Oncogene (2001) 20, 3076 ± 3085 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

ATP-dependent chromatin remodeling factors: Nucleosome shu‚ers with many missions

Patrick Varga-Weisz*,1

1Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 OTL, UK

This review addresses recent developments in the ®eld of tion and methylation; (ii) through nucleosome structure ATP-dependent chromatin remodeling factors. These alterations: They may change position with respect to factors use the energy of ATP hydrolysis to introduce the DNA sequence, the path of the DNA around superhelical torsion into DNA, which suggests a common nucleosomes may be altered, and histones may be mechanistic basis of action. Chromatin remodeling removed. The major activities that are involved in factors function both in transcriptional activation and nucleosome structure alterations use the energy repression, but they may have roles outside of transcrip- supplied by ATP hydrolysis to a€ect nucleosomes. tional regulation such as DNA repair. A study of the These enzymes are called ATP-dependent chromatin nucleosome dependent ATPase ISWI in yeast illustrates (or nucleosome) remodeling factors. Some of the recent the involvement of ATP-dependent chromatin remodeling insights regarding these activities are the topic of this in transcriptional repression by setting up inaccessible review as well as a discussion how they may be chromatin structures at promoters. However, factors involved in transcription regulation and other func- such as ISWI are also involved in the restructuring of tions. large chromatin domains and even whole chromosomes. Most ATP-dependent chromatin remodeling factors Transcriptional regulation by ATP-dependent chromatin are multi subunit complexes with an ATPase as the remodeling factors occurs in concert with histone catalytic center. These ATPase subunits can be modifying enzymes such as histone acetyltransferases classi®ed into three families; the SWI2/SNF2-, the and histone deacetylases: In yeast, SWI/SNF targeting Mi-2/CHD-, and the ISWI-ATPases (Eisen et al., is a requirement for histone acetyltransferases activity at 1995). A short summary of the ATPases and the promoters that are active at late stages of mitosis, when complexes is in Figure 1. The ATPases contain a highly the chromatin is still condensed. This demonstrates that conserved ATPase core domain, which is surrounded ATP-dependent remodeling factors facilitate covalent by N- and C-terminal domains, which di€er consider- histone modi®cations. However, they are also regulated ably between these ATPase families (see Figure 1). The by histone modi®cations and in some circumstances they complexes di€er in the number of subunits, ranging function in parallel with histone modi®cations towards from two in some ISWI complexes to 11 or more in the the same goal. Oncogene (2001) 20, 3076 ± 3085. SWI/SNF complexes (reviewed in Kingston and Narlikar, 1999; Vignali et al., 2000). The nucleosome Keywords: DNA superhelical torsion; chromatin remodeling ATPase CHD1 from budding yeast may domain; Bromodomain; histone code; DNA repair function in non-complexed form (Tran et al., 2000). The yeast SWI/SNF complex was the ®rst ATP- dependent chromatin remodeling factor to be identi- Introduction ®ed, both by genetics and biochemistry. In budding yeast there is a similar factor, called RSC, and there Nucleosomes are the building blocks of chromatin and are SWI/SNF homologues in all eukaryotes. ISWI- are composed of protein spools made from histone containing factors were identi®ed as major nucleosome octamers, around which the DNA wraps in almost two remodeling force in ¯y, yeast, frog and human cell turns. The nucleosomes form arrays, which are the extracts. Mi-2/CHD containing factors contain also basis for higher order chromatin structures. Nucleo- histone deacetylases and are involved in transcriptional somes play a major role in all aspects of eukaryotic repression. transcriptional regulation by limiting access to sites on the DNA, e.g., transcription factor binding sites. Nucleosomes play a regulatory role, because they can ATP-dependent chromatin remodeling factors alter the be altered in two principal ways: (i) through covalent positions of nucleosomes along DNA, rendering DNA modi®cations; most of these occur at the histone either accessible or inaccessible terminal `tails' and include acetylation, phosphoryla- The outcome of ATP-dependent chromatin remodeling on a nucleosome or a nucleosomal array varies *Correspondence: P Varga-Weisz depending on a number of factors: the enzyme in ATP-dependent chromatin remodeling factors P Varga-Weisz 3077

