The nucleosome-remodeling ATPase ISWI is regulated by poly-ADP-ribosylation

ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatin set the functional state of chromatin. However, how these enzymatic activities are coordinated in the nucleus is largely unknown. We found that the evolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribose polymerase PARP genetically interact. We present evidence showing that ISWI is target of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI function in vitro and in vivo. Our work suggests that ISWI is a physiological target of PARP and that poly-ADP-ribosylation can be a new, important post-translational modification regulating the activity of ATP-dependent nucleosome remodelers.     

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.     

dMi-2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties

Mi-2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP-dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. We have compared the remodelling activity of recombinant Drosophila Mi-2 (dMi-2) with that of recombinant ISWI. Both proteins are nucleosome-stimulated ATPases and promote nucleosome mobilization. However, dMi-2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. We have used antibodies to immobilize a complex containing dMi-2 and the dRPD3 histone deacetylase from Drosophila embryo extracts. This complex shares the nucleosome-stimulated ATPase and nucleosome mobilization properties of recombinant dMi-2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish dMi-2 from both SWI2/SNF2 and ISWI, defining a new family of ATP-dependent remodelling machines.     

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.     

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.     

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.     

Distinct strategies to make nucleosomal DNA accessible

One hallmark of ATP-dependent remodeling complexes is the ability to make nucleosomal DNA accessible to regulatory factors. We have compared two prominent human ATP-dependent remodelers, BRG1 from the SWI/SNF family and SNF2h from the ISWI family, for their abilities to make a spectrum of nucleosomal sites accessible. By measuring rates of remodeling at seven different sites on a mononucleosome and at six different sites on the central nucleosome of a trinucleosome, we have found that BRG1 opens centrally located sites more than an order of magnitude better than SNF2h. We provide evidence that this capability of BRG1 is caused by its ability to create DNA loops on the surface of a nucleosome, even when that nucleosome is constrained by adjacent nucleosomes. This specialized ability to make central sites accessible should allow SWI/SNF family complexes to facilitate binding of nuclear factors in chromatin environments where adjacent nucleosomes might otherwise constrain mobility.     

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.     

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.     

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.     

ISWI induces nucleosome sliding on nicked DNA.

The ATPase ISWI is the molecular motor of several remodeling factors that trigger nucleosome sliding in vitro. In search for the underlying mechanism, we found that unilateral binding of ISWI to a model nucleosome correlated with directional movement of the nucleosome toward the enzyme. It has been proposed that ISWI might loosen histone-DNA interactions through twisting DNA. However, nucleosome sliding assays on nicked DNA substrates suggest that propagation of altered twist is not involved. Surprisingly, nicks in the linker DNA in front of the nucleosome facilitate sliding. These data suggest that the rate of nucleosome sliding is limited by a conformational change other than twisting, such as the formation of a short loop, of DNA at the entry into the nucleosome.     

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.     

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.     

The DNA-binding domain of the Chd1 chromatin-remodelling enzyme contains SANT and SLIDE domains

The ATP-dependent chromatin-remodelling enzyme Chd1 is a 168-kDa protein consisting of a double chromodomain, Snf2-related ATPase domain, and a C-terminal DNA-binding domain. Here, we show the DNA-binding domain is required for Saccharomyces cerevisiae Chd1 to bind and remodel nucleosomes. The crystal structure of this domain reveals the presence of structural homology to SANT and SLIDE domains previously identified in ISWI remodelling enzymes. The presence of these domains in ISWI and Chd1 chromatin-remodelling enzymes may provide a means of efficiently harnessing the action of the Snf2-related ATPase domain for the purpose of nucleosome spacing and provide an explanation for partial redundancy between these proteins. Site directed mutagenesis was used to identify residues important for DNA binding and generate a model describing the interaction of this domain with DNA. Through inclusion of Chd1 sequences in homology searches SLIDE domains were identified in CHD6-9 proteins. Point mutations to conserved amino acids within the human CHD7 SLIDE domain have been identified in patients with CHARGE syndrome.     

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.     

1H, 13C and 15N resonance assignments of an N-terminal domain of CHD4

Chromatin-remodeling proteins have a pivotal role in normal cell function and development, catalyzing conformational changes in DNA that ultimately result in changes in gene expression patterns. Chromodomain helicase DNA-binding protein 4 (CHD4), the defining subunit of the nucleosome remodeling and deacetylase (NuRD) complex, is a nucleosome-remodeling protein of the SNF2/ISWI2 family, members of which contain two chromo domains and an ATP-dependent helicase module. CHD3, CHD4 and CHD5 also contain two contiguous PHD domains and have an extended N-terminal region that has not previously been characterized. We have identified a stable domain in the N-terminal region of CHD4 and report here the backbone and side chain resonance assignments for this domain at pH 7.5 and 25 °C (BMRB No. 18906).