627, 890–897 (2024).

    Article  CAS  PubMed  Google Scholar 

  8. Ramachandran, S., Ahmad, K. & Henikoff, S. Transcription and remodeling produce asymmetrically unwrapped nucleosomal intermediates. Mol. Cell 68, 1038–1053 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ehara, H., Kujirai, T., Shirouzu, M., Kurumizaka, H. & Sekine, S. Structural basis of nucleosome disassembly and reassembly by RNAPII elongation complex with FACT. Science 377, eabp9466 (2022).

    Article  CAS  PubMed  Google Scholar 

  10. Rhee, H. S., Bataille, A. R., Zhang, L. Y. & Pugh, B. F. Subnucleosomal structures and nucleosome asymmetry across a genome. Cell 159, 1377–1388 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hsieh, L. J. et al. A hexasome is the preferred substrate for the INO80 chromatin remodeling complex, allowing versatility of function. Mol. Cell 82, 2098–2112 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Clapier, C. R., Iwasa, J., Cairns, B. R. & Peterson, C. L. Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes. Nat. Rev. Mol. Cell Biol. 18, 407–422 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yan, L. & Chen, Z. A unifying mechanism of DNA translocation underlying chromatin remodeling. Trends Biochem. Sci. 45, 217–227 (2020).

    Article  CAS  PubMed  Google Scholar 

  14. Flaus, A., Martin, D. M., Barton, G. J. & Owen-Hughes, T. Identification of multiple distinct Snf2 subfamilies with conserved structural motifs. Nucleic Acids Res. 34, 2887–2905 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bantele, S. C. S. & Pfander, B. Nucleosome remodeling by Fun30 in the DNA damage response. Front. Mol. Biosci. 6, 78 (2019).

  16. Neves-Costa, A., Will, W. R., Vetter, A. T., Miller, J. R. & Varga-Weisz, P. The SNF2-family member Fun30 promotes gene silencing in heterochromatic loci. PLoS ONE 4, e8111 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Stralfors, A., Walfridsson, J., Bhuiyan, H. & Ekwall, K. The FUN30 chromatin remodeler, Fft3, protects centromeric and subtelomeric domains from euchromatin formation. PLoS Genet. 7, e1001334 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Rowbotham, S. P. et al. Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1. Mol. Cell 42, 285–296 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Sachs, P. et al. SMARCAD1 ATPase activity is required to silence endogenous retroviruses in embryonic stem cells. Nat. Commun. 10, 1335 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Taneja, N. et al. SNF2 family protein Fft3 suppresses nucleosome turnover to promote epigenetic inheritance and proper replication. Mol. Cell 66, 50–62 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lee, J. et al. Chromatin remodeller Fun30Fft3 induces nucleosome disassembly to facilitate RNA polymerase II elongation. Nat. Commun. 8, 14527 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hong, F. et al. Dissecting early differentially expressed genes in a mixture of differentiating embryonic stem cells. PLoS Comput. Biol. 5, e1000607 (2009).

    Article  MathSciNet  PubMed  PubMed Central  Google Scholar 

  23. Chen, X. F. et al. The Fun30 nucleosome remodeller promotes resection of DNA double-strand break ends. Nature 489, 576–580 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Costelloe, T. et al. The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection. Nature 489, 581–584 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Densham, R. M. et al. Human BRCA1-BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection. Nat. Struct. Mol. Biol. 23, 647–655 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lo, C. S. Y. et al. SMARCAD1-mediated active replication fork stability maintains genome integrity. Sci. Adv. 7, eabe7804 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Awad, S., Ryan, D., Prochasson, P., Owen-Hughes, T. & Hassan, A. H. The Snf2 homolog Fun30 acts as a homodimeric ATP-dependent chromatin-remodeling enzyme. J. Biol. Chem. 285, 9477–9484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Karl, L. A. et al. A SAM-key domain required for enzymatic activity of the Fun30 nucleosome remodeler. Life Sci. Alliance 6, e202201790 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Markert, J., Zhou, K. D. & Luger, K. SMARCAD1 is an ATP-dependent histone octamer exchange factor with de novo nucleosome assembly activity. Sci. Adv. 7, eabk2380 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Aboulache, B. L., Hoitsma, N. M. & Luger, K. Phosphorylation regulates the chromatin remodeler SMARCAD1 in nucleosome binding, ATP hydrolysis, and histone exchange. J. Biol. Chem. 300, 107893 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lim, M. et al. A ubiquitin-binding domain that binds a structural fold distinct from that of ubiquitin. Structure 27, 1316–1325 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhang, M. et al. Hexasome-INO80 complex reveals structural basis of noncanonical nucleosome remodeling. Science 381, 313–319 (2023).