Figure 1 The three major identi®ed nucleosome remodeling ATPases, their domains, homologues, and complexes in which they are found. Each ATPase core domain has seven, highly conserved subdomains. This ATPase core-domains are braced by domains that di€er considerably between the three types of nucleosome remodeling ATPases action, the underlying DNA sequence and other tional repression (Holstege et al., 1998; Sudarsanam et proteins present. One common result in in vitro assays al., 2000; Sudarsanam and Winston, 2000). Further- is the change of nucleosomal positions along the DNA. more, the analysis suggested that the repression by This may be achieved with minimal disruption of SWI/SNF was not due to reduced transcriptional histone-DNA interactions by a mechanism best activator expression (Sudarsanam et al., 2000). described as nucleosome sliding along the path of the DNA (LaÈ ngst et al., 1999; Hamiche et al., 1999; Whitehouse et al., 1999; Jaskelio€ et al., 2000). Some ATP-dependent chromatin remodeling activities generate remodeling factors are able to disrupt nucleosomes in a superhelical torsion, which translates to nucleosome way that leads to histone octamer transfer to a separate remodeling segment of DNA (Lorch et al., 1999; Phelan et al., 2000). The presence of histone chaperones, which Central to the function of ATP-dependent chromatin sequester histones, and histone modi®cations such as remodeling factors are ATPases with sequence similar- acetylation may have a role in modulating the ity to DNA dependent helicases, which are enzymes nucleosome remodeling reaction and they may drive that separate the strands of DNA. However, as it has such a reaction towards nucleosome disruption (Ito et been shown for the SWI/SNF and the ISWI-containing al., 2000). In all cases the movement of nucleosomes NURF complexes, these chromatin remodeling en- may either increase or reduce the accessibility of a site zymes do not function as helicase in DNA strand for DNA binding proteins such as transcription displacement assays in general (CoÃte et al., 1994; Shen factors. Therefore, the nucleosome remodeling reaction et al., 2000). To detect transient DNA structure may lead to transcriptional activation or repression alterations by chromatin remodeling factors a novel and, indeed, the same remodeling factor may operate assay was necessary that allowed the monitoring of in both ways (Tyler and Kadonaga, 1999; Sudarsanam superhelical torsion of DNA (Havas et al., 2000). and Winston, 2000). In this way, repression could be The assay relies on a DNA structure called cruci- mediated by a restructuring of the chromatin from an form that is produced at inverted repeats of DNA if open to a closed con®rmation, and also by allowing the repeated sequence anneals with its complement on transcriptional repressors to bind to chromatin or by the same strand (see Figure 2a). Cruciform formation facilitating other modi®cations of the chromatin such requires base unpairing and is, therefore, energetically as deacetylation of histones. Whole-genome expression costly. DNA cruciform formation is a measure for analysis of swi/snf mutants in yeast revealed that the superhelical tension in DNA and can be monitored mRNA levels for many genes are elevated in the with enzymes that speci®cally cleave this structure. The mutants indicating a role for SWI/SNF in transcrip- puri®ed SWI/SNF complex causes cruciform extrusion

Oncogene ATP-dependent chromatin remodeling factors P Varga-Weisz 3078 a

b

Figure 2 ATP-dependent chromatin remodeling factors generate superhelical torsion. (a) Cruciform formation of DNA with inverted repeats. (b) Models demonstrating how an ATP-dependent chromatin remodeling factor may introduce negative superhelical tension into linear DNA. In order to introduce superhelical torsion, the factor creates a closed domain within the linear DNA. This may be through the tight interaction with something that holds onto the DNA (`BLOCK'). The `block' may be another factor molecule or a nucleosome. The block could also be created through the tight interaction of the factor with a speci®c site of the DNA, for example the end of the DNA molecule. In the `tracking' model (left arrow) the factor holds tight to the `block' on one

