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Histone H4 tail mediates allosteric regulation of nucleosome remodelling by linker DNA

Abstract

Imitation switch (ISWI)-family remodelling enzymes regulate access to genomic DNA by mobilizing nucleosomes1. These ATP-dependent chromatin remodellers promote heterochromatin formation and transcriptional silencing1 by generating regularly spaced nucleosome arrays2,3,4,5. The nucleosome-spacing activity arises from the dependence of nucleosome translocation on the length of extranucleosomal linker DNA6,7,8,9,10, but the underlying mechanism remains unclear. Here we study nucleosome remodelling by human ATP-dependent chromatin assembly and remodelling factor (ACF), an ISWI enzyme comprising a catalytic subunit, Snf2h, and an accessory subunit, Acf1 (refs 2, 11, 12, 13). We find that ACF senses linker DNA length through an interplay between its accessory and catalytic subunits mediated by the histone H4 tail of the nucleosome. Mutation of AutoN, an auto-inhibitory domain within Snf2h that bears sequence homology to the H4 tail14, abolishes the linker-length sensitivity in remodelling. Addition of exogenous H4-tail peptide or deletion of the nucleosomal H4 tail also diminishes the linker-length sensitivity. Moreover, Acf1 binds both the H4-tail peptide and DNA in an amino (N)-terminal domain dependent manner, and in the ACF-bound nucleosome, lengthening the linker DNA reduces the Acf1-H4 tail proximity. Deletion of the N-terminal portion of Acf1 (or its homologue in yeast) abolishes linker-length sensitivity in remodelling and leads to severe growth defects in vivo. Taken together, our results suggest a mechanism for nucleosome spacing where linker DNA sensing by Acf1 is allosterically transmitted to Snf2h through the H4 tail of the nucleosome. For nucleosomes with short linker DNA, Acf1 preferentially binds to the H4 tail, allowing AutoN to inhibit the ATPase activity of Snf2h. As the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail to compete AutoN off the ATPase and thereby activating ACF.

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Figure 1: The linker DNA length and histone H4 tail regulate the remodelling pause phases but not the translocation phases.
Figure 2: Deletion of the NegC domain of the Snf2h catalytic subunit does not substantially affect linker DNA length sensing by the ACF complex.
Figure 3: The AutoN domain of Snf2h and the nucleosomal H4 tail are important for linker DNA length sensing by the ACF complex.
Figure 4: The N-terminal region of the Acf1 accessory subunit is important for linker DNA length sensing by the ACF complex.

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References

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

    Article  CAS  Google Scholar 

  2. Ito, T., Bulger, M., Pazin, M. J., Kobayashi, R. & Kadonaga, J. T. ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor. Cell 90, 145–155 (1997)

    Article  CAS  Google Scholar 

  3. Varga-Weisz, P. D. et al. Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Nature 388, 598–602 (1997)

    Article  CAS  ADS  Google Scholar 

  4. Tsukiyama, T., Palmer, J., Landel, C. C., Shiloach, J. & Wu, C. Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae. Genes Dev. 13, 686–697 (1999)

    Article  CAS  Google Scholar 

  5. Langst, G., Bonte, E. J., Corona, D. F. & Becker, P. B. Nucleosome movement by CHRAC and ISWI without disruption or trans-displacement of the histone octamer. Cell 97, 843–852 (1999)

    Article  CAS  Google Scholar 

  6. Yang, J. G., Madrid, T. S., Sevastopoulos, E. & Narlikar, G. J. The chromatin-remodeling enzyme ACF is an ATP-dependent DNA length sensor that regulates nucleosome spacing. Nature Struct. Mol. Biol. 13, 1078–1083 (2006)

    Article  CAS  Google Scholar 

  7. He, X., Fan, H. Y., Narlikar, G. J. & Kingston, R. E. Human ACF1 alters the remodeling strategy of SNF2h. J. Biol. Chem. 281, 28636–28647 (2006)