    Article  CAS  PubMed  Google Scholar 

  33. Levendosky, R. F., Sabantsev, A., Deindl, S. & Bowman, G. D. The Chd1 chromatin remodeler shifts hexasomes unidirectionally. eLife 5, e21356 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Yuan, J. J., Chen, K. J., Zhang, W. B. & Chen, Z. C. Structure of human chromatin-remodelling PBAF complex bound to a nucleosome. Nature 605, 166–171 (2022).

    Article  CAS  PubMed  Google Scholar 

  35. Nodelman, I. M. et al. Nucleosome recognition and DNA distortion by the Chd1 remodeler in a nucleotide-free state. Nat. Struct. Mol. Biol. 29, 121–129 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Li, L. F. et al. Structure of the ISW1a complex bound to the dinucleosome. Nat. Struct. Mol. Biol. 31, 266–274 (2024).

    Article  CAS  PubMed  Google Scholar 

  37. Wang, L., Chen, K. J. & Chen, Z. C. Structural basis of ALC1/CHD1L autoinhibition and the mechanism of activation by the nucleosome. Nat. Commun. 12, 4057 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wu, H. et al. Reorientation of INO80 on hexasomes reveals basis for mechanistic versatility. Science 381, 319–324 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Li, M. J. et al. Mechanism of DNA translocation underlying chromatin remodelling by Snf2. Nature 567, 409–413 (2019).

    Article  CAS  PubMed  Google Scholar 

  40. Liu, X. Y., Li, M. J., Xia, X., Li, X. M. & Chen, Z. C. Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure. Nature 544, 440–445 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Flaus, A. & Owen-Hughes, T. Mechanisms for ATP-dependent chromatin remodelling: the means to the end. FEBS J. 278, 3579–3595 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Yan, L. J., Wang, L., Tian, Y. Y., Xia, X. & Chen, Z. C. Structure and regulation of the chromatin remodeller ISWI. Nature 540, 466–469 (2016).

    Article  CAS  PubMed  Google Scholar 

  43. Xia, X., Liu, X. Y., Li, T., Fang, X. Y. & Chen, Z. C. Structure of chromatin remodeler Swi2/Snf2 in the resting state. Nat. Struct. Mol. Biol. 23, 722–729 (2016).

    Article  CAS  PubMed  Google Scholar 

  44. Clapier, C. R., Verma, N., Pamell, T. J. & Cairns, B. R. Cancer-associated gain-of-function mutations activate a SWI/SNF-family regulatory hub. Mol. Cell 80, 712–725 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Nousbeck, J. et al. Mutations in SMARCAD1 cause autosomal dominant adermatoglyphia and perturb the expression of epidermal differentiation-associated genes. Br. J. Dermatol. 171, 1521–1524 (2014).

    Article  CAS  PubMed  Google Scholar 

  46. Marks, K. C., Banks, W. R., Cunningham, D., Witman, P. M. & Herman, G. E. Analysis of two candidate genes for Basan syndrome. Am. J. Med. Genet. A 164, 1188–1191 (2014).