Oncogene ATP-dependent chromatin remodeling factors P Varga-Weisz 3079 from inverted repeats in linear DNA in an ATP- tation may result in a corkscrew type motion of the dependent fashion. Cruciform extrusion is normally DNA along the nucleosome surface and, therefore, a only observed in supercoiled plasmids, where the DNA relative sliding of the nucleosome along the DNA. linkage into a circle creates a closed domain. This There are several implications from the ®ndings means that the action of the SWI/SNF complex creates described above: Unlike ISWI and Mi-2, which need a domain within the linear DNA, where torsion cannot nucleosomal DNA, the SWI/SNF complex introduces be simply relieved by a rotation of the DNA around its superhelical torsion into naked DNA and this may not own axis. In this domain the DNA is suciently only a€ect the interaction of histones with DNA, but negatively supercoiled to support the stable existence of also the interaction of other proteins with DNA. The a cruciform. All known classes of nucleosome remodel- idea that SWI/SNF might act by dealing with non- ing ATPases function in this way, however, the ISWI histone proteins, such as transcriptional repressors ATPase and the Mi-2 chromatin remodeling complex Tup1/Ssn6p, Hir1/2 proteins and Sin1p, is indicated only on templates assembled into chromatin (Havas et from several studies in yeast (Gavin and Simpson, al., 2000). Because an energy-dependent change of 1997; Dimova et al., 1999; Perez-Martin and Johnson, DNA topology is common to all known chromatin 1998). Another possibility is that complexes such as remodeling ATPases, it is very likely at the mechanical SWI/SNF may introduce non-B type DNA structures basis of the activity of these enzymes. (such as cruciforms, Z-DNA, triplex DNA, and unwound DNA) in vivo (Havas et al., 2000). Since the discovery of these non-B DNA structures, it has How do ATP-dependent chromatin remodeling factors been suggested that they may have a role in regulatory generate DNA torsion? processes (reviewed in: van Holde and Zlatanova, 1994). For example, they may be involved in the To generate torsion the free rotation of the DNA must creation of accessible sites in chromatin by preventing be constrained to prevent the dissipation of the stress. nucleosome formation. Finally, a change of super- DNA loop formation is one way to achieve this, and, helical tension may have wide ranging e€ects through indeed, SWI/SNF dependent loops have been detected its impact on higher order chromatin folding (Travers, by microscopy (Bazett-Jones et al., 1999). Once 1992). constrained, rotation of the DNA creates torsion. Two mechanisms to twist DNA are illustrated in Figure 2b. One mechanism is tracking along of the ATP-dependent chromatin remodeling are recruited to helical DNA backbone against or from a ®xed point promoters and cooperate with histone modifying enzymes (e.g., another chromatin remodeller molecule or a nucleosome, Figure 2b, `tracking'), as has been All known classes of chromatin remodeling ATPases described for type I restriction enzymes (Janscak and are recruited to speci®c sites such as promoters by Bickle, 2000). The other mechanism is a molecular direct interaction with sequence speci®c DNA binding wrench action, as has been suggested for TFIIH in proteins, such as transcription factors. In addition, promoter melting (Kim et al., 2000), where two ATP-dependent chromatin remodeling factors coop- proteins change their positional interaction with one erate with histone modifying enzymes such as histone another whilst remaining bound to the DNA. (Figure acetyltransferases (HATs) and deacetylases in the 2b, `molecular wrench'). A sliding type of movement of promoter remodeling. HATs add acetyl groups to a protein along the helical path of DNA will lysines at the amino termini of the core histones, a necessitate rotation of the DNA helix around its axis reaction that is usually associated with activation of if the protein is simultaneously anchored to another gene expression (reviewed in: Brown et al., 2000; Howe site on the DNA (`BLOCK' in Figure 2b scheme). et al., 1999; Berger, 1999; Grant et al., 1998; Imhof and Indeed, all nucleosome remodeling factors are likely to Wol€e, 1998). Histone deacetylases reverse this mod- interact directly with nucleosomes. i®cation and have function in gene repression (reviewed It is not clear how the superhelical torsion in the in: Ng and Bird, 2000; Knoep¯er and Eisenman, 1999; DNA leads to nucleosome remodeling, but it is Ayer, 1999; Kuo and Allis, 1998; Struhl, 1998). Some plausible that it a€ects DNA-histone interactions. recent studies which illustrate this action on promoters Possible outcomes could range from simple rotation follow. of the DNA on the surface of the nucleosome to a In the NURD and related complexes the nucleosome peeling o€ the DNA from the nucleosome surface (for remodeling ATPase Mi-2 comes with a histone illustrative movies on this see: http://www.dundee.a- deacetylase in one package and the nucleosome c.uk/biochemistry/owenhughes/mech1.html). DNA ro- remodeling has been shown to assist histone deacetyla-

