ATP-dependent chromatin assembly factor large subunit

Acf1 confers unique activities to ACF/CHRAC and promotes the formation rather than disruption of chromatin in vivo

Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin assembly and remodeling factor) catalyzes the ATP-dependent assembly of periodic nucleosome arrays in vitro, and consists of Acf1 and the ISWI ATPase. Acf1 and ISWI are also subunits of CHRAC (chromatin accessibility complex), whose biochemical activities are similar to those of ACF. This study investigated the in vivo function of the Acf1 subunit of ACF/CHRAC in Drosophila. Although most Acf1 null animals die during the larval-pupal transition, Acf1 is not absolutely required for viability. The loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin in embryos. Biochemical experiments with Acf1-deficient embryo extracts further indicate that ACF/CHRAC is a major chromatin assembly factor in Drosophila. The phenotypes of flies lacking Acf1 suggest that ACF/CHRAC promotes the formation of repressive chromatin. The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes. Moreover, cells in animals lacking Acf1 exhibit an acceleration of progression through S phase, which is consistent with a decrease in chromatin-mediated repression of DNA replication. In addition, acf1 genetically interacts with nap1, which encodes the NAP-1 nucleosome assembly protein. These findings collectively indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus (Fyodorov, 2004).

Eukaryotic DNA is packaged into a periodic nucleoprotein complex termed chromatin. The nucleosome is the basic repeating unit of chromatin, and the nucleosomal core consists of 146 bp of DNA wrapped around an octamer of histones H2A, H2B, H3, and H4. In addition to the core histones, chromatin contains other components such as linker histones and high mobility group proteins. Chromatin is involved in the regulation of transcription and other DNA-directed processes via posttranslational modifications of core histones, the reorganization of nucleosomes by chromatin remodeling factors, and the alteration of higher-order structures (Fyodorov, 2004 and references therein).

The assembly of chromatin is a fundamental biological process that occurs in proliferating cells during DNA replication and in quiescent cells during maintenance and repair of chromosomes. During DNA replication, chromatin structure is transiently disrupted at the replication fork, and the preexisting nucleosomes are segregated randomly between the daughter DNA strands. Then, additional nucleosomes are formed with newly synthesized histones. In this process, it appears that histones H3 and H4 are deposited prior to the incorporation of histones H2A and H2B. Chromatin assembly also occurs in nonreplicating DNA, and several examples of replication-independent assembly of chromatin have been described. These latter processes may occur during histone replacement, DNA repair, and transcription (Fyodorov, 2004 and references therein).

The basic chromatin assembly process is mediated by core histone chaperones and an ATP-utilizing motor protein. The histone chaperones include CAF-1 (chromatin assembly factor-1), NAP-1 (nucleosome assembly protein-1), Asf1 (anti-silencing function-1), nucleoplasmin, N1/N2, and Hir (histone regulatory) proteins. These proteins appear to deliver the histones from the cytoplasm to the sites of chromatin assembly in the nucleus. The ATP-utilizing assembly factor ACF (ATP-utilizing chromatin assembly and remodeling factor) can catalyze the transfer of histones from the chaperones to the DNA to yield periodic nucleosome arrays. The assembly reaction can also be catalyzed by purified RSF (remodeling and spacing factor), which appears to possess both chaperone and motor activities (Fyodorov, 2004).

This work investigates the biological function of ACF. ACF was purified from Drosophila embryos as an activity that mediates the ATP-dependent assembly of regularly spaced nucleosome arrays in vitro. During the assembly process, ACF commits to and translocates along the DNA template. ACF consists of two subunits, Acf1 and ISWI, which cooperatively catalyze nucleosome assembly in conjunction with histone chaperone proteins NAP-1 or CAF-1. Acf1 is the larger subunit of ACF, and it possesses WAC, DDT, WAKZ, PHD finger, and bromo-domain motifs. ISWI belongs to the SNF2-like family of DNA-dependent ATPases, and is a subunit of the ACF, CHRAC (chromatin accessibility complex), NURF, and TRF2 complexes. NURF and TRF2 complexes share only the ISWI subunit with ACF, whereas CHRAC is closely related to ACF. CHRAC was purified on the basis of its ability to increase the access of restriction enzymes to DNA in chromatin, and it consists of Acf1, ISWI, and two small subunits, CHRAC-14 and CHRAC-16, which are detected only during early embryonic development. The biochemical activities of ACF and CHRAC are indistinguishable. These Acf1-containing species will be referred to as 'ACF/CHRAC'. To study the function of ACF/CHRAC in vivo, a genetic analysis of the Drosophila acf1 gene was performed. The results indicate that Acf1 programs ACF/CHRAC to perform functions that are distinct from those of the NURF complex, which shares a common ISWI ATPase subunit with ACF/CHRAC. In addition, the phenotypes of flies lacking Acf1 suggest that ACF/CHRAC does not disrupt chromatin, as might be expected for a nucleosome remodeling factor, but rather promotes the formation of chromatin, as would be expected for a chromatin assembly factor (Fyodorov, 2004).

Polycomb regulation is caused by chromatin-dependent transcriptional silencing. The identity of body segments in Drosophila is specified by homeotic genes of the Antennapedia and bithorax complexes, which are in turn subject to regulation by Polycomb and trithorax group (PcG and trxG) genes. PcG genes encode protein complexes that can maintain chromatin-dependent transcriptional silencing via cis-acting DNA elements termed Polycomb response elements, or PREs (Fyodorov, 2004).

To determine the influence of Acf1 on Polycomb regulation, whether the loss of Acf1 affects transcriptional repression by the Ubx PRE in a PRE-miniwhite reporter gene was examined. In the wild-type control background(acf1³/acf1³), the expression of the PRE-miniwhite reporter gene was strongly repressed, with pigments limited to a small part of the adult fly eye. In the absence of Acf1 (acf1¹/acf1¹), partial activation was observed of the PRE-miniwhite reporter gene with pigments distributed over a larger area of the eye. This observed derepression in the homozygous acf1¹ background is comparable to derepression in a heterozygous Pc background (Fyodorov, 2004).

