HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins

Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.     

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.     

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.     

Acetylation increases access of remodelling complexes to their nucleosome targets to enhance initiation of V(D)J recombination

Targeted chromatin remodelling is essential for many nuclear processes, including the regulation of V(D)J recombination. ATP-dependent nucleosome remodelling complexes are important players in this process whose activity must be tightly regulated. We show here that histone acetylation regulates nucleosome remodelling complex activity to boost RAG cutting during the initiation of V(D)J recombination. RAG cutting requires nucleosome mobilization from recombination signal sequences. Histone acetylation does not stimulate nucleosome mobilization per se by CHRAC, ACF or their catalytic subunit, ISWI. Instead, we find the more open structure of acetylated chromatin regulates the ability of nucleosome remodelling complexes to access their nucleosome templates. We also find that bromodomain/acetylated histone tail interactions can contribute to this targeting at limited concentrations of remodelling complex. We therefore propose that the changes in higher order chromatin structure associated with histone acetylation contribute to the correct targeting of nucleosome remodelling complexes and this is a novel way in which histone acetylation can modulate remodelling complex activity.     

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.     

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.     

The chromatin remodelers ISWI and ACF1 directly repress Wingless transcriptional targets

The highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of WG target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to WG targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that WG signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1.     

The ACF1 complex is required for DNA double-strand break repair in human cells

DNA double-strand breaks (DSBs) are repaired via?nonhomologous end-joining (NHEJ) or homologous?recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.     

Expansion of the ISWI chromatin remodeler family with new active complexes

ISWI chromatin remodelers mobilize nucleosomes to control DNA accessibility. Complexes isolated to date pair one of six regulatory subunits with one of two highly similar ATPases. However, we find that each endogenously expressed ATPase co‐purifies with every regulatory subunit, substantially increasing the diversity of ISWI complexes, and we additionally identify BAZ2B as a novel, seventh regulatory subunit. Through reconstitution of catalytically active human ISWI complexes, we demonstrate that the new interactions described here are stable and direct. Finally, we profile the nucleosome remodeling functions of the now expanded family of ISWI chromatin remodelers. By revealing the combinatorial nature of ISWI complexes, we provide a basis for better understanding ISWI function in normal settings and disease.