    Article  CAS  Google Scholar 

  8. Dang, W., Kagalwala, M. N. & Bartholomew, B. Regulation of ISW2 by concerted action of histone H4 tail and extranucleosomal DNA. Mol. Cell. Biol. 26, 7388–7396 (2006)

    Article  CAS  Google Scholar 

  9. Stockdale, C., Flaus, A., Ferreira, H. & Owen-Hughes, T. Analysis of nucleosome repositioning by yeast ISWI and Chd1 chromatin remodeling complexes. J. Biol. Chem. 281, 16279–16288 (2006)

    Article  CAS  Google Scholar 

  10. He, X., Fan, H. Y., Garlick, J. D. & Kingston, R. E. Diverse regulation of SNF2h chromatin remodeling by noncatalytic subunits. Biochemistry 47, 7025–7033 (2008)

    Article  CAS  Google Scholar 

  11. Ito, T. et al. ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev. 13, 1529–1539 (1999)

    Article  CAS  Google Scholar 

  12. LeRoy, G., Loyola, A., Lane, W. S. & Reinberg, D. Purification and characterization of a human factor that assembles and remodels chromatin. J. Biol. Chem. 275, 14787–14790 (2000)

    Article  CAS  Google Scholar 

  13. Poot, R. A. et al. HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins. EMBO J. 19, 3377–3387 (2000)

    Article  CAS  Google Scholar 

  14. Clapier, C. R. & Cairns, B. R. Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes. Nature 492, 280–284 (2012)

    Article  CAS  ADS  Google Scholar 

  15. Killian, J. L., Li, M., Sheinin, M. Y. & Wang, M. D. Recent advances in single molecule studies of nucleosomes. Curr. Opin. Struct. Biol. 22, 80–87 (2012)

    Article  CAS  Google Scholar 

  16. Eberharter, A., Vetter, I., Ferreira, R. & Becker, P. B. ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts. EMBO J. 23, 4029–4039 (2004)

    Article  CAS  Google Scholar 

  17. Clapier, C. R., Langst, G., Corona, D. F., Becker, P. B. & Nightingale, K. P. Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI. Mol. Cell. Biol. 21, 875–883 (2001)

    Article  CAS  Google Scholar 

  18. Hamiche, A., Kang, J. G., Dennis, C., Xiao, H. & Wu, C. Histone tails modulate nucleosome mobility and regulate ATP-dependent nucleosome sliding by NURF. Proc. Natl Acad. Sci. USA 98, 14316–14321 (2001)

    Article  CAS  ADS  Google Scholar 

  19. Shogren-Knaak, M. et al. Histone H4–K16 acetylation controls chromatin structure and protein interactions. Science 311, 844–847 (2006)

    Article  CAS  ADS  Google Scholar 

  20. Ferreira, H., Flaus, A. & Owen-Hughes, T. Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms. J. Mol. Biol. 374, 563–579 (2007)

    Article  CAS  Google Scholar 

  21. Valouev, A. et al. Determinants of nucleosome organization in primary human cells. Nature 474, 516–520 (2011)

    Article  CAS  Google Scholar 

  22. Kagalwala, M. N., Glaus, B. J., Dang, W., Zofall, M. & Bartholomew, B. Topography of the ISW2-nucleosome complex: insights into nucleosome spacing and chromatin remodeling. EMBO J. 23, 2092–2104 (2004)

    Article  CAS  Google Scholar 

  23. Yamada, K. et al. Structure and mechanism of the chromatin remodelling factor ISW1a. Nature 472, 448–453 (2011)

    Article  CAS  ADS  Google Scholar 

  24. Blosser, T. R., Yang, J. G., Stone, M. D., Narlikar, G. J. & Zhuang, X. Dynamics of nucleosome remodelling by individual ACF complexes. Nature 462, 1022–1027 (2009)

    Article  CAS  ADS  Google Scholar 

  25. Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl Acad. Sci. USA 93, 6264–6268 (1996)

    Article  CAS  ADS  Google Scholar 

  26. Deindl, S. et al. ISWI remodelers slide nucleosomes with coordinated multi-base-pair entry steps and single-base-pair exit steps. Cell 152, 442–452 (2013)