Whether acf1 interacts genetically with the segmentation function of Pc was investigatede. The appearance of extra sex combs on distal portions of the second and third legs in F1 males was scored in the progeny from a cross between males with a heterozygous deficiency for Pc (Df(3L)Asc) and females homozygous for acf1 alleles. The mutation of acf1 significantly enhanced this Pc phenotype in a manner similar to that seen with other enhancers of the Pc gene. Whereas only about 18% or 17% of the Df(3L)Asc/+; acf1³/+ or Df(3L)Asc/+; acf1⁴/+ males had extra sex combs on second and/or third pairs of legs (from the total number of male progeny scored, 61% or 58% of the Df(3L)Asc/+; acf1¹/+ or Df(3L)Asc/+; acf1²/+ male flies had the extra sex comb phenotype). In addition, >50% of males in the latter two crosses exhibited ectopic pigmentation of their A3 and A4 abdominal tergites, which was never observed in crosses with acf1³ or acf1⁴ mothers. These results, combined with the derepression of PRE-mediated miniwhite silencing, demonstrate that acf1 is a Polycomb enhancer and suggest that ACF/CHRAC is involved in the assembly and/or maintenance of repressive chromatin in Polycomb-responsive loci (Fyodorov, 2004).

The identity of Drosophila abdominal segments A5-A8 is determined by homeotic selector genes of the bithorax complex. For instance, in Pc/acf1 males, the posteriorly directed homeotic transformation may be caused by an increase in the expression of the bithorax complex gene Abd-B on loss of Acf1. In contrast, the anterior transformation phenotype of ISWI/+; acf1/acf1 and nap1/nap1; acf1/acf1 animals is reminiscent of mutations in various trithorax group genes, which include the brm and kis genes that encode ATPase subunits of chromatin remodeling complexes. This anterior transformation is likely to result from a decrease in expression of Abd-B on loss of Acf1. These data suggest that Acf1 may be involved in repression or activation of Abd-B in different contexts. Transcriptional repression of Abd-B by Acf1 is consistent with its function in the assembly of repressive chromatin. In fact, genetic evidence in yeast as well as polytene chromosome localization studies in Drosophila primarily implicate ISWI-containing complexes in transcriptional repression in vivo. Transcriptional activation of Abd-B by Acf1 could be due to its chromatin remodeling function, which could potentially facilitate transcription, or to an indirect effect, such as the repression of a transcriptional repressor of Abd-B (Fyodorov, 2004).

Surprisingly, Acf1 is not absolutely required for viability. Chromatin from homozygous acf1 mutant embryos exhibits less nucleosomal periodicity as well as a shorter repeat length than chromatin from wild-type embryos. Extracts from Acf1-deficient embryos assemble nucleosomes in vitro much less efficiently than wild-type extracts, and also that the deficiency in chromatin assembly can be rescued on addition of purified recombinant ACF or Acf1. These findings indicate that ACF/CHRAC is a major chromatin assembly activity in Drosophila, but also that Acf1-deficient flies contain other ATP-utilizing chromatin assembly factor(s) that are able to sustain partial viability (Fyodorov, 2004).

The analysis of the Acf1 null flies revealed that ACF/CHRAC performs different biological functions than NURF, even though ACF/CHRAC and NURF both share a common ISWI ATPase. Hence, the unique subunits of ACF/CHRAC and NURF can program the basic motor function of ISWI to perform specific biological tasks in vivo (Fyodorov, 2004).

ATP-utilizing motor proteins could potentially assemble or disrupt chromatin structure. Through multiple lines of investigation, the function of ACF/CHRAC was studied in vivo. (1) Whether there are genetic interactions between acf1 and nap1 was investigated, because the ACF/CHRAC motor protein and the NAP-1 histone chaperone function together in chromatin assembly in vitro. Double mutant nap1/nap1; acf1/acf1 flies exhibit a homeotic transformation that is not seen in the corresponding single mutant flies. These results are consistent with the biochemical activities of ACF/CHRAC and NAP-1 in the chromatin assembly process (Fyodorov, 2004).

(2) The effect of Acf1 on heterochromatic transcriptional silencing was tested. In these experiments, suppression of pericentric position-effect variegation was detected on loss of Acf1. It was additionally found that Acf1-deficient flies exhibit reduced levels of Polycomb-mediated transcriptional silencing. These findings indicate that ACF/CHRAC is important for the establishment and/or maintenance of repressive chromatin states (Fyodorov, 2004).

(3) Whether Acf1 enhances or disrupts chromatin-mediated repression of DNA replication was investigated. Shortening of S phase was observed in Acf1-deficient embryos and larval neuroblasts, consistent with a role of ACF/CHRAC in the assembly rather than disruption of chromatin in vivo. The effect of chromatin structure on the duration of S phase in larvae was investigated with a deficiency that uncovers the histone gene cluster. These animals contain reduced levels of histones and exhibit an acceleration of late S phase progression in larval neuroblasts relative to that in wild-type flies. Thus, the mutation of acf1 as well as the reduction in the level of histones each correlate with an increase in the rate of S phase progression. These data collectively support a role of Acf1 in the assembly of histones into chromatin (Fyodorov, 2004).

In summary, several independent lines of experimentation implicate Acf1 in the formation of chromatin in vivo. These experiments provide evidence for the function of ACF/CHRAC (and other ATP-utilizing factors) in the assembly of chromatin in conjunction with the NAP-1 histone chaperone. They also include the unexpected finding of a role of ACF/CHRAC in Polycomb-mediated silencing as well as the discovery of mutations (acf1 and Df(2L)DS6) that result in an unusual increase in the rate of S phase. Lastly, the loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin, which support a role of Acf1 in the assembly of repressive chromatin. Hence, the collective biochemical and genetic data indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus (Fyodorov, 2004).

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

The ATPase ISWI is the molecular motor of several nucleosome remodeling complexes including ACF. The ACF-nucleosome interactions have been studied, and the characteristics of ACF-dependent nucleosome remodeling have been determined. In contrast to ISWI, ACF interacts symmetrically with DNA entry sites of the nucleosome. Two-color fluorescence cross-correlation spectroscopy (FCCS) 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 (Strohner, 2005).