side, but tracks along one of the DNA strand backbones on the other side (from the base pair marked with a black dot away from the `block' to the base marked with an empty circle). At the end more DNA base pairs are between the tracking site of the factor and the `block', the DNA is negatively overwound. In the `wrench action' model the ATP-dependent chromatin remodeling factor constantly holds tight to a speci®c site on the DNA (black dot), but changes its position with respect to the block, with which it interacts, too. This necessitates a rotation of the DNA axis, which introduces torsion into the DNA

Oncogene ATP-dependent chromatin remodeling factors P Varga-Weisz 3080 tion in vitro (Wade et al., 1998; Zhang et al., 1998; The requirement of SWI/SNF dependent chromatin Tong et al., 1998). The Drosophila homologue of Mi-2 remodeling for Gcn5 HAT function of a subset of interacts with the transcriptional repressor Hunchback, yeast genes is the result of their activation at late stages which binds directly to regulatory sequences of HOX of mitosis, when the chromatin is still condensed genes (Kehle et al., 1998). The zinc ®nger protein (Krebs et al., 2000). Interestingly, several of the above Ikaros, important for normal B- and T-cell develop- mentioned studies on the relationship of SWI/SNF and ment, interacts with Mi-2 complexes in lymphoid and Gcn5, show that both activities are required at steps erythroid cells (Kim et al., 1999; O'Neill et al., 2000). subsequent to transcriptional activator binding (Cosma The yeast SWI/SNF complex interacts directly with a et al., 1999; Krebs et al., 2000; Gregory et al., 1999). It variety of transcriptional activators (reviewed in: is not known, what this step may be, it could be Peterson and Workman, 2000). A non-exhaustive list recruitment of the basal transcriptional machinery or of factors that are involved in recruitment of SWI/SNF transcriptional elongation. SWI/SNF has been shown (Table 1) illustrates the great variety of DNA sequence to facilitate elongation through chromatin (Brown and speci®c factors with which SWI/SNF may interact. The Kingston, 1997). fact that SWI/SNF is a huge complex (*2 Mda) with The budding yeast has two ISWI ATPase isoforms, many subunits that provide a variety of interaction Isw1 and 72, that form di€erent complexes (Tsukiya- surfaces could explain the diversity of interaction ma et al., 1999). The Isw2-chromatin remodeling factor partners. represses transcription of early meiotic genes during The recruitment of SWI/SNF may lead to transcrip- mitotic growth (Goldmark et al., 2000). This function tional activation, as in the case of the HO endonu- of the Isw2 complex is dependent upon the sequence clease promoter, where the Swi5p transcription factor speci®c DNA-binding factor Ume6p, which recruits the recruits SWI/SNF (Cosma et al., 1999; Krebs et al., complex to target genes, after which ISW2 establishes 1999). The cooperation of SWI/SNF and the HAT and/or maintains a nuclease-inaccessible chromatin Gcn5 is required at several promoters in yeast for structure near the Ume6p binding site. This remodeling chromatin remodeling leading to transcriptional activa- by ISWI results in transcriptional repression of a set of tion (Pollard and Peterson, 1997; Gregory et al., 1999; yeast genes. Another transcriptional repressor that is Holstege et al., 1998). An analysis of the timing of recruited by Ume6p is the histone deacetylase Rpd3 in binding at the HO endonuclease promoter indicates a complex with a protein called Sin3. The Isw2 that the SWI/SNF complex is recruited to the complex and Rpd3 work, therefore, towards the same promoter before Gcn5 (Cosma et al., 1999) and its goal of transcriptional repression, however, their action activity is required for later histone acetylation by is independent and not epistatic (Goldmark et al., Gcn5 (Cosma et al., 1999; Krebs et al., 1999, 2000). 2000).

