A 'loop recapture' mechanism for ACF-dependent nucleosome remodeling

The ATPase ISWI is the molecular motor of several nucleosome remodeling complexes including ACF. We analyzed the ACF-nucleosome interactions and determined the characteristics of ACF-dependent nucleosome remodeling. In contrast to ISWI, ACF interacts symmetrically with DNA entry sites of the nucleosome. Two-color fluorescence cross-correlation spectroscopy measurements show that ACF can bind four DNA duplexes simultaneously in a complex that contains two Acf1 and ISWI molecules. Using bead-bound nucleosomal substrates, nucleosome movement by mechanisms involving DNA twisting was excluded. Furthermore, an ACF-dependent local detachment of DNA from the nucleosome was demonstrated in a novel assay based on the preferred intercalation of ethidium bromide to free DNA. The findings suggest a loop recapture mechanism in which ACF introduces a DNA loop at the nucleosomal entry site that propagates over the histone octamer surface and leads to nucleosome repositioning.     

Spatial contacts and nucleosome step movements induced by the NURF chromatin remodeling complex

The nucleosome remodeling factor NURF is a four-subunit, ISWI-containing chromatin remodeling complex that catalyzes nucleosome sliding in an ATP-dependent fashion, thereby modulating the accessibility of the DNA. To elucidate the mechanism of nucleosome sliding, we have investigated by hydroxyl radical footprinting how NURF makes initial contact with a nucleosome positioned at one end of a DNA fragment. NURF binds to two separate locations on the nucleosome: a continuous stretch of linker DNA up to the nucleosome entry site and a region asymmetrically surrounding the nucleosome dyad within the minor grooves, close to residues of the histone H4 tail that have been implicated in the activation of ISWI activity. Kinetic analysis reveals that nucleosome sliding occurs in apparent increments or steps of 10 bp. Furthermore, single nucleoside gaps as well as nicks about two helical turns before the dyad interfere with sliding, indicating that structural stress at this region assists the relative movement of DNA. These findings support a sliding model in which the position-specific tethering of NURF forces a translocating ISWI ATPase to pump a DNA distortion over the histone octamer, thereby changing the translational position of the nucleosome.     

A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI

The ATPase ISWI is the catalytic core of several nucleosome remodeling complexes, which are able to alter histone–DNA interactions within nucleosomes such that the sliding of histone octamers on DNA is facilitated. Dynamic nucleosome repositioning may be involved in the assembly of chromatin with regularly spaced nucleosomes and accessible regulatory sequence elements. The mechanism that underlies nucleosome sliding is largely unresolved. We recently discovered that the N-terminal ‘tail’ of histone H4 is critical for nucleosome remodeling by ISWI. If deleted, nucleosomes are no longer recognized as substrates and do not stimulate the ATPase activity of ISWI. We show here that the H4 tail is part of a more complex recognition epitope which is destroyed by grafting the H4 N-terminus onto other histones. We mapped the H4 tail requirement to a hydrophilic patch consisting of the amino acids R₁₇H₁₈R₁₉ localized at the base of the tail. These residues have been shown earlier to contact nucleosomal DNA, suggesting that ISWI recognizes an ‘epitope’ consisting of the DNA-bound H4 tail. Consistent with this hypothesis, the ISWI ATPase is stimulated by isolated H4 tail peptides ISWI only in the presence of DNA. Acetylation of the adjacent K₁₂ and K₁₆ residues impairs substrate recognition by ISWI.     

Acf1, the largest subunit of CHRAC, regulates ISWI-induced nucleosome remodelling.

The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP-dependent 'nucleosome remodelling' activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. We have now identified the p175 subunit of CHRAC as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI qualitatively by altering the directionality of nucleosome movements and the histone 'tail' requirements of the reaction. The Acf1-ISWI heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC-14 and CHRAC-16. Whether topoisomerase II is an integral subunit has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be molecularly defined as a complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16.     

The DNA chaperone HMGB1 facilitates ACF/CHRAC-dependent nucleosome sliding

Nucleosome remodelling complexes CHRAC and ACF contribute to chromatin dynamics by converting chemical energy into sliding of histone octamers on DNA. Their shared ATPase subunit ISWI binds DNA at the sites of entry into the nucleosome. A prevalent model assumes that DNA distortions catalysed by ISWI are converted into relocation of DNA relative to a histone octamer. HMGB1, one of the most abundant nuclear non-histone proteins, binds with preference to distorted DNA. We have now found that transient interaction of HMGB1 with nucleosomal linker DNA overlapping ISWI-binding sites enhances the ability of ACF to bind nucleosomal DNA and accelerates the sliding activity of limiting concentrations of remodelling factor. By contrast, an HMGB1 mutant with increased binding affinity was inhibitory. These observations are consistent with a role for HMGB1 as a DNA chaperone facilitating the rate-limiting DNA distortion during nucleosome remodelling.     

Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI

The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.     

ISWI is an ATP-dependent nucleosome remodeling factor

The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin. We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions. The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes. The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs.     

Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme

The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5- or 10-bp gaps in the 3'-5' strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3'-5'-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.     

Nucleosome movement by CHRAC and ISWI without disruption or trans-displacement of the histone octamer.