    Article  CAS  Google Scholar 

  47. Valentin, M. N., Solomon, B. D., Richard, G., Ferreira, C. R. & Kirkorian, A. Y. Basan gets a new fingerprint: mutations in the skin-specific isoform of SMARCAD1 cause ectodermal dysplasia syndromes with adermatoglyphia. Am. J. Med. Genet. A 176, 2451–2455 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Belotserkovskaya, R. et al. FACT facilitates transcription-dependent nucleosome alteration. Science 301, 1090–1093 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Kemble, D. J., McCullough, L. L., Whitby, F. G., Formosa, T. & Hill, C. P. FACT disrupts nucleosome structure by binding H2A-H2B with conserved peptide motifs. Mol. Cell 60, 294–306 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Chen, P. et al. Functions of FACT in breaking the nucleosome and maintaining its integrity at the single-nucleosome level. Mol. Cell 71, 284–293 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Belikov, S., Åstrand, C., Holmqvist, P. H. & Wrange, Ö. Chromatin-mediated restriction of nuclear factor 1/CTF binding in a repressed and hormone-activated promoter in vivo. Mol. Cell. Biol. 24, 3036–3047 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Belikov, S., Gelius, B., Almouzni, G. & Wrange, Ö. Hormone activation induces nucleosome positioning. EMBO J. 19, 1023–1033 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Henikoff, J. G., Belsky, J. A., Krassovsky, K., MacAlpine, D. M. & Henikoff, S. Epigenome characterization at single base-pair resolution. Proc. Natl Acad. Sci. USA 108, 18318–18323 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Adkins, N. L. et al. Nucleosome-like, single-stranded DNA (ssDNA)-histone octamer complexes and the implication for DNA double strand break repair. J. Biol. Chem. 292, 5271–5281 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Navarro, C., Lyu, J., Katsori, A. M., Caridha, R. & Elsasser, S. J. An embryonic stem cell-specific heterochromatin state promotes core histone exchange in the absence of DNA accessibility. Nat. Commun. 11, 5095 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Xu, M. et al. Partitioning of histone H3-H4 tetramers during DNA replication-dependent chromatin assembly. Science 328, 94–98 (2010).

    Article  CAS  PubMed  Google Scholar 

  57. Iyengar, S. & Farnham, P. J. KAP1 protein: an enigmatic master regulator of the genome. J. Biol. Chem. 286, 26267–26276 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Yu, J. et al. A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance. Cell 187, 5010–5028 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Wang, N. et al. Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates. Cell 184, 370–383 (2021).

  63. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the staff at the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing), Beijing Frontier Research Center for Biological Structure and Tsinghua–Peking Joint Center for Life Sciences for support from the cryo-EM facility and the computational facility support on the cluster of Bio-Computing Platform. This work was supported by the National Key Research and Development Program (2022YFA1302700 to Z.C.), the National Natural Science Foundation of China (32130016 and 31825016 to Z.C.; W2442015 to Y.S.). Q.X. is supported by the National Key R&D Program of China (2022YFA1302704) and the National Natural Science Foundation of China (32370753); and H.S. by the Postdoctoral Fellowship Program of CPSF (GZC20231333).

Author information

Author notes

  1. These authors contributed equally: Pengjing Hu, Jingxi Sun, Hongyao Sun, Kangjing Chen

Authors and Affiliations

  1. MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing, P.R. China

    Pengjing Hu, Jingxi Sun, Hongyao Sun, Kangjing Chen, Youyang Sia, Qiaoran Xi & Zhucheng Chen

  2. Tsinghua-Peking Joint Center for Life Sciences, Beijing, P.R. China

    Pengjing Hu, Jingxi Sun, Kangjing Chen, Youyang Sia & Zhucheng Chen

  3. Beijing Frontier Research Center for Biological Structure, Beijing, P.R. China

    Pengjing Hu, Jingxi Sun, Kangjing Chen, Youyang Sia & Zhucheng Chen

  4. State Key Laboratory of Molecular Oncology, Beijing, P.R. China

    Hongyao Sun & Qiaoran Xi

  5. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA

    Xian Xia

Authors
  1. Pengjing Hu
    View author publications

    Search author on:PubMed Google Scholar

  2. Jingxi Sun
    View author publications

    Search author on:PubMed Google Scholar

  3. Hongyao Sun
    View author publications

    Search author on:PubMed Google Scholar

  4. Kangjing Chen
    View author publications

    Search author on:PubMed Google Scholar

  5. Youyang Sia
    View author publications

    Search author on:PubMed Google Scholar

  6. Xian Xia
    View author publications

    Search author on:PubMed Google Scholar

  7. Qiaoran Xi
    View author publications

    Search author on:PubMed Google Scholar

  8. Zhucheng Chen
    View author publications

    Search author on:PubMed Google Scholar

Contributions

P.H. and J.S. prepared the samples and performed the biochemical analysis. K.C. performed the EM analysis. H.S. and Q.X. conducted the mES cell analyses. Y.S. and X.X. performed the initial studies. Z.C. wrote the manuscript with help from all of the authors. Z.C. directed and supervised all of the research.