Table 1 Factors involved in recruitment of yeast, ¯y and mammalian SWI/SNF complexes to speci®c sites Organism (ATPase) Reference Factors involved in targeting Comments

S. cerevisiae Neely et al., 1999 Gcn4, Swi5, Hap4, VP16 (Swi2/Snf2) Natarajan et al., 1999 Gcn4 Swi5 Krebs et al., 1999; Cosma et al., 1999 Yudkovsky et al., 1999 GAL4-AH/GAL4-VP16 Dimova et al., 1999 Hir1/Hir2 Recruitment by transcriptional repressors Drosophila Kal et al., 2000 Zeste Trithorax group protein (Brahma) Mammalian Kwon et al., 1994 GAL4-VP16/AH (brg1/brm) Brown and Kingston, 1997 Heat shock factor 1 Fryer and Archer, 1998 Glucocorticoid receptor Oestlund Farrants et al., 1997 Glucocorticoid receptor Trouche et al., 1997 E2F RB and hbrm cooperate to repress the activation functions of E2F1 Ichinose et al., 1997 Estrogen receptor Ligand dependent interaction Armstrong et al., 1998; EKLF SWI/SNF-related E-RC1 Lee et al., 1999; (Erythroid Kruppel-like complex Kadam et al., 2000 Factor) Wu et al., 2000 Epstein ± Barr virus nuclear protein 2 Cheng et al., 1999 C-MYC c-MYC interacts with SWI/SNF subunit INI1/hSNF5 Kowenz-Leutz and Leutz, 1999 C/EBP beta Myeloid gene activation Ito et al., 2001 Fos/Jun SWI/SNF subunit BAF60a as a determinant of transactivation Bochar et al., 2001 BRCA1