This study examined the molecular mechanism of nucleosome remodeling by the ISWI-containing ACF chromatin remodeling complexes. It has been observed that ISWI binds asymmetrically to the nucleosome, thereby moving nucleosomes to the extremes of a linear DNA fragment. In contrast, ACF moves nucleosomes to central positions of the same DNA fragment. Accordingly, symmetrical contact sites of ACF on the nucleosome have been observed, in contrast to the single-sided ISWI-nucleosome interaction. Contacts of ACF with the nucleosome resemble the interactions of ISWI, yISW2 and dNURF with the nucleosome, in that these complexes bind to the DNA linker and the DNA entry-exit sites of the nucleosome. ACF-nucleosome interactions extend ~40 bp into the nucleosome up to superhelix location 2 (SHL2) and the EB-binding assay detects ACF-dependent DNA accessibility close to SHL2. The histone H4 tail is required for the ATPase activity of ISWI and the nucleosome crystal structure and crosslinking data suggest that the histone H4 tail interacts with DNA around the SHL2 site. Hence, the proximity of the histone H4 tail with this region is consistent with its critical function in the remodeling process (Strohner, 2005).

The FCCS analysis indicates that ACF consists of a heterotetramer of two ISWI (119 kDa) and two Acf1 (170 kDa) subunits with an apparent molecular mass of 690-730 kDa, similar to CHRAC. In addition, size-exclusion chromatography and glycerol gradients of ACF revealed an apparent complex size of ~600-700 kDa in the absence of DNA. Previously, the molecular mass of ACF was estimated to be between ~220 and 400 kDa in glycerol gradients; this would correspond to a heterodimer of one molecule of Acf1 and ISWI. However, recent data and biochemical experiments (P. Becker, personal communication to Strohner, 2005) support these findings since they show the presence of more than one ISWI and Acf1 molecule in ACF. The ACF complex harbors four independent DNA-binding sites, whereas only one DNA duplex is bound to ISWI. The FCCS and footprinting data suggest that ACF places two ISWI molecules symmetrically at the DNA entry site of the nucleosome. This remodeler-nucleosome topology would not favor nucleosome repositioning to the borders of the DNA fragment because ISWI could introduce DNA into the nucleosome from either side, eliminating the directionality of nucleosome repositioning. Symmetrical binding of the motor molecules to the DNA entry site would probably position nucleosomes at thermodynamically favored sites. Such a site seems to be the center of the rDNA fragment that contains highly structured DNA (Strohner, 2005).

The mechanism by which nucleosomes are moved over DNA is still under discussion and the different families of remodeling ATPases may use the same mechanism, irrespective of the different outcomes in various remodeling assays. Data show the acquirement of DNA twist with all remodeling complexes tested. However, remodeling has been shown not to be affected by DNA nicks and DNA flaps in the path of the moving nucleosome. Although these nucleosome modifications do not have substantial effects, interpretation of the experiments is complicated and a possible role for twist defect diffusion in nucleosome remodeling can not be excluded. However, recent data show Rsc-dependent nucleosome remodeling on DNA molecules exhibiting gaps in one DNA strand, suggesting a looping mechanism for SWI/SNF-like remodeling complexes. Other studies mapping the increments of nucleosome movements indicate that different classes of ATPases move nucleosomes in steps that are multiples of ~10 bp. In these experiments nucleosome movements in single base pair steps were not observed, which would be expected for a mechanism that involves DNA twisting. However, the fast remodeling kinetics and the asynchronous remodeling reaction pose a challenge for the detection of remodeling intermediates. Therefore modified nucleosomes were generated that allowed elucidation of the remodeling mechanism. The data exclude a mechanism that involves DNA rotation, because this process would disrupt nucleosomes coupled to magnetic beads (Strohner, 2005).

SWI/SNF-type remodeling complexes have been shown to generate accessible DNA during nucleosome remodeling, whereas DNA accessibility was not obvious for ISWI-containing remodeling complexes. This study applied a novel assay, in which ethidium bromide binding and crosslinking was used to detect a transient site-specific detachment of the nucleosomal DNA. As predicted by the loop recapture model, ACF-dependent incorporation of EB at the borders of the nucleosome at two regions, overlapping with the ACF-nucleosome interactions, was observed. At these sites binding of ACF increases the accessibility of DNA. Thus, for the first time direct evidence is presented that remodelers of the ISWI class remove DNA locally from the nucleosome, indicative of DNA loop formation. Nucleosome remodeling ATPases belong to the helicase superfamily 2 (SF2) suggesting that Snf2p-related ATPases would be ATP-dependent DNA translocases. Like these, ISWI may bind or hydrolyze ATP at steps subsequent to substrate binding. In agreement with these data, a recent report analyzing the yeast Isw2 complex suggests that ATP is hydrolyzed subsequent to the nucleosome relocation step. It was concluded that ATP hydrolysis would lead to DNA release from the DNA-binding site of the remodeler and change its conformation to an extended form. This is in agreement with the finding that for ACF both the substrate-binding step and the introduction of the DNA loop do not require ATP (Strohner, 2005).

In summary, the data presented in this study strongly suggest that nucleosome repositioning by ISWI-containing remodelers follows the loop recapture model. Based on the symmetric footprinting pattern, subunit composition of ACF and the FCCS data, a model is favored in which the multiple DNA-binding sites per ACF complex allow the formation of a topological domain. Changing their spatial arrangement upon substrate binding induces the formation of a DNA loop. A subsequent coordinated release of the DNA-binding sites would then lead to migration of the DNA loop over the histone octamer surface and result in nucleosome movement (Strohner, 2005).

The histone fold subunits of Drosophila CHRAC facilitate nucleosome sliding through dynamic DNA interactions

The chromatin accessibility complex (CHRAC) is an abundant, evolutionarily conserved nucleosome remodeling machine able to catalyze histone octamer sliding on DNA. CHRAC differs from the related ACF complex by the presence of two subunits with molecular masses of 14 and 16 kDa, whose structure and function have been unknown. The structure of Drosophila CHRAC14-CHRAC16 has been determined by X-ray crystallography at 2.4-A resolution and it was found that they dimerize via a variant histone fold in a typical handshake structure. In further analogy to histones, CHRAC14-16 contains unstructured N- and C-terminal tail domains that protrude from the handshake structure. A dimer of CHRAC14-16 can associate with the N terminus of ACF1, thereby completing CHRAC. Low-affinity interactions of CHRAC14-16 with DNA significantly improve the efficiency of nucleosome mobilization by limiting amounts of ACF. Deletion of the negatively charged C terminus of CHRAC16 enhances DNA binding 25-fold but leads to inhibition of nucleosome sliding, in striking analogy to the effect of the DNA chaperone HMGB1 on nucleosome sliding. The presence of a surface compatible with DNA interaction and the geometry of an H2A-H2B heterodimer may provide a transient acceptor site for DNA dislocated from the histone surface and therefore facilitate the nucleosome remodeling process (Hartlepp, 2005).