Corresponding authors

Correspondence to Qiaoran Xi or Zhucheng Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Blaine Bartholomew, Hitoshi Kurumizaka and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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 Additional biochemical analyses of SMARCAD1.

(a) Representative native gels of the tetrasome, hexasome and nucleosome assembled with the “601” sequence, and SDS-PAGE gels of various proteins used. Similar results were obtained in more than three independent experiments. (b) Multi-angle light scattering analysis of full-length SMARCAD1. The estimated molecular weight is ~113 kDa and the theoretical molecular weight is 119.7 kDa. (c) Mobility of full-length SMARCAD1 after dephosphorylation by CIP in 6% SDS-PAGE. The protein is detected by His-tag antibody (Abclonal). RT, incubation at room temperature. Similar results were obtained in three independent experiments. (d) ATPase activities of the full length SMARCAD1 with and without CIP treatment. P, phosphorylated SMARCAD1; dP, dephosphorylated SMARCAD1. Data are presented as mean values +/- SDs from three independent experiments. (e) ATPase activities of SMARCAD1 motor expressed in HEK293F and E.coli. Data are presented as mean values +/- SDs from three independent experiments. (f) Representative gels of tetrasome remodelling by the SMARCAD1 motor. Quantification of the cut fraction is shown at the right. Data are presented as mean values +/- SDs from three independent experiments. (g) Representative gels of nucleosome and hexasome remodelling by the full-length SMARCAD1. Quantification of the cut fractions is shown at the right. Data are presented as mean values +/- SDs from three independent experiments. (h) Representative gels of the remodelling activity towards the 100N100 nucleosome by Snf2 and SMARCAD1. Quantifications of the cut fractions are shown. Data are presented as mean values +/- SDs from three independent experiments. (i) Representative gels of the sliding activity towards the 20N40 nucleosome (left) and 20H40 hexasome (right) by full-length SMARCAD1. Quantifications of the remaining substrate are shown. Data are presented as mean values +/- SDs from three independent experiments. (j) Representative gels of the dimer exchange activity towards the 20N40 nucleosome (up) and 20H40 hexasome (bottom) by full-length SMARCAD1. Quantifications of the exchanged product are shown. Data are presented as mean values +/- SDs from three independent experiments.

Source Data

Extended Data Fig. 2 Cryo-EM analysis of the SMARCAD1-hexasome complex in ADP.BeFx – bound state.

(a-c) Representative cryo-EM micrographs collected from three independent samples. Scale bar: 50 nm. (d) Flowchart of the cryo-EM data processing. (e) 2D class averages of characteristic projection views of cryo-EM particles. (f) Angular distributions of the cryo-EM particles in the final round of refinement. (g) Resolution estimation of the EM maps. Gold standard Fourier shell correlation (FSC) curves, showing the overall nominal resolutions of 3.1 Å for the overall complex. (h) Local density maps of the motor in SMARCAD1-Hexasome complex. (i) Local density maps of the hexasome. (j) Local density maps of the H4 tail bound by SMARCAD1.

Extended Data Fig. 3 Comparison of the SMARCAD1, Snf2 and Ino80 motors.

