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

The packaging of DNA into nucleosomes presents a substantial energy barrier that restricts access to the genomic DNA¹⁵. ISWI-family remodellers use the energy from ATP hydrolysis to disrupt histone-DNA contacts and reposition nucleosomes¹. The catalytic subunits of ISWI enzymes possess an SF2-like ATPase that translocates DNA across the nucleosome¹. The nucleosome translocation activity is further regulated by the accessory subunits of ISWI complexes^6,10,16. Many ISWI remodellers exhibit a nucleosome spacing activity^2-5. Critical to this spacing activity are two features of the nucleosome that modulate the activity of ISWI remodellers: (1) the N-terminal tail of histone H4^8,17-20, and (2) the length of the extranucleosomal linker DNA^6-10. The unmodified H4 tail stimulates ISWI activity by relieving the auto inhibitory effect of the AutoN domain within the catalytic subunit¹⁴. H4 tail acetylation associated with transcriptionally active chromatin is thought to help prevent ISWI-induced nucleosome spacing at actively transcribed genes^17-19. Regulation by the extranucleosomal linker DNA is responsible for generating the regularly-spaced nucleosome arrays important for heterochromatin formation. Shortening the linker DNA reduces the remodelling activity of nucleosome-spacing ISWI enzymes^6-10. As a result, nucleosomes are preferentially moved towards longer linkers to promote uniform spacing on nucleosome arrays. Interestingly, the catalytic activity of many ISWI-family enzymes is sensitive to linker DNA length sup to ∼60-70 bp^6-10, consistent with the inter-nucleosome spacing of heterochromatin observed in human cells²¹. This linker-length sensing range substantially exceeds the binding footprint (20-30 bp) of the catalytic subunit^22,23, whereas the accessory subunits of ISWI complexes can bind linker DNA as far as ∼60 bp from the nucleosome edge²². However, it is unknown how accessory subunits communicate linker length information to the catalytic subunit to regulate remodelling activity. In this work, we investigate the mechanism underlying DNA linker-length sensing by a prototypical ISWI-family enzyme, human ACF.

To examine how linker DNA regulates nucleosome translocation by ACF, we reconstituted mononucleosomes with varying linker lengths (n = 20-78 bp) on the entry side but a constant exit-side linker length of 3 bp (Fig. 1a). We also constructed mononucleosomes with wildtype (wt) histone H4 and two H4 mutants: (1) H4 tail deletion (H4Δ1-19), and (2) H4 with K16A mutation (H4K16A). We refer to nucleosome constructs with the following nomenclature: [wt H4/H4Δ1-19/H4K16A, n bp] for nucleosomes with n bp of DNA on the entry side and an octamer containing wt H4, H4Δ1-19, or H4K16A. We detected ACF-catalysed nucleosome translocation using fluorescence resonance energy transfer (FRET) by labelling the end of the exit-side linker DNA with the FRET acceptor Cy5, and the histone H2A with the FRET donor Cy3 (Fig. 1a)²⁴.

We first compared the remodelling kinetics of [wt H4, 78 bp], [wt H4, 40 bp], [wt H4, 20 bp], and [H4Δ1-19, 78 bp] nucleosomes using an ensemble FRET assay⁶. Upon addition of ACF and ATP, the FRET efficiency decreased as DNA was translocated towards the exit side (Fig. 1b and Extended Data Fig. 1a). As expected, the remodelling rate decreased as the linker DNA was shortened and deletion of the H4 tail drastically reduced the remodelling activity (Fig. 1b).

To identify which step (s) of the remodelling process are regulated, we monitored the remodelling of individual nucleosomes using single-molecule FRET^24,25. Single-nucleosome remodeling traces featured incremental translocation of DNA to the exit side interrupted by kinetic pauses (Fig. 1c and Extended Data Fig. 2a,b). The first pause occurred after ∼7 bp of DNA translocation and the second pause occurred after an additional ∼3 bp of translocation, consistent with previous findings^24,26. Moreover, the step sizes did not change with linker DNA length or histone H4 modification (Extended Data Fig. 2a,b). We divided the remodelling time trace into two translocation phases (T1, T2), during which the FRET efficiency decreased, and two pause phases (P1, P2) without appreciable FRET change (Fig. 1c). Notably, the DNA translocation rates between pauses did not change, whereas the pause-phase exit rates decreased dramatically when the linker DNA was shortened (Fig. 1d and Extended Data Fig. 2c). Moreover, the dependence of remodelling kinetics on entry-side linker lengths of mononucleosomes was quantitatively similar to the dependence on inter-nucleosome linker lengths of dinucleosomes (Extended Data Fig. 3), validating the use of mononucleosomes as a model system to study linker-length sensitivity. Interestingly, the H4 tail appeared to regulate the same phase of the remodelling process as the linker DNA (Fig. 1e). The H4K16A mutation and H4 tail deletion (H4Δ1-19) decreased the pause-phase exit rate by ∼2 and ∼20 fold, respectively (Fig. 1e). In contrast, neither modification had any appreciable effect on the translocation rates between pauses (Fig. 1e).