An enhancement of ACF-dependent nucleosome sliding by HMGB1, an abundant structure-specific DNA binding protein, has been documented (Bonaldi, 2002). Because the activating function of HMGB1 did not correlate with the strength of DNA binding but depended on a dynamic (i.e., weak) interaction with DNA, it was suggested that HMGB1 might act as a DNA chaperone that promotes the distortion of DNA at its entry into the nucleosome. Manipulating DNA-histone interactions at this strategic site is likely to be a rate-limiting step in the current prevailing models of nucleosome mobilization. Analysis of the properties of the p14-p16 heterodimer reveals a number of similarities to HMGB1. Both entities enhance the catalysis of nucleosome sliding at limiting ACF concentrations. Both bind DNA weakly and nonspecifically. In either case, the dynamics of DNA interaction is assured by the presence of highly acidic C termini (on HMGB1 and on p16). Deletion of these charged C termini leads to a dramatic increase of DNA binding by the remaining histone folds and HMG domains, respectively. Under those conditions, ACF-dependent nucleosome sliding is not stimulated but repressed, indicating that tight binding leads to a locking of nucleosomal positions, as has been observed upon interaction of linker histone with nucleosomes (Hartlepp, 2005).

The striking analogies between the properties of HMGB1 and the p14-p16 heterodimer led to a speculation that the small histone fold subunits of CHRAC may serve as a built-in DNA chaperone that aids the disruption of DNA-histone interactions during the remodeling process by transiently providing a DNA binding surface. The histone fold heterodimer of p14-p16 resembles the geometry of H2A-H2B but lacks the tight grip of their interacting side chains. p14-p16 thus provide a surface that may lend itself for transient deposition of a segment of DNA stripped off the histone octamer surface. Furthermore, the acidic C-terminal tail of CHRAC16 might be in place to serve as a transient acceptor for a positively charged histone surface, such as the N terminus of histone H3 that reaches out into the linker DNA (Hartlepp, 2005).

It has been suggested that some nucleosome remodeling enzymes use H2A-H2B heterodimer exchange to facilitate remodeling. In this respect, the presence of histone-fold subunits in CHRAC with an overall structure and charge distribution similar to histones H2A-H2B is worth noting. Replacement of H2A-H2B with p14-p16 would lead to significant nucleosome destabilization. In the nucleosome, the region following helix alphaC in histone H2A forms a two-stranded ß-sheet with the C-terminal end of a neighboring H4 histone, which stabilizes the octamer structure. In p16, this region corresponds to helix alphaC, which packs against helices alpha2 and alpha3. In a hypothetical model where the p14-p16 heterodimer would replace H2A-H2B in the nucleosome, the p16 helix alphaC would prevent a similar interaction with the histone H4 C terminus and might considerably destabilize the nucleosome. However, the fact that a destabilization of nucleosomes is never observed during CHRAC-induced remodeling argues against such a scenario. In addition, the observed differences in the core structures argue against a possible exchange during remodeling (Hartlepp, 2005).

CHRAC is an evolutionarily conserved machinery. Recently, the stimulatory role of the human homologues of p14 and p16 on human ACF has been reported, but that study did not provide a mechanistic explanation. The human p14-p16 homologues, hCHRAC17 and hCHRAC15, also contain acidic glutamate- and aspartate-rich C termini of different length. However, in unresolved contrast to the current findings, Kukimoto (2005) reported a reduced DNA binding upon deletion of the negatively charged tail domains (Hartlepp, 2005).

The question of whether ACF and CHRAC exist as independent entities in living cells is still unanswered. The lack of suitable mutations in metazoan cells or reagents to localize the histone fold subunits in nuclei with confidence have hindered the exploration of their physiologic functions. Under these circumstances, the existence of homologous proteins in yeast provides valuable information. Recently, the Saccharomyces cerevisiae histone fold proteins Dls1p and Dpb4p were shown to associate with the Isw2 remodeling complex to form an entity reminiscent of CHRAC (Iida, 2004; McConnell, 2004). The similarity between the yeast Isw2 and the metazoan ACF complexes was previously not appreciated due to the very limited similarity between ACF1 and Itc1p, the largest subunit of the Isw2 complex. Strikingly, the two proteins only show similarity in their very N terminus with a recognizable WAC motif, which has been shown to be involved in the interaction with the histone fold subunits. The precise role of Dls1p and Dpb4p is still unclear, since yeast CHRAC appears to counteract telomeric silencing but, in contrast, is involved in the repression of some target genes. The situation is complicated by the fact that Dpb4p is also a subunit of a DNA polymerase epsilon complex, and mutant phenotypes may therefore reflect composite functions. Isw2-dependent repression of transcription and nucleosome repositioning is variably effected by mutation of the DLS1 gene at different gene loci, suggesting the possibility that two complexes related to ACF and CHRAC also exist in yeast, differing only by the presence of the histone fold subunits. Resolution of these issues will be facilitated by localization of all CHRAC components in living cells (Hartlepp, 2005 and references therein).

ACF catalyses chromatosome movements in chromatin fibres

Nucleosome-remodelling factors containing the ATPase ISWI, such as ACF, render DNA in chromatin accessible by promoting the sliding of histone octamers. Although the ATP-dependent repositioning of mononucleosomes is readily observable in vitro, it is unclear to which extent nucleosomes can be moved in physiological chromatin, where neighbouring nucleosomes, linker histones and the folding of the nucleosomal array restrict mobility. In this study arrays were assembled consisting of 12 nucleosomes or 12 chromatosomes (nucleosomes plus linker histone) from defined components and subjected to remodelling by Drosophila ACF or the ATPase CHD1. Both factors increased the access to DNA in nucleosome arrays. ACF, but not CHD1, catalysed profound movements of nucleosomes throughout the array, suggesting different remodelling mechanisms. Linker histones inhibited remodelling by CHD1. Surprisingly, ACF catalysed significant repositioning of entire chromatosomes in chromatin containing saturating levels of linker histone H1. H1 inhibited the ATP-dependent generation of DNA accessibility by only about 50%. This first demonstration of catalysed chromatosome movements suggests that the bulk of interphase euchromatin may be rendered dynamic by dedicated nucleosome-remodelling factors (Maier, 2008).