(a) Structural comparison of the motor domains of SMARCAD1 (coloured coded) and Snf2 (grey, PBD code 5Z3U) in ADP.BeFX–bound state39. ADP.BeFX is shown as spheres. The motors domains are aligned. (b) Comparison of the different DNA-binding elements of SMARCAD1 (colour coded) and Snf2 (coloured grey). The structures of the motor domains are aligned. (c) Multiple sequence alignments of SMARCAD1 proteins and ScSnf2. The helixes (boxes) and sheets (arrows) of SMARCAD1 are coloured according to Fig. 1a. The helicase motifs are highlighted at the bottom of corresponding residues. The point mutants used in this study are highlighted with dots in magenta (NPD mutants), grey (DNA-binding mutants), orange (H4-binding mutants) and blue (V473D). (d) Structural comparison of the motor domains of SMARCAD1 (coloured coded) and Ino80 (grey, PBD code 8OO7)32. The motors domains are aligned. (e) Structure of Snf2 bound the H4 tail (PDB code 5Z3U)39. (f) Comparison of the structures of SMARCAD1 (colour coded) and Snf2 (coloured grey) around the regulatory hub region. The motor domains are aligned.

Extended Data Fig. 4 Supplemental data of the SMARCAD1 mutants.

(a) The ATPase activities of WT and DNA-binding mutants of SMARCAD1 in the presence of free DNA, nucleosome ad hexasome. 3 M, S609K/I639K/C951W. Data are presented as mean values +/- SDs from three independent experiments. The activities in the presence of free DNA and nucleosome are enlarged to show in the inset. (b-c) Representative gels of nucleosome (b) and hexasome (c) remodelling by the WT and DNA-binding mutants. (d) Quantification of the cut fraction in chromatin remodelling by WT and DNA-binding mutants. Data are presented as mean values +/- SDs from three independent experiments. (e) Representative gels of hexasome remodelling by the WT and H4-binding mutants. (f) Quantification of the cut fraction in chromatin remodelling. Data are presented as mean values +/- SDs from three independent experiments. (g) The ATPase activities of WT and NPD mutant SMARCAD1. Data are presented as mean values +/- SDs from three independent experiments. (h-i) Representative gels of nucleosome (h) and nucleosome intermediate (i) remodelling by the WT and NPD mutants. (j) Quantification of the cut fraction in chromatin remodelling by the WT and of NPD mutants. Data are presented as mean values +/- SDs from three independent experiments. (k) The ATPase activities of WT and CTH-binding mutants SMARCAD1. Data are presented as mean values +/- SDs from three independent experiments. (l-m) Representative gels of nucleosome (l) and nucleosome intermediate (m) remodelling by the WT and CTH mutants. (n) Quantification of the cut fraction in chromatin remodelling by the WT and of CTH mutants. Data are presented as mean values +/- SDs from three independent experiments.

Source Data

Extended Data Fig. 5 Analyses of the activities of SMARCAD1 in mESCs.

(a) Western blot assays to examine the expression profiles of both endogenous and green fluorescent protein (GFP)-tagged forms of SMARCAD1, encompassing wild-type (WT), D821K mutant, and ΔCTH truncation variants in mESCs. (b) Quantification of the relative expression levels of Smarcad1, along with two heterochromatin marker genes, in the indicated conditions. Specifically, comparisons were made between control cells (Con) and cells with SMARCAD1 KD and subsequently rescued with either an empty vector (KD + EV), wild-type SMARCAD1 (KD + WT), the D821K mutant (KD + D821K), or the ΔCTH truncation mutant (KD + ΔCTH). Data presented as mean ± SD of three independent experiments, statistical significance was determined by a two-tailed unpaired Student’s t-test, ****p < 0.0001, ***p < 0.001. Experiments were repeated three times independently with similar results (c) Representative images of the morphology and AP staining of mESCs under indicated conditions. The boxed regions are showed in Fig. 3j. Scale bar: 50 μm. Similar results were obtained in two independent experiments. (d) Co-IP of endogenous SSRP1 and SMARCAD1 from mESCs. Similar results were obtained in three independent experiments. (e) Co-IP of GFP-tagged WT and two FACT-binding mutant SMARCAD1 by SSRP1 from mESCs. Endogenous SMARCAD1 is knockdown (KD) as described above. Similar results were obtained in three independent experiments.

Source Data

Extended Data Fig. 6 Additional analyses of substrate preferences by SMARCAD1 and Snf2.

