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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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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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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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