Oncogene ATP-dependent chromatin remodeling factors P Varga-Weisz 3081 NURF (Georgel et al., 1997) and the N-terminal histone H4 tail is essential for ISWI ATPase function and nucleosome mobilization by the CHRAC complex (Clapier et al., 2001). Nucleosome remodeling by the SWI/SNF and the related yeast RSC complexes is not sensitive to the hyperacetylation of the histone N- terminal tails (Logie et al., 1999). However, the histone tails are required to carry out multiple rounds of nucleosomal array remodeling and hyperacetylation of histone tails interferes with multiple round reactions (Logie et al., 1999). An interpretation of these results is that the histone N-termini and their acetylation status control the rate of the dissociation of SWI/SNF and RSC from nucleosomal arrays. Many chromatin remodeling factors have domains in one or more of their subunits that may be involved in recognizing modi®ed histones. One such domain is the bromodomain, which is found in many chromatin remodeling factors, including the Swi2 ATPase of the SWI/SNF complex, the Sth1 ATPase of the RSC complex, and ACF1, a protein which interacts with the ISWI ATPase. This domain has been studied in several Figure 3 A role for ATP-dependent chromatin remodeling HATs, where it interacts speci®cally with acetylated factors in resetting promoter chromatin to the repressed ground histone tails (Dhalluin et al., 1999; Jacobson et al., state. The major function of the SWI/SNF complex may be to set 2000; Ornaghi et al., 1999). Therefore, it may serve in up a speci®c promoter structure by allowing sequence speci®c the communication between histone acetylation and factors to bind to the promoter (the horseshoe shaped protein is chromatin remodeling in general. A recent study in the TATA-binding protein, TBP, but could stand for any factor). An ISWI-complex may have its major function in the reversion of yeast provides in vivo evidence for the regulation of this process, back to the initial `closed' promoter structure SWI/SNF chromatin remodeling by histone acetylation and demonstrates the importance of the bromodomain for this dependence (Syntichaki et al., 2000). A ATP-dependent chromatin remodeling factors may synthetic promoter was used to monitor gene activa- translate the `nucleosomal code' tion by the Gcn4 transcription factor. Here transcrip- tion activation is dependent on the remodeling of The many modi®cations of histones, such as acetyla- positioned nucleosomes by both the HAT activity of tion, phosphorylation, or methylation may have a Gcn5 as well as the nucleosome remodeling activity of direct impact on chromatin structure, e.g., by altering the SWI/SNF complex. Gcn4 independently recruits nucleosome-nucleosome interactions. Alternatively, both SWI/SNF and Gcn5. The SWI/SNF mediated they may direct or regulate activities to the nucleosome nucleosome remodeling is dependent on histone that in turn alter its structure (Strahl and Allis, 2000). acetylation by Gcn5, whereas histone acetylation by Histone modi®cation may serve as a code that ATP- Gcn5 is independent of SWI/SNF at this promoter. dependent chromatin remodeling factors translate. In Moreover, the intact bromodomain of Gcn5 is required that way chromatin remodeling events could be for the stable association of SWI/SNF at this promoter targeted indirectly to speci®c sites via the action of a (the bromodomain is not required for ecient histone modifying enzyme, which is tethered to speci®c acetylation of histones). SWI/SNF associates in a sites by DNA sequence speci®c factors and modi®es stable fashion with Gcn4 in the absence of Gcn5, but histones locally. Histone modi®cations may also serve not in the presence of bromodomain mutant deriva- as a trigger to initiate nucleosome alterations, e.g., by tives of Gcn5. One possible explanation for these destabilizing the nucleosome. observations is that acetylation interferes with the If histone modi®cations regulate the action of ATP- stable interaction of SWI/SNF with the promoter and dependent chromatin remodeling factors, it is likely the interaction of the bromodomain with acetylated that the latter interact with the histone tail domains, as histone N-termini may antagonize this e€ect. One these are the major sites of histone modi®cations. The might conclude that acetylation of nucleosomes by histone tails constitute likely contact points, because Gcn5 is necessary for subsequent ®nely tuned control they protrude from the otherwise compact nucleosome of nucleosome remodeling events. Both the SWI/SNF core particle and may reach out far beyond the complex and the Gcn5-containing complex (the SAGA nucleosome. Indeed, several studies demonstrate the complex, Grant et al., 1998) are huge molecular entities importance of the histone tails for nucleosome and it is dicult to imagine that both these factors are remodeling by ATP-dependent chromatin remodeling able to associate with a transcriptional activator at the factors: The histone tails are essential for the action of same time. There is rather going to be a `dance of the the ISWI-containing nucleosome remodeling factor elephants' at the promoter, and some of the histone