    Article  CAS  Google Scholar 

  27. Mueller-Planitz, F., Klinker, H., Ludwigsen, J. & Becker, P. B. The ATPase domain of ISWI is an autonomous nucleosome remodeling machine. Nature Struct. Mol. Biol. 20, 82–89 (2013)

    Article  CAS  Google Scholar 

  28. Fyodorov, D. V. & Kadonaga, J. T. Binding of Acf1 to DNA involves a WAC motif and is important for ACF-mediated chromatin assembly. Mol. Cell. Biol. 22, 6344–6353 (2002)

    Article  CAS  Google Scholar 

  29. Gelbart, M. E., Rechsteiner, T., Richmond, T. J. & Tsukiyama, T. Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates. Mol. Cell. Biol. 21, 2098–2106 (2001)

    Article  CAS  Google Scholar 

  30. Narlikar, G. J. A proposal for kinetic proof reading by ISWI family chromatin remodeling motors. Curr. Opin. Chem. Biol. 14, 660–665 (2010)

    Article  CAS  Google Scholar 

  31. Lowary, P. T. & Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42 (1998)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  33. Luger, K., Rechsteiner, T. J. & Richmond, T. J. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999)

    Article  CAS  Google Scholar 

  34. Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods 3, 891–893 (2006)

    Article  CAS  Google Scholar 

  35. Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008)

    Article  CAS  Google Scholar 

  36. Kerssemakers, J. W. et al. Assembly dynamics of microtubules at molecular resolution. Nature 442, 709–712 (2006)

    Article  CAS  ADS  Google Scholar 

Download references

Acknowledgements

We thank G. Narlikar, T. Tsukiyama, J. Vaughan, J. Moffitt and E. Sun for discussions, S. Mukherji for assistance in designing the yeast experiments, S. Trauger, K. L. Hwang, R. Magnusson, G. Hilinski and J. McGee for assistance with protein characterization and biochemistry, and G. Narlikar for providing Snf2h and Acf1 expression vectors and purification protocols. This work was supported in part by the National Institutes of Health (GM105637 to X.Z.). W.L.H. acknowledges support from the National Institutes of Health T32G007753 Training Grant. S.D. was a Merck Fellow of the Jane Coffin Childs Foundation. X.Z. is a Howard Hughes Medical Investigator.

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Authors and Affiliations

Authors

Contributions

W.L.H., S.D. and X.Z. designed the experiments, with input from B.T.H. W.L.H. and S.D. performed the experiments and data analysis. B.T.H. helped prepare the histones and nucleosomes. S.D., W.L.H. and X.Z. wrote the paper, with input from B.T.H. X.Z. oversaw the project.

Corresponding author

Correspondence to Xiaowei Zhuang.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Control experiments to test the effect of ATP and surface-anchoring on nucleosome remodelling.

a, Ensemble remodelling time courses of [WT H4, 78 bp] nucleosomes by 10 nM ACF with 5 µM ATP (filled symbols) or without ATP (open symbols). Nucleosome translocation was monitored by the emission intensity of the FRET acceptor Cy5 under constant 532 nm illumination that excited the FRET donor Cy3. b, Comparison of the average remodelling kinetics for surface-anchored [WT H4, 40 bp] nucleosomes (measured by the single-molecule assay, more than 250 nucleosomes) and freely diffusing [WT H4, 40 bp] nucleosomes in solution (measured by the ensemble assay). [ACF] = 5 nM and [ATP] = 20 μM.