The chromatin accessibility complex (CHRAC) belongs to the class of nucleosome remodeling factors that increase the accessibility of nucleosomal DNA in an ATP-dependent manner. We found that CHRAC induces movements of intact histone octamers to neighboring DNA segments without facilitating their displacement to competing DNA or histone chaperones in trans. CHRAC-induced energy-dependent nucleosome sliding may, in principle, explain nucleosome remodeling, nucleosome positioning, and nucleosome spacing reactions known to be catalyzed by CHRAC. The catalytic core of CHRAC, the ATPase ISWI, also mobilized nucleosomes at the expense of energy. However, the directionality of the CHRAC- and ISWI-induced nucleosome movements differed drastically, indicating that the geometry of the native complex modulates the activity of its catalytic core.     

Chromatin remodeling in vivo: evidence for a nucleosome sliding mechanism

Members of the ISWI family of chromatin remodeling factors exhibit ATP-dependent nucleosome sliding, loading, and spacing activities in vitro. However, it is unclear which of these activities are utilized by ISWI complexes to remodel chromatin in vivo. We therefore sought to identify the mechanisms of chromatin remodeling by Saccharomyces cerevisiae Isw2 complex at its known sites of action in vivo. To address this question, we developed a method of identifying intermediates of the Isw2-dependent chromatin remodeling reaction as it proceeded. We show that Isw2 complex catalyzes nucleosome sliding at two different classes of target genes in vivo, in each case sliding nucleosomes closer to the promoter regions. In contrast to its biochemical activities in vitro, nucleosome sliding by Isw2 complex in vivo is unidirectional and localized to a few nucleosomes at each site, suggesting that Isw2 activity is constrained by cellular factors.     

No need for a power stroke in ISWI‐mediated nucleosome sliding

Nucleosome remodelling enzymes of the ISWI family reposition nucleosomes in eukaryotes. ISWI contains an ATPase and a HAND‐SANT‐SLIDE (HSS) domain. Conformational changes between these domains have been proposed to be critical for nucleosome repositioning by pulling flanking DNA into the nucleosome. We inserted flexible linkers at strategic sites in ISWI to disrupt this putative power stroke and assess its functional importance by quantitative biochemical assays. Notably, the flexible linkers did not disrupt catalysis. Instead of engaging in a power stroke, the HSS module might therefore assist DNA to ratchet into the nucleosome. Our results clarify the roles had by the domains and suggest that the HSS domain evolved to optimize a rudimentary remodelling engine.     

ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts

The nucleosome remodelling ATPase ISWI resides in several distinct protein complexes whose subunit composition reflects their functional specialization. Association of ISWI with ACF1, the largest subunit of CHRAC and ACF complexes, improves the efficiency of ISWI-induced nucleosome mobilization by an order of magnitude and also modulates the reaction qualitatively. In order to understand the principle by which ACF1 improves the efficiency of ISWI, we mapped their mutual interaction requirements and generated a series of ACF complexes lacking conserved ACF1 domains. Deletion of the C-terminal PHD finger modules of ACF1 or their disruption by zinc chelation profoundly affected the nucleosome mobilization capability of associated ISWI in trans. Interactions of the PHD fingers with the central domains of core histones contribute significantly to the binding of ACF to the nucleosome substrate, suggesting a novel role for PHD modules as nucleosome interaction determinants. Connecting ACF to histones may be prerequisite for efficient conversion of ATP-dependent conformational changes of ISWI into translocation of DNA relative to the histones during nucleosome mobilization.     

Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the HAND-SANT-SLIDE (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.     

Control of nucleosome movement: To space or not to space nucleosomes?

A key feature of ATP-dependent chromatin remodeling complexes is how they control the ability of the complex to translocate along DNA within the context of a nucleosome. Although these complexes generally initiate DNA translocation near the dyad axis of the nucleosome, the progression and eventual termination is regulated in quite distinct ways. The best studies examples of these are the ISWI type which has strong extranucleosomal DNA dependent activity or the SWI/SNF type which has no linker DNA requirement. Recent data provide more insights into the mechanism of regulation of DNA translocation by the ISWI type complexes and how the structure of the ISWI-nucleosome complex changes during chromatin remodeling.     

Expression of ISWI and its binding to chromatin during the cell cycle and early development

We have characterized Xenopus ISWI, a catalytic subunit of a family of chromatin-remodeling complexes. We show that ISWI is expressed constitutively during development but poorly expressed in adult tissues except oocytes which contain a large store of maternal protein. We further analyzed its localization both in vivo and in vitro in Xenopus cell cycle extracts and identified that ISWI binds to chromatin at the G1-S period. However, its association to chromatin does not require ongoing DNA replication. Immunodepletion of ISWI has no effect on either sperm chromatin decondensation or the kinetics and efficiency of DNA replication. Nucleosome assembly also occurs in ISWI-depleted extracts, but nucleosome spacing is disturbed. From these results, we conclude that ISWI is not necessary for sperm chromatin decondensation and the accelerated rates of DNA replication that characterize early development.     

hSWI/SNF-catalyzed nucleosome sliding does not occur solely via a twist-diffusion mechanism

Nucleosome remodeling by the hSWI/SNF complex and other chromatin remodeling complexes can cause translocation (sliding) of the histone octamer in cis along DNA. Structural and biochemical evidence suggest that sliding involves a DNA twist-diffusion process whereby the DNA rotates about the helical axis without major displacement from the surface of the nucleosome and that this process may be driven by torsional stress within the DNA. We report that hSWI/SNF efficiently catalyzes sliding of nucleosomes containing branched DNAs as steric blocks to twist-diffusion and a nick to allow dissipation of torsional stress within the nucleosome. These results suggest that SWI/SNF-catalyzed nucleosome sliding does not occur exclusively via a simple twist-diffusion mechanism and support models in which the DNA maintains its rotational orientation to and is at least partially separated from the histone surface during nucleosome translocation.