Due to the abundance of linker histones in interphase chromatin, H1-containing nucleosome arrays are probably the most common and physiological substrate for ATP-dependent chromatin remodelling factors. It is therefore important to understand whether and how these complexes can deal with the linker histone. So far, the literature mostly suggested that linker histones hinder chromatin remodelling. Residual remodelling activity has largely been attributed to incomplete loading of the substrate with linker histones. Attempts were made to rule out this experimental shortcoming by tightly controlling the stoichiometric incorporation of linker histones into chromatin arrays. Yet, ACF was able to induce the movement of entire chromatosome units throughout extended arrays. Importantly, the inability of CHD1 to remodel H1-containing chromatin confirms the inhibitory nature of the chromatosome array. These data are in accordance with previous findings in a crude, undefined system that nucleosome movements can occur within H1-containing chromatin, but they present the first direct demonstration of ATP-dependent chromatosome mobility in a defined chromatin array (Maier, 2008).

The results are surprising in light of the documented impediments of linker histones on nucleosome remodelling. First, H1 binding limits the amount of free linker DNA, which is known to determine the efficiency of ACF-dependent remodelling (Yang, 2006; Gangaraju, 2007). Second, H1 is likely to compete with ISWI-type remodellers for nucleosomal binding sites. In addition, H1 is believed to constrain the path of DNA entering and exiting the nucleosome and may therefore hinder DNA translocation. Finally, the increased compaction promoted by linker histones might restrict the access of remodelling factors towards the chromatin fibre. According to both currently favoured models for the structure of the 30-nm fibre, the linker DNA and hence all points of access for remodelling enzymes are located inside the chromatin fibre. The cation concentrations in these experiments promote the compaction of the nucleosomal array (Maier, 2008).

In spite of these possible constraints, a considerable ACF- and ATP-dependent repositioning of chromatosomes was observed. It is considered that H1 purified from Drosophila embryos might carry modifications, decreasing its affinity for chromatin. For example, the extensive phosphorylation of linker histone C-termini interferes with DNA binding and relieves its inhibitory impact on SWI/SNF-dependent chromatin remodelling. However, mass spectrometrical analysis of histone H1 purified from Drosophila embryos did not reveal extensive phosphorylation. It is therefore considered unlikely that phosphorylation impacted the outcome of these experiments (Maier, 2008).

The inhibitory effect of histone H1 on nucleosome remodelling was apparent when CHD1 was used as a remodelling enzyme. Notably, CHD1's activity on nucleosome arrays was equal to that of ACF, ruling out a defective activity of CHD1. Rather, ACF appears particularly suited for coping with linker histones. This is supported by the observation that ACF can assist the assembly of H1-containing chromatin arrays, whereas CHD1 can only promote assembly of H1-free chromatin. Recently, the ISWI-containing remodelling factor NURF has been suggested to be involved in modulating the association of H1 with chromosomes in vivo (Corona, 2007). The ability to slide chromatosomes may thus be a more widespread property of remodelling enzymes (Maier, 2008).

How might ACF achieve chromatosome repositioning? ACF may directly catalyse the eviction of H1 before nucleosome sliding, and a number of reports indicate that nucleosome-remodelling factors can, in principle, disrupt the DNA interactions of other proteins than core histones. Although it was not possible to detect free linker histones during remodelling, the analysis does not exclude that a fraction of H1 is transiently dislocated to secondary sites on the nucleosome array or an acceptor site on ACF. In vivo, linker histone displacement may be facilitated by cooperating histone chaperones. ACF and the histone chaperone NAP1 can act in concert towards the assembly of H1-containing chromatin, and it is thus conceivable that in cells ACF may cooperate with chaperones to catalyse the reverse reaction, which is the eviction of linker histones. However, since no chaperone was included in this experiment, alternative mechanisms have to be considered (Maier, 2008).

Chromatosome movements might already be facilitated if only the linker histone's globular domain was transiently detached from the nucleosome, while the C-terminal tail remained associated with the linker DNA. Such a scenario is reminiscent of documented changes on H1 interaction due to transcription, where selective crosslinking in Drosophila showed that the globular domain but not the C-terminal tail of linker histones was reversibly displaced from chromatin. In line with these considerations, the C-terminal tail contributes to H1 binding to DNA and determines its residence time on chromatin in living cells (Maier, 2008).

The analysis of chromatosome positions by primer extension revealed that in the arrays H1 protects DNA from nuclease digestion only on one side of the nucleosome, suggesting an asymmetrical binding of H1. This asymmetrical interaction, combined with the repetitive nature of the 601 array, endows the entire array with directionality. Although the precise topography of the ACF-nucleosome complex is not known at present, it has suggested on the basis of site-directed DNA affinity labelling that the related ISW2 complex interacts with linker DNA only on one side of the nucleosome (Kagalwala, 2004; Dang, 2006). It is thus speculated that ACF may interact with nucleosomal linker on the side that is not contacted by the globular domain of H1, in order to initiate the remodelling reaction. Propagation of a 'looped segment' of DNA around the histone octamer would then lead to movement of the histone octamer and concomitant displacement of the globular domain. The domain would then have to relocate and bind to the new nucleosome dyad and DNA entry point. A testable prediction of this hypothesis is that nucleosome sliding in presence of H1 would be unidirectional (Maier, 2008).

It is not knowm at this point whether ACF distributively targets individual nucleosomes within a nucleosome array or rather remodels neighbouring nucleosomes processively. In the latter case the fibre ends may provide points of entry. However, restriction enzyme accessibility assays did not reveal a gradient of increased accessibility towards the ends of the array, as might be expected from such a scenario. In contrast ACF is known to remain bound to its initial substrate during chromatin assembly, and it was observed earlier that nucleosomes within extended arrays were repositioned by Drosophila embryonic extract in apparent synchrony. Further experiments are required to clarify this issue (Maier, 2008).

This study provides the first evidence that ATP-dependent nucleosome-remodelling factors can mobilize entire chromatosomes, even if they reside in extensive arrays. Hence, the majority of euchromatin might be characterized by mobile nucleosomes and chromatosomes (Maier, 2008).