Extended Data Figure 2 Translocation step sizes and dwell time distributions of translocation and pause phases during nucleosome remodelling.

a, Histogram of FRET levels for the initial position and pause positions of [WT H4, 40 bp] nucleosomes upon remodelling by ACF. The histogram was fitted by multiple Gaussian peaks (black line) and the peak values were used to compute the average translocation distances between pauses. The translocation distances could be quantified using a calibration curve of FRET efficiency versus exit-side linker DNA length24,26, yielding a 7.0 bp step size between the initial position and the first pause and a 3.4 bp step size between the first pause and the second pause. b, Step sizes for various mononucleosomes and dinucleosomes. The dinucleosome constructs, [WT H4, 40 bp, WT H4] and [WT H4, 78 bp, WT H4], are each composed of one FRET-labelled nucleosome and one unlabelled nucleosome, spaced by 40 bp and 78 bp of internucleosomal linker DNA, respectively. The flanking linker DNA is 3 bp on the side of the FRET-labelled nucleosome and 40 bp on the side of the unlabelled nucleosome. The unlabelled nucleosome contains 2-nt ssDNA gaps at the SHL ± two sites to prevent translocation. Data are mean ± s.e.m. derived from at least 100 remodelling traces from three independent experiments. c, Dwell-time distributions for the first translocation phase, tT1, and the first pause phase, tP1, for nucleosomes with different lengths of linker DNA. [ACF] = 10 nM and [ATP] = 20 μM.

Extended Data Figure 3 DNA linker-length sensing by ACF is quantitatively similar for mononucleosomes and dinucleosomes.

a, The dinucleosomes contain a distal FRET-labelled nucleosome and a proximal unlabelled nucleosome connected by n bp of internucleosomal linker DNA. The flanking linker DNA is 3 bp and 40 bp on the side of the FRET-labelled and the unlabelled nucleosome, respectively. To facilitate the study of translocation of the FRET-labelled nucleosome, we placed 2-nt ssDNA gaps at the SHL ± two sites of the proximal nucleosome to prevent its repositioning. b, Comparison of translocation and pause-phase exit rates for mononucleosomes (filled bars) and dinucleosomes (hashed bars) with 40 bp and 78 bp linker lengths. [ACF] = 10 nM and [ATP] = 20 μM. Data are mean ± s.e.m. derived from at least 100 individual nucleosome remodelling traces from three independent experiments.

Extended Data Figure 4 SDS–PAGE analysis of WT, ΔNegC, AutoN-2RA, ΔN-term and ΔC-term ACF complexes.

a, WT Snf2h, ΔNegC Snf2h or AutoN-2RA Snf2h was co-expressed with Acf1-Flag in Sf9 insect cells and purified by affinity chromatography. b, ΔN-term Acf1 or ΔC-term Acf1 was co-expressed with Snf2h-Flag and purified by affinity chromatography. The presence of both Acf1 and Snf2h in each case indicated that WT SNF2h, ΔNegC SNF2h and AutoN-2RA SNF2h can all form complexes with Acf1 and that ΔN-term Acf1 and ΔC-term Acf1 can both form complexes with Snf2h.

Extended Data Figure 5 Short-range linker-length sensing by the isolated Snf2h catalytic subunit requires the NegC domain.

Ensemble remodelling time courses of [WT H4, 78 bp], [WT H4, 40 bp] and [WT H4, 20 bp] nucleosomes by 130 nM WT (a) or ΔNegC Snf2h (b) at 2 mM ATP.

Extended Data Figure 6 Ensemble remodelling time courses of nucleosomes by WT and AutoN-2RA ACF at two different enzyme concentrations.

a, Remodelling of [WT H4, 78 bp] and [WT H4, 40 bp] nucleosomes by 40 nM (top) and 10 nM (bottom) WT ACF. b, Remodelling of [WT H4, 78 bp] and [WT H4, 40 bp] nucleosomes by 40 nM (top) and 10 nM (bottom) AutoN-2RA ACF.