The above results indicate that both linker DNA and the H4 tail regulate the remodelling rate by changing the duration of pause phases, suggesting that these nucleosome features may impinge on an inhibitory mechanism that prevents the initiation of the DNA translocation phases. It has been shown that although the ISWI ATPase domain can translocate nucleosomes autonomously²⁷, the catalytic subunit contains two well-conserved autoregulatory domains, AutoN and NegC, which inhibit ATP hydrolysis and its coupling to DNA translocation, respectively¹⁴. The AutoN inhibition can be relieved by the H4 tail whereas the NegC inhibition can be relieved by binding of the HAND-SANT-SLIDE module to linker DNA¹⁴. Could the regulation of remodelling by linker DNA length occur through these inhibitory domains?

To address this question, we first examined the role of the NegC domain. Surprisingly, deletion of the NegC domain in the ACF complex (ΔNegC ACF) did not substantially affect the dependence of remodelling kinetics on linker DNA lengths ranging from 20 to 78 bp (Fig. 2a, b and Extended Data Fig. 4a). Removing the H4 tail dramatically reduced the remodelling rates of both wt and ΔNegC ACF (Fig. 2b). These results suggest that the NegC domain does not play a substantial role in linker length sensing by the ACF complex. In contrast, the isolated Snf2h catalytic subunit exhibited a short-range (20-40 bp) linker length sensitivity that depended on NegC (Extended Data Fig. 5), in a manner similar to the Drosophila ISWI, which lacks any accessory subunit¹⁴.

Next, we mutated the AutoN domain with two point substitutions (R142A and R144A) in the ACF complex (AutoN-2RA ACF; Fig. 3a and Extended Data Fig. 4a). AutoN bears sequence homology to the H4 tail, which can compete the inhibitory AutoN domain off the ATPase, and the 2RA mutation in AutoN is expected to diminish the H4 tail dependence of remodelling by ISWI enzymes¹⁴. Remarkably, this mutation not only increased the remodelling rate of nucleosomes lacking the H4 tail, but also completely abolished the linker-length dependence of remodelling by specifically increasing the remodelling rate of short-linker nucleosomes (Fig. 3b,c and Extended Data Fig. 6). These results suggest an essential role for AutoN in linker length sensing by the ACF complex.

Since AutoN competes with the H4 tail for binding to the ATPase¹⁴, we considered the possibility that this competition is involved in sensing linker DNA length and hypothesized that the H4 tail is only available to compete AutoN off the ATPase when the linker DNA is sufficiently long. Consistent with this hypothesis, adding exogenous H4-tail peptide, which should help compete AutoN off the ATPase when the nucleosomal H4 tail is unavailable, specifically increased the remodelling rate of short-linker nucleosome ([wt H4, 40 bp]) by wt ACF (Fig. 3d). Furthermore, deletion of the nucleosomal H4 tail, in addition to slowing down remodelling, abolished the dependence of remodelling rate on linker DNA length (Fig. 3e). These results indicate that the H4 tail is indeed involved in linker DNA sensing.

Because the catalytic subunits of ISWI-family enzymes only interact with ∼20-30 bp of extranucleosomal DNA, the linker-length sensitivity of ACF cannot be accounted for by the catalytic subunit alone. Our findings raise the intriguing possibility of a linker-length sensing mechanism where the accessory subunit Acf1 interacts with the H4 tail in a linker-length dependent manner, which modulates the H4 tail availability for competing with AutoN. To test this possibility, we generated two Acf1 mutants, ΔC-term Acf1 and ΔN-term Acf1, in which 134 residues at the C-terminus or 371 residues at the N-terminus were deleted, respectively (Extended Data Fig. 7a and Fig. 4a). Because the central region of Acf1 required for Snf2h binding^16,28 was not deleted, both mutants were able to form complexes with Snf2h, which are referred to as ΔC-term and ΔN-term ACF (Extended Data Fig. 4b).

We first probed which region of Acf1 interacts with the H4 tail by comparing the binding affinities of wt, ΔC-term and ΔN-term Acf1 for the H4-tail peptide using a fluorescence anisotropy assay. Interestingly, wt Acf1 exhibited specific, nanomolar affinity for the H4-tail peptide (Fig. 4b and Extended Data Fig. 7b) that was not substantially altered upon deletion of the C-terminal region (Extended Data Fig. 7c), but was completely lost upon deletion of the N-terminal portion (Fig. 4b). These results indicate that Acf1 interacts with the H4 tail likely through its N-terminal region. Acf1 also bound double-stranded DNA and deletion of the N-terminal region abolished this interaction too (Extended Data Fig. 8), consistent with the previous finding that the WAC motif within the N-terminal region is important for binding of ACF to the linker DNA²⁸. Given the distinct properties of DNA and the H4 tail, their specific binding interfaces within Acf1 N-term are likely distinct.