Protein Interactions

To determine whether the majority of Acf1 exists in the ACF complex, the native form of Acf1 was purified from Drosophila embryos using a Western blot assay with antibodies against Acf1. In these experiments, the relation between Acf1 and two other Drosophila ISWI-containing complexes [nucleosome remodeling factor (NURF) and chromatin-assembly complex (CHRAC)] were investigated. NURF consists of ISWI (Tsukiyama, 1995), NURF-55 (Martínez-Balbás, 1998 [note: NURF-55 is identical to dCAF-1 p55; Tyler, 1996]), inorganic pyrophosphatase (Gdula, 1998), and a 215-kD subunit. CHRAC is a complex of 670 kD native mass (as determined by gel filtration) that contains ISWI, topoisomerase II, and proteins with apparent molecular masses of ~175, 20, and 18 kD (Varga-Weisz, 1997). Therefore, in the course of the purification of native Acf1, Western blot assays were carried out for Acf1 and ISWI as well as for dCAF-1 p55/NURF-55 and topoisomerase II to test for the potential copurification of Acf1 with NURF or CHRAC (Ito, 1999).

Acf1 (p170-p185) and ISWI coelute at a lower potassium phosphate concentration than topoisomerase II or dCAF-1 p55/NURF-55. The chromatin assembly activity of the protein fractions was also determined in a standard ACF assay in conjunction with micrococcal nuclease digestion analysis (Ito, 1997). ACF chromatin assembly activity comigrates precisely with Acf1 protein through the last three purification steps. Analysis of the protein composition of the most purified Acf1-containing fractions by SDS-PAGE and silver staining reveals that Acf1 (p170 and p185) and ISWI are the only polypeptides that comigrate with ACF activity. Notably, the p47 polypeptide that was seen previously in ACF preparations (Ito, 1997) is now not detected. Consistent with this observation, partial amino acid sequence of p47 was determined and found to be identical to Drosophila yolk protein 2. Thus, p47 appears to have been a contaminant in the earlier ACF preparations (Ito, 1999).

These results show that the purification of the native form of Acf1 leads to the isolation of the ACF complex, which comprises Acf1 (p170 and p185) and ISWI. Given that the ACF complex exhibits an apparent molecular mass of 220 kD (Ito, 1997) and the calculated molecular masses of Acf1 and ISWI are 170 and 119 kD, respectively, it appears likely that an ACF protomer consists of heterodimers of either p170 + ISWI or p185 + ISWI. These studies also suggest that the majority of Acf1 appears to exist as a subunit of ACF. It is therefore possible that Acf1 is a unique component of ACF, but it cannot be excluded that Acf1 may be present in other less abundant forms. Finally, the distinct purification properties of dCAF-1 p55/NURF-55 and topoisomerase II (a component of CHRAC) relative to ACF support the conclusion that ACF is distinct from NURF and CHRAC (Ito, 1999).

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 a set of nucleosomes lacking individual histone N termini were generated from recombinant histones. The conserved N termini (the N-terminal tails) of histone H4 were found to be essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, is 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. It is concluded that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate (Clapier, 2001).

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. The p175 subunit of CHRAC has been identified 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 (Eberharter, 2001).

A heterodimeric complex of Acf1 and ISWI previously had been termed 'ACF'. In this context, Acf1 significantly increases the activity of ISWI in chromatin assembly. Since Acf1 has been identified as a component of CHRAC, the impact of Acf1 on ISWI-induced nucleosome sliding was examined. The directionality of nucleosome sliding differs depending on whether the reaction is catalysed by ISWI alone or by CHRAC. Flag-tagged ISWI and Acf1 were expressed from baculovirus vectors in insect cells, affinity purified and assayed for nucleosome sliding. In agreement with previous results, catalytic amounts (2-3 fmol) of ISWI move a mononucleosome from the center of a 248 bp rDNA fragment to the fragment end. No mobility was observed when the end-positioned nucleosome is exposed to ISWI. In contrast to the movement generated by ISWI, CHRAC catalyses nucleosome sliding from the end to the center of the DNA fragment. Strikingly, CHRAC-type directionality of nucleosome sliding is also obtained if Acf1 is added to ISWI, either after separate expression or by co-expression of both proteins in Sf9 cells. While Acf1 alone is inactive for nucleosome sliding, it boosts ISWI activity by at least an order of magnitude such that 10-fold lower enzyme concentrations (0.3-0.5 fmol) are required for nucleosome mobilization. Most importantly, Acf1 changes the directionality of sliding such that end-positioned nucleosomes move to central positions (Eberharter, 2001).

In order to determine whether Acf1 has an additional effect on the kinetics of nucleosome mobility under these conditions, a time course of nucleosome mobility was performed. The amounts of enzymes were chosen such that complete mobilization of the nucleosome was expected after 90 min (10-fold less ACF than ISWI). At any given time point throughout the reaction, the ratio of nucleosomes that had been mobilized to those that had not moved was determined. Nucleosome movement in the two reactions proceeds with similar speed, indicating that ACF is about an order of magnitude more efficient in nucleosome mobilization than ISWI alone. This could be explained most readily if Acf1 stimulates the ATPase activity of ISWI. To determine whether this was the case, the enzymes were compared in standard ATPase assays. ISWI alone shows a robust (7-fold) nucleosome stimulation of ATPase. This response to a nucleosomal structure remains unaltered if Acf1 is added, either after separate expression or through co-expression. While Acf1 alone does not show any sign of ATPase activity, it also does not stimulate the ATPase of ISWI significantly (7-fold stimulation over the free DNA level in all cases) (Eberharter, 2001).

Deletion of the H4 N-termini completely abolishes the ability of CHRAC to slide nucleosomes, whereas removal of any other histone tail has only minor effects. In contrast, ISWI-induced sliding not only requires the histone H4 N-termini (like CHRAC), but is also impaired if any of the other tails are deleted. Since Acf1 modulates the directionality of nucleosome sliding to resemble that of CHRAC, the histone tail dependence of ACF-induced nucleosomal sliding was tested. As expected, deletion of the H4 tail completely abolishes the remodelling activity of ACF. Removal of any other histone tail, however, has only little influence on the sliding activity of ACF. This result reinforces the notion of a qualitative alteration of the nucleosome remodelling activity of ISWI by Acf1, which points to altered interaction with the nucleosomal substrate (Eberharter, 2001).

Acf1 is a member of a growing family of proteins with similar domain architecture including WSTF, one of the genes invariantly deleted in William-Beuren syndrome patients. In addition to several other sequence similarities, these factors contain a prominent C-terminal bromodomain and one or two PHD fingers. Acf1-like factors so far have been found exclusively in association with ISWI. Complexes consisting of just ISWI and an Acf1-like factor were purified from Drosophila, human and yeast cells. One of the ISWI-containing remodelling factors in Xenopus extracts contains an unknown protein (p175) in addition to ISWI and Acf1. CHRAC, defined as a four-subunit complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16, has been identified so far in human and fly cells. Although ISWI can function as a remodelling ATPase in certain in vitro assays, it has never been purified from a native source on its own. These data suggest that ISWI and an Acf1-like factor constitute a functional core module with which other proteins may associate to generate a family of diverse remodelling machines, potentially tailored to specific functions (Eberharter, 2001).

Acf1 was first identified as a protein associated with ISWI to form the nucleosome assembly and spacing factor ACF. While either recombinant ISWI or Acf1 alone is only poorly active in an assembly system, the reconstitution of ACF from the two subunits increases the in vitro nucleosome assembly activity by some 30-fold. ISWI, expressed in a bacterial system, can, in principle, function autonomously in various cell-free remodelling assays. The direct comparison of the activity of factors expressed under similar conditions from baculovirus vectors shows that Acf1 enhances ISWI-induced nucleosome mobility by about an order of magnitude. In addition, the association of Acf1 has a striking qualitative effect as it alters the directionality of nucleosome sliding triggered by ISWI and affects the sensitivity of the ATPase towards deletion of the histone N-termini on the nucleosomal substrate. ISWI and Acf1 approach the nucleosome in a co-ordinated manner, leading to a new quality of interaction, such that Acf1 does not simply enhance the action of ISWI. Whether Acf1 interacts with DNA directly, effectively hindering the sliding of the nucleosome to the fragment end, remains to be explored. Upon association of ISWI with Acf1, 10-fold lower enzyme concentrations and correspondingly fewer ATP hydrolysis events are required to move a nucleosome as compared with free ISWI alone. It is possible that ACF has a higher affinity for the nucleosomal substrate, due to interaction domains contributed by Acf1. A decreased off-rate may lead to a higher processivity of the enzyme, converting the energy of ATP hydrolysis more effectively into directional nucleosome sliding. Testing this and alternative hypotheses will require more quantitative measurements of the parameters of the nucleosome sliding reaction (Eberharter, 2001).

The PHD finger and bromodomain are likely to be involved in Acf1 activity. PHD fingers are protein interaction surfaces found in many chromatin-bound regulators. Bromodomains are equally abundant among nuclear regulators. They are a hallmark of the remodelling ATPases of the SWI2/SNF2 type, but are absent in ISWI. Bromodomains are known interactors of acetylated histone H4 N-termini. Nucleosome remodelling by ISWI critically depends on the integrity of the H4 tail on the nucleosomal substrate. It is possible that tandem PHD fingers and a bromodomain form a cooperative interaction unit, as has been suggested recently for the KRAB proteins. It will be interesting to see whether Acf1 interacts directly with the H4 tail during nucleosome mobilization and whether histone acetylation modulates this process. The function of the N-terminal WAC domain of Acf1, which has been implicated in targeting proteins to heterochromatin, is unknown (Eberharter, 2001).

Although CHRAC was first perceived due to its activity to render nucleosomal DNA accessible, it was soon discovered that CHRAC and the related ACF may have an important role in the assembly of regular nucleosomal arrays in vitro. Nucleosome mobility is not restricted to the assembly phase, but can also be observed within an ordered nucleosomal array. CHRAC may be involved mainly in the assembly and maintenance of nucleosomal arrays with dynamic properties. Several observations are in line with such a function. (1) The in vitro phenomenology shows that CHRAC and ACF can catalyse the assembly of dynamic nucleosomal arrays. (2) The restricted expression of ISWI, Acf1 and the CHRAC-14/16 pair during Drosophila embryonic development correlates with the time of most intense nuclear division. (3) Proteins with similarity to CHRAC-14 and CHRAC-16 have been found to associate with human DNA polymerase epsilon. (4) Mutation of ISWI in male flies leads to a striking abnormality of the structure of the male X chromosome, which is marked and perhaps sensitized by specific acetylation of the histone H4 N-terminus, although the additional presence of ISWI in NURF complicates the interpretation of the mutant phenotype. The outcome of rendering nucleosomes mobile may depend on the circumstances: in vitro, CHRAC can facilitate SV40 replication by promoting the access of T antigen to a nucleosomal origin. In contrast, an ACF-like complex contributes to the targeted repression in yeast, presumably by modulating nucleosome positions in the promoters of meiosis-specific genes (Eberharter, 2001 and references therein).

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, their mutual interaction requirements were mapped and a series of ACF complexes were generated lacking conserved ACF1 domains. Deletion of the C-terminal PHD finger modules of ACF1 or their disruption by zinc chelation profoundly affects 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 (Eberharter, 2004).

Therefore, binding of ACF1 to ISWI leads to a remarkable increase in energy efficiency of the nucleosome remodelling reaction catalysed by ISWI: while the amount of ATP hydrolysed in response to the nucleosome substrate remains unchanged upon ACF1 interaction, the complex moves nucleosomes an order of magnitude more efficiently than ISWI alone. This study finds that the PHD modules in the C-terminus of ACF1 are crucially involved in this activation. Previously this boost of activity has been found only in conjunction with a change of nucleosome sliding directionality. Deletion of the PHD domain selectively affects the efficiency of the sliding reaction, but does not alter the type of nucleosome movement. It appears, therefore, that structures in the N-terminus of ACF1, or simply the fact that ACF1 interacts with ISWI, determine the qualitative outcome of nucleosome mobilization (Eberharter, 2004).

PHD fingers have been proposed to serve as protein interaction surfaces, but the demonstration of contacts with the central parts of core histones in the context of a nucleosome remodelling factor is novel. Disruption of the PHD structure through zinc chelation destroys the interaction of ACF1 with histones and at the same time abolishes the stimulatory effect of ACF1 on the efficiency of nucleosome sliding. Evidently, ACF1-histone contacts are crucial for efficient nucleosome remodelling. The results are in conflict with those obtained earlier by Fyodorov (2002), who did not observe a detrimental effect of deleting the PHD or bromodomain modules in an ACF-dependent chromatin assembly system. This discrepancy may be due to the different functional assays employed (chromatin assembly versus nucleosome sliding) and perhaps also to differences in the protein expression protocol: whereas Fyodorov was unable to express soluble ACF1, the current procedure yielded soluble protein that could be functionally characterized (Eberharter, 2004).

According to the favourite model, ISWI-containing remodelling factors lift DNA off the histone surface at the edge of the nucleosome and distort it into a bulge or loop. Propagation of this distortion over the surface of the histone octamer leads to nucleosome relocation. It is considered that efficient displacement of nucleosomal DNA relative to the histone octamer requires contacts of the remodelling enzyme with both the DNA and histone moieties. ISWI alone binds nucleosomes mainly through interactions of its C-terminal SLIDE domain with nucleosomal DNA (Grüne, 2003). Although a segment of the H4 N-terminus is absolutely required for ISWI function (Clapier, 2001), no stable interactions of ISWI with histones are known. ISWI may be a relatively inefficient remodelling enzyme because it lacks a stable anchoring point on the histone body. The PHD-histone contacts documented in this study may provide such an anchoring point for the enzyme, assuring that the presumed conformational changes triggered by ATP binding and hydrolysis are efficiently converted into positional shifts of DNA relative to histones. According to a variation of this model, ACF plays an active role in the propagation of the DNA distortion ('the loop') around the histone octamer, which would necessitate changing contacts of the remodelling machinery with the histone octamer surface as it traverses around the particle. The observation that the PHD fingers of ACF1 interact with all four histones suggests that they recognize a common structural feature on the histone pairs, and is compatible with models involving multiple, different contacts of the remodeller on the histone octamer (Eberharter, 2004).

ACF1 is a prominent member of a family of related proteins in various species, which interact with ISWI to form several distinct nucleosome remodelling complexes such as ACF, NURF, WCRF and NoRC. These proteins share with ACF1 a similar domain organization including C-terminal PHD and bromodomains. A region of NURF301, the largest subunit of NURF, containing two PHD fingers and the adjacent bromodomain binds to all four core histones, but the functional consequences of these interactions have not been determined. Interestingly, human ACF1 (alias WCRF180) and the related human WSTF, which associate with the human homologue of ISWI, SNF2H, function with only one PHD module and the hSNF2H-interacting protein RSF1 does not share any sequence similarity with ACF1, except for one PHD module. It is therefore likely that one PHD module may be sufficient for function (Eberharter, 2004).

PHD fingers are diverse in sequence and may connect different proteins in various contexts. The observation that PHD modules of ACF1 serve to tether a nucleosome remodelling enzyme to its substrate adds a new function to the list that should be tested for the known or suspected modifiers of chromatin structure, such as the remodelling ATPase Mi-2, the epigenetic regulators Trithorax, Polycomb-like, Ash1, Ash2 and Lid (Eberharter, 2004).

PHD modules are frequently found in the direct neighbourhood of a bromodomain. Examples include the ACF1-related proteins discussed here, and also the histone acetyltransferases CBP and p300 and the KAP1 repressor. From their functional analysis of domains involved in KAP-1 repression, it has been concluded that both domains form an integrated, cooperative unit involved in binding the Mi2alpha subunit of the NuRD complex. In the context of histone binding, the idea of cooperativity between PHD fingers and bromodomains is attractive. While it is well established that bromodomains interact preferentially with acetylated N-termini of histones H3 and H4, this study shows that the PHD modules of ACF1 interact with the central domains of the core histones. This interaction may thus be further modulated by additional contacts of bromodomain with appropriately modified N-termini, as a means of fine-tuning nucleosome remodelling activity in response to the histone modification status. Whether this principle applies to ACF remains to be seen. However, the Bromo-PHD modules of the histone acetyltransferase p300 have been functionally characterized and both domains have been found to cooperate for preferential binding to highly acetylated nucleosomes in a stringent assay (Eberharter, 2004).

Interestingly, deletion of the ACF1 bromodomain alone does not diminish nucleosome sliding, but consistently improves it. Given the limited knowledge on the structure of the ACF1 C-terminus as an entity, it is difficult to interpret this observation. Conceivably, the interaction of bromodomain with another domain of the remodelling machinery dampens its activity until a conformational change triggered by its interaction with an acetylated histone N-terminus unleashes full remodelling potential. This scenario suggests a strategy by which histone acetylation could regulate remodelling activity other than by simply increasing the affinity of the remodelling machinery for the substrate (Eberharter, 2004).

This study mapped AID, of ISWI, required for ACF1 binding, to the very C-terminus of ISWI, directly adjacent to the SANT/SLIDE module, the nucleosome interaction determinant of ISWI. Through this interaction, ACF1 is brought close to the nucleosome surface, but this interaction also leads to the apposition of several domains on both ACF subunits: the SANT domain of ISWI, which may be involved in contacting histone tails; the SLIDE domain of ISWI, which contacts nucleosomal DNA (Grüne, 2003); the PHD fingers of ACF1, which binds the histone octamer surface; and finally bromodomain with its potential for interactions with appropriately modified histone N-termini. Unravelling the sequence and dynamics of enzyme-substrate contacts during the remodelling process remains a major challenge for future research (Eberharter, 2004).

DEVELOPMENTAL BIOLOGY

Embryonic

To determine whether or not the levels of Acf1 vary in the course of the growth and development of the organism, Western blot analyses of Acf1 have been carried out throughout Drosophila development. In these experiments affinity-purified antibodies against Acf1, ISWI, and dNAP-1 were used in conjunction with approximately equivalent amounts of total protein derived from Drosophila at each of several stages of development. Acf1 (both p170 and p185 forms) and ISWI are readily detected in embryos, but their levels are significantly lower in larvae, pupae, and adults. However, dNAP-1 is present at high levels throughout development, except in adult males. With Acf1, it is also notable that there does not appear to be any significant variation in the relative amounts of the p170 versus p185 forms of the protein throughout embryogenesis. The presence of the highest levels of Acf1 and ISWI in embryos roughly correlates with the high amounts of DNA replication and chromatin assembly as well as transcription and chromatin remodeling that occurs during embryogenesis (Ito, 1999).

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date revised: 15 May 2008

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