Oncogene ATP-dependent chromatin remodeling factors P Varga-Weisz 3082 modi®cations may have a role in choreographing this role in chromatin assembly in vitro and possibly in vivo. `dance'. ACF1, a protein that interacts with ISWI in the Drosophila and human ACF and CHRAC complexes contains a heterochromatin targeting domain and is a ATP-dependent chromatin remodeling may change the member of a family of proteins that all may be targeted chromatin structure of whole domains to heterochromatin (Ito et al., 1999; LeRoy et al., 2000; Bochar et al., 2000a; Poot et al., 2000). Indeed, ACF1 Chromatin is subjected to various global remodeling localizes to pericentromeric heterochromatin in mouse events, including chromosome condensation and de- cells (Nadine Collins, Custodia Garcia-Jimenez and condensation at mitosis, remodeling of chromatin of PVW, submitted). This presents the possibility that developing sperm cells, the remodeling of the sperm these factors may be involved in setting up hetero- chromosomes after fertilization and the chomosome chromatin or heterochromatin-like structures in vivo. condensation at spore formation in yeast. Heterochro- The biochemical characterization of the CHRAC and matin is a chromosome domain that stays condensed in ACF enzymes suggested that they may have global interphase. It has functions in chromosome stability e€ects on chromatin: for example, both CHRAC and and segregation and in gene silencing (Wallrath, 1998; ACF remodel whole arrays of nucleosomes in catalytic Hennig, 1999). A dramatic example of a conversion of amounts (with respect to nucleosomes) in vitro (Varga- an entire chromosome into heterochromatin occurs at Weisz and Becker, 1998; Ito et al., 1997). ISWI- X-chromosome inactivation during early mammalian containing remodeling factors alter chromatin structure embryo development. The duplication of the chromo- in vitro without evicting nucleosomes. Instead they somes during DNA replication is a major chromatin mobilize the nucleosomes to alter their position (LaÈ ngst remodeling event. et al., 1999; Hamiche et al., 1999). Such a property Chromatin remodeling factors have been shown to may be required to change progressively the chromatin a€ect gene expression by remodeling the chromatin structure of a whole domain, e.g., after nucleosome structure at promoters. However, some of these deposition in S phase. The analysis of ISWI function in factors, such as the ISWI complexes in Drosophila Drosophila reveals a function of ISWI in the main- and Xenopus oocytes (reviewed in Varga-Weisz and tenance of higher order chromatin structure in vivo: Becker, 1998; Guschin et al., 2000) are abundant ISWI mutations caused striking alterations in the molecules, and they may also have a global role. Such structure of the male X chromosome (Deuring et al., factors may in¯uence gene regulation by altering the 2000). This links ISWI-function to dosage compensa- chromatin structure of whole chromatin domains or tion in Drosophila, whereby the single male X- even whole chromosomes. Examples in favor of this chromosome is transcriptionally hyperactive compared idea are given below. to each of the two female X-chromosomes. Animal cloning by transplantation of a somatic cell The situation as described above for some of the nucleus into an unfertilized enucleated egg (the key ATP-dependent chromatin remodeling factors may be process that resulted in cloned sheep `Dolly', Wilmut et similar as with certain HATs and deacetylases in that al., 1997) leads to a dramatic remodeling of the somatic they may have both a targeted as well as a global role: chromosomes. In this process many proteins are For example, the yeast HAT Gcn5 is part of a complex speci®cally lost from the nucleus and others are taken that is recruited to certain promoters by transcription up from the egg cytoplasm. The result is a dedi€er- activators. The histone deacetylase RPD3 complex is entiation of the somatic nucleus with loss of epigenetic recruited to promoters by the repressor Ume6p. marks. This remodeling can be reconstituted in vitro However, these two enzymes also e€ect global histone with a Xenopus egg extract. The reaction requires acetylation over large chromosomal regions (Vogelauer energy in form of ATP and can be monitored by the et al., 2000). Therefore, targeted histone modi®cations essentially complete release of the TATA binding occur in a background of global acetylation and protein (TBP) from the somatic chromatin. An ISWI deacetylation and this may be important for the rapid containing remodeling factor is a key molecule in this reversal to the ground state of transcriptional repres- large scale chromatin remodeling in vitro (Kikyo et al., sion, after a stimulus for activation has disappeared. In 2000). The activity of ISWI cannot be replaced by a similar manner, some ATP-dependent chromatin other similar chromatin remodeling factors. This remodeling factors (perhaps those that contain the suggests that ISWI may also have a role in reverting ATPase ISWI) could have a role in maintaining a epigenetic marks in vivo by remodeling chromosomes. global chromatin structure and in resetting nucleoso- Furthermore, this ®nding suggests a possible role for mal arrays to the ground state after remodeling by ISWI in removing TBP in general, resetting the another factor such as SWI/SNF occurred (Figure 3). chromatin structure of promoters to a repressed ground state (Figure 3), consistent with the observation in yeast, where an ISWI-complex is involved in ATP-dependent chromatin remodeling may have transcriptional repression (Goldmark et al., 2000). functions outside of transcriptional regulation ISWI-containing chromatin remodeling factors such as ACF, CHRAC (reviewed in: Varga-Weisz and The packaging of the genome in chromatin presents Becker, 1998) and RSF (LeRoy et al., 1998) have a barriers that restrict the access of all kinds of enzymes