Extended Data Figure 7 The C-terminal region of Acf1 is not required for specific binding to the H4 tail or for linker-length sensing by the ACF complex.

a, Domain maps of WT and ΔC-term Acf1 (residues 1423–1556 deleted). b, Fluorescence anisotropy of TMR-labelled WT or 2RA H4-tail peptide in the presence of varying amounts of WT Acf1. In the 2RA H4-tail peptide, two charged arginines in the basic patch of the H4-tail peptide corresponding to the 2RA mutation in AutoN were replaced with alanines. Kd for the 2RA H4-tail peptide (41 ± 29 nM) is substantially higher than that of the WT H4-tail peptide (3 ± 9 nM). Data are presented as mean ± s.e.m. (error bars, 95% confidence intervals, n = 3 three independent titration experiments). When excess unlabelled H4-tail peptide was added to compete the TMR-labelled peptide off Acf1, the fluorescence anisotropy was reduced to the background level observed in the absence of Acf1. c, Fluorescence anisotropy of TMR-labelled H4-tail peptide in the presence of varying amounts of WT or ΔC-term Acf1. The measured Kd for ΔC-term Acf1 is 2 ± 7 nM, which is similar to that for WT Acf1 (3 ± 9 nM). Data are presented as mean ± s.e.m. (error bars, 95% confidence intervals, n = 3 independent titration experiments). d, Ensemble remodelling time courses of [WT H4, 78 bp], [WT H4, 40 bp] and [H4Δ1–19, 78 bp] nucleosomes by 40 nM WT (black/grey lines) and ΔC-term ACF (purple/light purple symbols) at 5 µM ATP.

Extended Data Figure 8 Binding of dsDNA to Acf1 depends on its N-terminal region.

Electrophoretic mobility of dsDNA (225 bp, 8 nM) in the presence or absence of 22 nM WT or ΔN-term Acf1. As a comparison, lanes 2 and 4 show Acf1 samples without the dsDNA.

Extended Data Figure 9 α-histone H4 immunoblot analysis validates the formation of the Acf1-H4 crosslinked product.

a, Left: SDS–PAGE analysis of samples containing ACF alone or ACF with nucleosomes (20 bp linker DNA) that do not possess the cysteine-reactive crosslinker on the H4 tail. Both samples yield two distinct bands corresponding to the Acf1 and Snf2h subunits (180 kDa and 122 kDa, respectively). Additionally, histone bands at low molecular masses are present in the lane for the sample containing nucleosomes. Right: corresponding immunoblot using α-H4 antibody. In the presence of nucleosomes without crosslinker, a single H4 band is visible at 11 kDa corresponding to the histone itself. b, Top: incubation of ACF and nucleosomes that contain a crosslinker at the H4 tail yield Acf1-H4 and Snf2h-H4 crosslinking bands. These bands are absent for ACF without addition of nucleosomes (‘−nucleosomes’) or upon addition of nucleosomes without a crosslinker (‘+nucleosomes (−crosslinker)’). Proteolytic degradation of Acf1 gave rise to a fainter band immediately below Acf1. Bottom: α-histone H4 immunoblotting reveals specific Acf1-H4 and Snf2h-H4 bands that are absent for ACF without addition of nucleosomes or upon addition of nucleosomes without a crosslinker.

Extended Data Figure 10 DNA constructs used for mononucleosomes and dinucleosomes.

The 601 nucleosome positioning sequence is shown in green (601* represents the introduction of 2 nt gaps at nucleotides 53 and 54 in the top and bottom strands of the 601 positioning sequence, respectively). For mononucleosome DNA constructs, the ssDNA spacer used to circumvent surface effects in single-molecule FRET measurements is underlined. Constructs referred to as ‘mononucleosome [or dinucleosome] with n bp linker DNA’ were used in single-molecule and bulk remodelling experiments. For ensemble remodelling experiments with mononucleosomes, the ssDNA spacer was omitted without any appreciable change in the overall remodelling kinetics. Constructs referred to as ‘symmetric mononucleosomes with n bp linker DNA’ were used in crosslinking experiments. Asymmetric constructs with the same linker length on one side of the nucleosome but only 3 bp of linker DNA on the other side displayed quantitatively similar crosslinking behaviour.

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Hwang, W., Deindl, S., Harada, B. et al. Histone H4 tail mediates allosteric regulation of nucleosome remodelling by linker DNA. Nature 512, 213–217 (2014). https://doi.org/10.1038/nature13380

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