Next, we investigated nucleosome remodelling by the ΔC-term and ΔN-term ACF complexes. Notably, the ΔC-term mutation did not substantially alter the dependence of remodelling kinetics on linker DNA length (Extended Data Fig. 7d), whereas the linker-length sensitivity was eliminated in the ΔN-term ACF complex (Fig. 4c,d). This finding is consistent with the specific affinity of Acf1 N-term for the H4 tail (Fig. 4b). Furthermore, if the loss of linker-length sensitivity was simply a result of losing the linker DNA binding affinity of Acf1, ΔN-term ACF should demonstrate inefficient remodelling for all linker DNA lengths. Instead, ΔN-term ACF remodelled both short- and long-linker nucleosomes at rates close to the rate with which wt ACF remodelled long-linker nucleosomes (Fig. 4c,d), suggesting that deletion of Acf1 N-term disabled a mechanism that inhibits remodelling at short linker lengths. ΔN-term ACF also maintained the H4-tail requirement in remodelling (Fig. 4c).

Since Acf1 has affinity to both DNA and the H4 tail, a plausible interpretation of the above observations is that nucleosomal linker DNA and H4 tail are in competition for binding to the N-terminal region of Acf1 and that this competition is modulated by the length of the linker DNA. Only when the linker is sufficiently short does Acf1 preferentially bind to the H4 tail, making it unavailable to compete with the inhibitory AutoN. Deletion of Acf1 N-term diminishes the Acf1-H4 tail interaction such that the H4 tail is equally available to activate the ATPase at both short and long linker DNA lengths. We therefore probed the linker-length dependence of the proximity between Acf1 and the H4 tail in ACF-bound nucleosomes featuring a cysteine-reactive crosslinker on the H4 tail. Specific H4-Acf1 crosslinking product was clearly observed as a band with reduced electrophoretic mobility compared to non-crosslinked Acf1 (Fig. 4e and Extended Data Fig. 9). Remarkably, the Acf1-H4 crosslinking efficiency decreased substantially with increasing linker DNA length (Fig. 4e), supporting our model that the Acf1-H4 tail interaction is modulated by the linker DNA length. In contrast, the H4-Snf2h crosslinking efficiency did not change substantially with linker DNA length, likely because Snf2h remains sufficiently close to the H4 tail regardless of the linker DNA length, which allows crosslinking even when the H4 tail was not specifically bound to its binding pocket on Snf2h.

Finally, we tested the physiological importance of the N-terminal region of Acf1 by studying the role of its homologue in yeast²⁹. Yeast ISW2 is functionally similar to ACF. It is composed of a catalytic subunit (Isw2) that is homologous to Snf2h and three accessory subunits (Itc1, Dpb4 and Dls1), among which Itc1 is homologous to Acf1. We generated three mutant yeast strains: (1) deletion of the entire itc1 gene (Δitc1), (2) deletion of only the portion of itc1 that encodes the N-terminal region of Itc1 equivalent to Acf1 N-term (Δitc1-Nterm), and (3) a rescue strain that was derived from the Δitc1-Nterm strain by deleting the remainder of itc1 (rescue-Δitc1). Both Δitc1 and rescue-Δitc1 showed growth rates similar to that of the wt strain (Fig. 4f), consistent with previous observations²⁹. In contrast, the Δitc1-Nterm strain displayed dramatically slower growth (Fig. 4f), consistent with an aberrant chromatin-misregulation phenotype.

Taken together, our results suggest a nucleosome-spacing mechanism for ACF in which the linker DNA length is sensed by the Acf1 accessory subunit and allosterically transmitted to the Snf2h catalytic subunit through the H4 tail of the nucleosome (Fig. 4g). Surprisingly, Acf1 and the AutoN domain of Snf2h function collectively in DNA linker-length sensing. When the linker DNA is short, Acf1 preferentially binds to and sequesters the H4 tail, making it unavailable to compete its sequence homologue, AutoN, off the ATPase. Hence, the ATPase activity is inhibited by AutoN. As the linker DNA length increases, Acf1 shifts its binding preference to the linker DNA and releases the H4 tail, allowing it to compete AutoN off the ATPase and activate ACF. This competition between the H4 tail and linker DNA for Acf1 binding likely involves the N-terminal region of Acf1. It is interesting to note that linker DNA sensing occurs during the pause phases when the ATPase domain is not actively translocating DNA, suggesting that AutoN engages the ATPase domain during the pauses. In order to exit the pauses, the H4 tail is required to relieve the inhibitory effect of AutoN. The re-engagement of AutoN with the ATPase domain after each translocation phase would give ACF an opportunity to periodically sense the linker DNA length. Such frequent sensing may allow a more efficient nucleosome spacing, as previously hypothesized³⁰. The linker DNA and the H4 tail are two important substrate features that regulate nucleosome remodelling by ISWI-family enzymes, the former enabling uniform nucleosome spacing for heterochromatin formation and the latter specifying regions of chromatin for silencing. Our results now reveal an unexpected convergence of the regulatory pathways defined by these two distinct nucleosome features.