Oncogene ATP-dependent chromatin remodeling factors P Varga-Weisz 3083 that process DNA. Therefore, one would expect that some remodeling ATPases for which a chromatin ATP-dependent chromatin remodeling factors are remodeling activity has not yet been proven. This important in processes besides transcriptional regula- includes the transcriptional regulator ATR-X, a protein tion, for example in DNA damage repair, recombina- involved in the X-linked alpha-thalassemia/mental tion and replication. Moreover, ATP-dependent retardation syndrome (Picketts et al., 1996). In plants chromatin remodeling factors may have a role in there are many putative nucleosome remodeling chromatin assembly itself. ATPases, which have a role in epigenetic gene Recently, a nucleosome remodeling complex puri®ed regulation, including DDM1 (Decrease in DNA from yeast was shown to contain an ATPase, Ino80p, Methylation) (Jeddeloh et al., 1999) and MOM1 with amino acid sequence similarity to ISWI (Shen et (Amedeo et al., 2000). al., 2000). This ATPase forms a complex with proteins There are many open or only partially answered related in amino acid sequence to the bacterial RuvB questions about ATP-dependent chromatin remodeling DNA helicase, which catalyses branch migration of factors: What are the mechanisms of ATP-dependent Holliday junctions (A Holliday junction is a four-way chromatin remodeling? How is a distortion of DNA junction DNA intermediate formed during the process structure channeled into a nucleosome remodeling of homologous genetic recombination). Interestingly, event? How do the various complexes di€er in the INO80 complex has DNA strand displacement mechanisms and biological function? The interaction activity and is, therefore, an ATP-dependent helicase ± of chromatin remodellers with sequence speci®c factors unlike other chromatin remodeling factors that have poses the question whether the recruiting factors been tested for this activity. This activity depends on interact with the remodeller in solution prior to the ATPase function of the Ino80 protein. Cells with chromatin remodeling, or does the sequence speci®c mutations in INO80 show hypersensitivity to DNA factor already bound to the DNA target the remodeler, damage, which may suggest a function in DNA as has been shown in the case with SWI/SNF at the damage repair. However, ino80 mutants show reduced HO and PHO8 promoters? Are ATP-dependent transcription of several genes, and, therefore, the DNA chromatin remodeling complexes all targeted to the damage sensitive phenotype may be the result of sites of action, and if so, how? How is the size of the reduced transcription of a gene induced by DNA remodeled chromatin domain regulated? The biological damage. function of most these complexes is unclear. For Another protein that may couple nucleosome example, ISWI is essential in Drosophila (but not in remodeling with DNA damage repair is the Cockayne yeast). What is the basis for this? How do these syndrome B protein (CSB). This DNA-dependent activities relate to each other and to other chromatin ATPase of the SWI2/SNF2 family is required for remodeling activities, such as histone acetylation? transcription-coupled DNA excision repair (which removes UV light-induced DNA lesions such as cyclobutane pyrimidine dimers of transcribed genes). This enzyme interacts directly with core histones and Acknowledgments remodels chromatin structure in an ATP-dependent The work in my laboratory is supported by Marie Curie manner in vitro (Citterio et al., 2000). Cancer Care and a grant from the Association of International Cancer Research (AICR), St Andrews. I am grateful for comments which improved the manuscript from Drs Raymond Poot, Ludmila Bozhenok, Nadine Outlook Collins, Jacob Dalgaard, Thomas OelgeschlaÈ ger, Graham Currie and Irina Tsaneva. I am grateful to many The number of known ATP-dependent chromatin researchers who sent me preprints, especially Tom Owen- remodeling factors will most likely increase, since there Hughes and Peter Becker for sending manuscripts before are many factors with sequence similarity to nucleo- publication.

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