(a) Representative EMSA gels of SMARCAD1 bound to the 20N40 nucleosome and 20H40 hexasome in the presence of ADP-BeFx (top) and in the Apo state (bottom). (b) Quantifications of the bound factions of SMARCAD1 to nucleosome (NCP) and hexasome (Hex) under different nucleotide conditions. The disassociation constants (Kd, nM) estimated from curve fitting are shown at the bottom. Data are presented as mean values +/- SDs from three independent experiments. (c) The DNA sequences and the oligoes used to generate the gapped hexasome. Boxed regions, the 601 sequence. (d) Representative gels of gapped hexasome sliding by Snf2.

Source Data

Extended Data Fig. 7 Cryo-EM analysis of the SMARCAD1-nucleosome complex in ADP.BeFx – bound state.

(a) Representative cryo-EM micrograph among 11,129 images collected from one sample. Scale bar: 50 nm. (b) 2D class averages of characteristic projection views of cryo-EM particles. (The diameter of the circular mask: 28 nm). (c) Flowchart of the cryo-EM data processing. (d) Angular distributions of the cryo-EM particles in the final round of refinement. (e) Cryo-EM density map of the SMARCAD1-nucleosome complex coloured on the basis of Local Resolution Estimation in cryosparc. (f) Resolution estimation of the EM map. Gold standard Fourier shell correlation (FSC) curves.

Extended Data Fig. 8 Cryo-EM analysis of the SMARCAD1-nucleosome complex in Apo state.

(a) A representative cryo-EM micrograph from 11,451 micrographs collected from one sample. Scale bar: 50 nm. (b) 2D class averages of characteristic projection views of cryo-EM particles. (c) Flowchart of the cryo-EM data processing. (d) Angular distributions of the cryo-EM particles in the final round of refinement. (e) Cryo-EM density map of the SMARCAD1-nucleosome complex coloured on the basis of the Local Resolution Estimation in cryosparc. The boxed regions are enlarged to show in (g). (f) Resolution estimation of the EM map. Gold standard Fourier shell correlation (FSC) curves, showing the overall nominal resolutions of 3.1 Å for the overall complex. (g) Local density map of the DNA around SHL2 of nucleosome. The structure of the canonical “601” nucleosome (PBD code 3MVD) was fit into map.

Extended Data Fig. 9 SMARCAD1-FACT cooperation in chromatin remodelling.

(a) AlphaFold prediction of the SMARCAD1-SSRP1 interaction. The electrostatics of the CUE2 domain is shown in the middle; the PAE values of prediction are shown on the right. (b) Superposition of the structures of SMARCAD1 and FACT bound the hexasome (PBD code 6UPK)6. The structures of the hexasome are aligned. (c) Representative gels of nucleosome sliding by full-length SMARCAD1 and FACT-H2A-H2B (FAB). The specific proteins used are indicated on the top. (d) The ATPase activity towards the canonical nucleosome by WT SMARCAD1 alone, FACT-H2A-H2B (FAB), and in combination (WT + FAB). Data are presented as mean values +/- SDs from three independent experiments. (e) Activation of the SMARCAD1 remodelling activity towards the nucleosome by FACT-H2A-H2B in a dosage-dependent manner. A fixed concentration of WT SMARCAD1 (100 nM) was used, with increased concentrations of FACT-H2A-H2B (FAB), FACT, H2A-H2B added to the nucleosome sliding assays in vitro. Representative gels are showed under indicated conditions. Right panel: quantification of the nucleosome sliding activities. Data are presented as mean values +/- SDs from three independent experiments. (f) Representative gels of hexasome remodelling by the full-length WT and mutant SMARCAD1 (100 nM). Right pane, observed initial restriction site accessibility rates (KOBS) from quantification of the cut fraction. Data are presented as mean values +/- SDs from three independent experiments.

Source Data

Extended Data Fig. 10 Structural comparison of SMARCAD1 bound to the hexasome and nucleosome.

The structures of the histone octamer are aligned. The boxed region is enlarged to show at the bottom.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Information

The uncropped gels for Figs. 1, 4 and 5, and Extended Data Figs. 1, 4–6 and 9.

Reporting Summary

Peer Review File

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hu, P., Sun, J., Sun, H. et al. Subnucleosome preference of human chromatin remodeller SMARCAD1. Nature 644, 818–826 (2025). https://doi.org/10.1038/s41586-025-09100-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41586-025-09100-0

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing