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Dynamic nucleosomes. Karolin Luger. Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, Colorado State. University, Fort ...
Chromosome Research (2006) 14:5Y16 DOI : 10.1007/s10577-005-1026-1

# Springer 2006

Dynamic nucleosomes Karolin Luger Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA; Tel: +1-970-4916405; Fax: +1-970-4915113; E-mail: [email protected]

Key words: chromatin remodelling, histone, histone chaperone, nucleosome

Abstract It is now widely recognized that the packaging of genomic DNA together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, repair and recombination. How chromatin structure modulates the expression and maintenance of knowledge encoded in eukaryotic genomes, and how these processes take place within the context of a highly complex and compacted genomic chromatin environment remains a major unresolved question in biology. Here we review recent advances in our understanding of how nucleosome and chromatin structure may have to adapt to promote these vital functions.

Introduction Virtually all studies of processes that require access to eukaryotic DNA have to take into account its packaging with a roughly equal mass of proteins to form large macromolecular assemblages, collectively termed chromatin. Histones, by forming nucleosomes, are responsible for the first level of structural organization (Luger et al. 1997). Hundreds of thousands of nucleosomes are further compacted into hierarchical structures of increasing complexity, yet largely unknown architecture (Luger & Hansen 2005). It has become clear that any mechanism that has the potential to alter the level of compaction must have an inherent role in regulating DNA accessibility. The nucleosome core particle (NCP) is the universally repeating unit in chromatin with a molecular weight of 210 kDa. High-resolution crystal structures of the NCP from different organisms (Luger et al. 1997, White et al. 2001, Chantalat et al. 2003, Tsunaka et al. 2005), or containing histone variants

(Suto et al. 2000, Chakravarthy et al. 2005) and mutants (Muthurajan et al. 2004), have been determined. These structures reveal an octameric histone core around which 147 base pairs of DNA are wrapped in 1.65 superhelical turns (Figure 1). The histone octamer itself is composed of two copies each of the four histone proteins, H2A, H2B, H3, and H4 (see Luger & Richmond 1998a, 1998b, for reviews of nucleosome structure). The massive distortion of the DNA is brought about by the tight interaction between the rigid framework of the histone proteins with the DNA at fourteen independent DNA-binding locations (Luger & Richmond 1998a). With respect to nucleosome and chromatin dynamics, several aspects of nucleosome structure are particularly noteworthy. First, the nucleosome exhibits a modular assembly in which the two H2AYH2B dimers (shown in yellow/red in Figure 1) can be removed while interaction between the DNA and the (H3YH4)2 tetramer is maintained (Akey & Luger 2003). This reflects in-vivo and in-vitro assembly (and perhaps also disassembly) pathways.

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Figure 1. Structure of the nucleosome core particle. (A) H3 is shown in blue, H4 in green, H2A in yellow and H2B in red. The particles are viewed down the superhelical axis of the DNA (grey). Three particles from the crystal lattice are shown to emphasize end-to-end stacking of the ends of nucleosomal DNA. Note that the histone tails are for the most part too disordered to be included in the structure. (B) Magnification of boxed area in (A).

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Figure 2. Surface representation of the histone octamer. Blue surfaces indicate basic residues, red surfaces are acidic. (A) Only the histone octamer is shown in approximately the same view as in Figure 1. (B) The nucleosome is viewed down the dyad axis, with the superhelical axis in y (obtained by a rotation of the above orientation around the x-axis). The DNA is shown in ball- and-stick representation.

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8 Second, rather drastic changes in the amino acid sequence of histones (for example, as seen in histone variants) are for the most part accommodated without significant changes in nucleosome structure and stability (Chakravarthy et al. 2004). Third, the nucleosomal surface (which is to a large part defined by the histone octamer), is a highly contoured landscape with a very distinctive charge distribution (Chakravarthy et al. 2004) (Figure 2). It is this surface that is implicated in nucleosomeYnucleosome contacts to promote higher-order structure formation. Fourth, all crystal structures of mono-nucleosomes reported to date have been determined with particles reconstituted onto 146 or 147 base pairs of DNA. The ends of the DNA are engaged in base-stacking interactions that are vital for crystal packing (Figure 1). Thus, it is possible that the penultimate 10Y20 base pairs of DNA, as viewed in the crystal structure, may actually be more variable in their conformation in the context of long nucleosomal arrays or higherorder structure. The histones within the nucleosome have evolved to accomplish two conflicting yet vital tasks: first, the õ2 m of eukaryotic DNA have to be packaged within the confines of the nucleus, preventing knots and tangles and protecting the genome from physical damage. Second, the information that is encoded within the DNA needs to be accessed at appropriate times, and this is to a large part regulated by local changes in nucleosome and chromatin structure by complex mechanisms that are only now emerging. The concept that chromatin and nucleosomes are inherently dynamic and highly malleable is not entirely new (see, for example, Glotov et al. 1982, Pennings et al. 1991) but does go against the intuitive

Table 1. The effect of chromatin modifications on nucleosome and chromatin structure.

Histone modifications Histone variants Chromatin remodelling factors Histone chaperones Nucleosome binding proteins

DNA breathing

Nucleosome sliding remodelling

? +/j ?

? +/j +

+/j +/j +/j

+ + / (j)

+ (+)

? +/j

Higher-order structure

+ and j indicate positive and negative effects respectively.

knowledge that an assembly in which DNA is tethered to a protein spool via õ240 direct and indirect contacts along its entire length (Davey et al. 2002) is unlikely to undergo spontaneous rearrangements. Histones have evolved to highly constrain DNA to achieve approximately five-fold compaction at the first level and to promote the formation of higher-order structure; yet, a variety of pathways ensure highly regulated access to the packaged DNA (Table 1).

DNA accessibility is regulated by several mechanisms Several mechanisms exist to make nucleosomal DNA more accessible to the cellular machinery: the transient dissociation and re-binding of the ends of nucleosomal DNA; nucleosome sliding and Fremodelling_ (spontaneous or catalysed); and changes in chromatin higher-order structure (Figure 3). All of these are affected directly by modifications in the amino acid sequence of histones (either by posttranslational modifications, or by the introduction of histone variants). Additionally, remodelling factors, histone chaperones, and chromatin-binding proteins all contribute in a combinatorial manner to the structural changes that are necessary to allow access to the DNA template.

Transient site-exposure by dissociation and re-binding (breathing) of the ends of nucleosomal DNA Transient site-exposure by dissociation and re-binding (breathing) of the ends of nucleosomal DNA (Figure 3A; first proposed by Widom and coworkers: Polach & Widom 1995, Anderson & Widom 2000, Li & Widom 2004) has been studied mostly in mononucleosomes; however, it is likely to occur at least to a certain extent under in-vivo conditions. It provides an explanation for the relatively higher affinity of a transcription factor for its binding site near the nucleosomal entry and exit point, and for the observed co-operative binding of eukaryotic regulatory proteins to nucleosomal target sites. It is conceivable that DNA breathing also facilitates the invasion of nucleosomal DNA by RNA polymerases. We have recently shown that accessibility of tran-

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Figure 3. Different mechanisms for exposing nucleosomal DNA. (A) Schematic for transient site exposure and its effect on binding sites near the end or near the nucleosomal dyad. Transcription factor binding sites are shown in black; the corresponding factor is shown as a red sphere. The yellow star indicates a histone chaperone. (B) Nucleosome sliding; note how binding of a transcription factor can lead to nucleosome positioning. (C) Chromatin higher-order structures are indicated schematically; linker histones are shown in blue.

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scription factor binding sites that are near the nucleosomal dyad may also be regulated by this phenomenon (Figure 3A, middle panel; White & Luger 2004). From a structural viewpoint, it is likely that the nature of proteinYDNA interactions (for example, by the introduction of a histone H3 variant or by posttranslational modifications) at the penultimate one or two attachment points affects the dynamic equilibrium between the partially bound and partially unbound state. This equilibrium is also likely to be shifted towards the unbound state by the transient removal of H2AYH2B dimers by histone chaperones (Figure 3A, bottom panel).

Fremodelling_ was originally coined to explain the effects of ATP-dependent chromatin remodelling factors on defined nucleosomal substrates, such as changes in DNase footprinting and changes in DNAYprotein contacts (Peterson & Tamkun 1995). It was suggested that the path of the DNA on the histone octamer was changed in an undefined manner, and that the very nature of histoneYDNA interactions was altered, resulting in increased transcription levels. To date, it is unclear whether this is only observed under relatively restricted in-vitro conditions, or whether it is a physiologically relevant outcome of ATP-dependent chromatin remodelling.

Nucleosome sliding

Changes in chromatin higher-order structure

Nucleosome sliding is a phenomenon that has unfortunately been misnamed: it is not the nucleosome that slides (this would be impossible, since the nucleosome actually includes the DNA) but rather the position of the histone octamer with respect to the DNA is altered; thus, a more appropriate name would be histone octamer sliding (Figure 3B). Irrespective of nomenclature, this process frees up DNA sequences that were previously bound by the histone octamer. Under certain in-vitro conditions, nucleosome sliding occurs spontaneously in a temperaturedependent manner (Pennings et al. 1991). The temperatures required for sliding in defined model systems allow for a comparison of the relative stability of histoneYDNA interactions in nucleosomes reconstituted with histone mutants or histone variants (Flaus et al. 2004). While it is likely that spontaneous nucleosome sliding occurs in vivo at least to some extent, it is also promoted by large and highly complex ATP-dependent chromatin remodelling machines (recently reviewed in Smith & Peterson 2005), and by histone chaperones (Park et al. 2005).

Most of the phenomena discussed above have been studied mainly on mono-nucleosomes or on defined nucleosomal arrays. It is unclear whether and how they take place in the context of various levels of chromatin higher-order structure. Thus, it is likely that changes in chromatin higher-order structure need to precede many, if not all of the steps listed above. In and of itself, any change in the degree of compaction potentially has the most pronounced effect on DNA accessibility (Figure 3C). Until recently, not very much was known about the molecular details of higher-order structure beyond the nucleosome, and therefore accounts of changes in the degree of compaction have lacked a molecular basis. Recent breakthroughs in elucidating the structure of a tetra-nucleosome give hope that we will eventually be able to understand the different levels of chromatin higher-order structure and the mechanisms that promote their interconversion (Dorigo et al. 2004, Schalch et al. 2005).

Post-translational modifications of histones Remodelling of chromatin and nucleosomes The concept of Fremodelling_ of chromatin and nucleosomes in the true sense of the word has gone out of fashion in recent years as the nucleosome sliding activity of ATP-dependent chromatin remodelling factor has taken centre stage. The term

Post-translational modifications of histones affect chromatin structure at various levels. Numerous review articles address the ever-growing number of combinations of post-translational histone modifications and the cross-talk between the involved enzymatic activities (see Cosgrove & Wolberger 2005, for a recent review). The long-standing view

Dynamic nucleosomes that the flexible histone tails are the main targets of post-translational modifications has recently been revised, and we now know of a multitude of residues within the structured portions of the histones that are targets for modifying enzymes (Freitas et al. 2004). There are now about 150 known histone modifications but the numbers are expected to increase at a steady rate. Post-translational modifications of histones appear to be involved in nearly all aspects of DNA biology, and an exhaustive review would clearly be beyond the scope of this article. However, two points are important to note. First, all of the post-translational modifications known to date are reversible. The last bastion of irreversible histone modification, the methylation of lysines, was recently found to be reversed by the action of a demethylase (Shi et al. 2004, and reviewed in Wysocka et al. 2005). The enzymes that are responsible for attachment and removal of post-translational modifications are often present at the same time. Thus, the ensemble of modifications at any given time and location is likely to undergo constant dynamic change. Second, histone modifications quite dramatically alter the character and reactivity of the particular amino acid. This is most obviously true for the Fbulky_ modifications such as ADP ribosylation, ubiquitylation, and sumoylation; but also for acetylation and methylation (monoYdi, and Ytrimethylation) of lysines, which generates strange amino acids indeed.

Histone tails The precise mechanism by which any combination of histone modifications results in the observed effects on transcription and repair has remained elusive. An obvious, but perhaps somewhat simplistic hypothesis is that changes in the charge of the histone tails alter their ability to bind and constrain nucleosomal DNA. Earlier evidence has suggested that histone tails do regulate access to nucleosomal DNA (Lee et al. 1993), although their effect on DNA breathing is predicted to be weak (Polach et al. 2000, Mutskov et al. 1998). Circumstantial evidence from our own lab indicates that histone tails do contribute to a certain extent to the binding of the DNA ends, and their removal appears to lower the energy barrier for repositioning of the

11 DNA with respect to the histone octamer (Gottesfeld et al. 2001, Edayathumangalam & Luger manuscript in preparation). It is likely that these effects will be modulated by the presence of post-translational modifications within the histone tails. However, it is important to note that all the experiments cited above were done in the context of a mononucleosome. It is quite possible that the histone tails assume their true function(s) only in the context of chromatin higherorder structure, or at the very least in a polynucleosomal environment. Another commonly held view is that the histone tails with their varying degree of modifications contribute to the recruitment of specific protein factors that Fread the histone code,_ with various outcomes for DNA accessibility. For example, heterochromatin protein 1 (HP1), a structural component of silent (highly compacted) chromatin at telomeres and centromeres, requires methylation at lysine 9 of histone H3 (Jacobs & Khorasanizadeh 2002, and references therein); similarly, the interaction of Sir3 with chromatin requires its own set of histone tail modifications (reviewed in Millar et al. 2004). There are many more such examples, amply supported by structural studies that provide a basis for our understanding of selectivity. Post-translational modifications, specifically in the structured regions of the four core histones, may also directly affect DNA accessibility by altering proteinYDNA interactions. Several of the residues that were recently found to be modified are involved in interaction with the DNA, and we have shown earlier that subtle changes at a single proteinYDNA interface near the nucleosomal dyad can alter the in-vitro sliding properties of nucleosomes by disrupting three to five of the total 120 hydrogen bonds between histones and DNA (Muthurajan et al. 2003, Flaus & Owen-Hughes 2003). Lastly, post-translational modifications of histone tails and surface residues have the potential to alter nucleosomeYnucleosome interactions. The formation of higher-order structures, by definition, requires the close packing of nucleosomal surfaces, and posttranslational modifications are expected to alter the surface shape and charge of nucleosomes. Additionally, the surface of the histone octamer within the nucleosome is likely to act as a binding platform for histone tails (Dorigo et al. 2003, 2004, Gordon et al. 2005) or other protein factors; again, interactions may be altered depending on the set of existing

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12 modifications on either histone tails or structured regions of the histone proteins. Crystallographic studies have shown that stacking of nucleosomes is achieved by a close packing of the surfaces mainly defined by the histone octamer, and that several energetically favourable modes of interaction are possible (White et al. 2001). Histone variants: subtle and not-so-subtle effects on nucleosome and chromatin structure Substitution of one or more of the core histones with the corresponding histone variant has the potential to exert considerable influence on the structure and function of nucleosomes and chromatin (reviewed in Chakravarthy et al. 2004). Histone variants are distinct non-allelic forms of major-type histones whose expression pattern is not restricted to S-phase. They are found in most eukaryotic organisms, and are expressed in all tissue types. Histone variants exhibit moderate to significant degrees of sequence homology with their corresponding major-type counterparts: H2A.X (82%) and H3.3 (õ96%) are the least divergent of all histone variants; they differ in only a few amino acids from their replicationdependent counterparts. H2A.Z (õ60%), macroH2A (õ65%), H2A.Bbd (40%), and CenpA which has a 93 amino acid domain that is 62% identical to H3 are increasingly divergent in their histone moiety from H2A and H3, respectively. As is the case with histones in general, the structured regions of the histones (encompassing histone folds and extensions) are more conserved than the histone tails. In many cases, histone variants are even more conserved than their highly conserved major-type paralogues (Sullivan et al. 2002), indicating that they all have evolved to fulfil important functions that cannot be accomplished by major-type H2A and H3. The replacement of histones H2A or H3 with their corresponding variants can have several outcomes on chromatin structure. First, subtle structural variations that result from sequence differences have been described in two instances where the crystal structure of variant-containing nucleosomes has been solved (Suto et al. 2000, Chakravarthy et al. 2005). DNA binding and stability of variant-containing nucleosomes may be altered, as found for nucleosomes containing H2A.Z (Abbott et al. 2001, Park et al. 2004) and H2A.Bbd (Bao et al. 2004, Gautier et al.

2004). In vivo, this may result in facilitated or decreased histone eviction during transcription processes. Evidence for this has recently been presented for yeast H2A.Z (Zhang et al. 2005). Second, nucleosome sliding (Flaus et al. 2004) or ATPdependent chromatin remodelling may be affected (Angelov et al. 2000, Santisteban et al. 2000). Third, the nucleosomal surface may be altered significantly (Chakravarthy et al. 2004), with profound implications for nucleosomeYnucleosome interactions, as shown for H2A.Z (Fan et al. 2004b). Third, the availability of sites for post-translational modifications within the tails are altered (perhaps most drastically for H2A.Bbd; Bao et al. 2004), but also for other histone variants, most notable for H2A.X) and this may confer profound changes on chromatin structure that are more or less independent of nucleosome structure.

ATP-dependent chromatin remodelling factors: many machines, many products Many comprehensive reviews have been published on the mechanisms by which ATP-dependent chromatin remodelling machines non-covalently alter nucleosome structure (for example, Fan et al. 2004a, Smith & Peterson 2005). It has become clear that different chromatin remodelling factors generate different Fremodelled_ substrates, and that these differences can be ascribed to the type of ATPase present in the complex (Fan et al. 2004a). The observed outcomes also appear to depend to a certain extent on the model system that is used to study these activities. Given the multitude of different remodelling complexes and the variety of experimental systems, it is not surprising that there is evidence for the promotion of nucleosome sliding, histone exchange, histone eviction, and changes in higherorder structure. Just as with many histone modifications, the link between chromatin remodelling and transcription regulation has been firmly established; however, mechanistic details remain to be filled in.

Histone chaperones join the dance In the cell, histones are usually found in complex with a diverse group of histone-binding proteins,

Dynamic nucleosomes the so-called histone chaperones (Akey & Luger 2003). These are chaperones in the true sense of the word, since they prevent improper interactions (for example, non-native interactions between histones and DNA, or between histones and other proteins), and promote proper ones that result in the formation of the nucleosome. Their predominant role was thought to be in nucleosome assembly; however, recent data implies that they have a role in regulating DNA accessibility in the absence of DNA replication. For example, yeast nucleosome assembly protein 1 (yNAP-1) is capable of removing histone H2AYH2B dimers from folded nucleosomes, and, depending on availability, replaces them with canonical or variant dimers in vitro (Bruno et al. 2004, Park et al. 2005). yNAP-1 has also been shown to be a component of the remodelling complex Swr1 that is responsible for the exchange of the H2A variant H2A.Z into yeast chromatin (Mizuguchi et al. 2004). The transient removal of H2AYH2B dimers from mononucleosomes also results in nucleosome sliding (Park et al. 2005) and promotes DNA breathing (Park et al. unpublished observations). It remains to be seen how general a phenomenon this is. For example, do histone chaperones other than yNAP-1 also promote nucleosome sliding? Is this activity relevant in vivo? Can NAP-1-mediated histone exchange and sliding also be observed in nucleosomal arrays? The observation that H2AYH2B dimers (and, to a certain extent, also (H3YH4)2 tetramers) appear to be in rapid exchange in most regions of compacted chromatin (Jackson 1990, Kimura & Cook 2001) and that histone variants are incorporated in replication-independent assembly pathways (Ahmad & Henikoff 2002) is certainly consistent with the notion that histone chaperones play an important role in promoting chromatin fluidity in vivo.

Nucleosome-binding proteins An increasing number of non-histone proteins are known to directly bind to nucleosomes, with consequences on nucleosome and chromatin structure and dynamics. These include the highly abundant linker histones and high-mobility group proteins, architectural proteins, and an increasing number of transcription factors. A common emerging theme is that many

13 of these factors are only transiently associated with chromatin (see, for example, Nagaich et al. 2004, Agresti et al. 2005). Surprisingly, this holds true even for histone H1 which appears to exchange rapidly in euchromatin as well as in heterochromatin (Misteli et al. 2000, Lever et al. 2000). H1 is referred to as the Flinker histone_ due to its association with the DNA that links two neighbouring nucleosomes in an array (linker DNA; Hansen 2002). One linker histone is associated with nearly every nucleosome in mammalian cells. H1 promotes the organization of chromatin into the 30 nm filament and increases the repeat length of nucleosomes in vitro. In addition to a structural role, H1 subtypes and variants also have gene-specific effects on transcription (Thomas 1999, Lee et al. 2004), and impinge on cell cycle regulation (Khochbin 2001). The mechanism by which H1 exerts these functions remains unclear, and the location of H1 in the filament, its role in filament formation, and the structure of the 30-nm filament itself have all been controversial. Some studies suggest that H1 organizes linker DNA in a stem-like organization towards the nucleosome (Bednar et al. 1998, Toth et al. 2001), however, other models also exist (reviewed in Travers 1999). The general location of H1 near the entry and exit point of nucleosomal DNA, and the fact that it organizes linker DNA imply that H1 affects the breathing of DNA ends. In vitro, the binding of H1 clearly has an inhibitory effect on nucleosome sliding (Ragab & Travers 2003) and on ATP-dependent chromatin remodeling (Hill & Imbalzano 2000). Finally, linker histone binding to nucleosomal sites stabilizes folded chromatin fibers, but does not direct their formation (Georgel & Hansen 2001). A review of chromatin architectural proteins with clear effects on chromatin higher-order structure is presented in this issue (McBryant et al. 2006). Additionally, the three families of high-mobility group proteins or HMGs (after histones, the second most abundant chromatin proteins with distinct effects on chromatin structure; Bianchi & Agresti 2005) are worth mentioning. HMGNs (formerly HMG14/17) are among the few nuclear proteins known to specifically recognize the generic structure of the nucleosome (Bustin 1999). Their binding has no effect on nucleosome remodelling or on the compaction of defined nucleosomal arrays in vitro

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14 (Hill et al. 2005); however, earlier reports describe that they unfold chromatin higher-order structure by competing with linker histones, a characteristic that seems to be common to all three groups of HMG proteins (Catez et al. 2004). Chromatin modifying activities are recruited to chromatin regions of interest by transcriptional activators and repressors. This requires the ability of at least some of these regulatory factors to recognize their binding site in the structural context of a nucleosome. Despite the identification of an increasing number of regulatory factors that are capable of targeting chromatin, the molecular details of such interactions have been largely uncharacterized. Not surprisingly, transcription factor binding to the NCP is affected greatly by the rotational and translational position of the binding site with respect to the histone octamer. Binding sites that face inwards on the DNA superhelix are occluded from solvent by the tight interaction with histone proteins, thus providing an obvious explanation for the dependence on rotational position (Gottesfeld et al. 2001). Binding sites that are located towards the end of nucleosomal DNA are usually bound with higher affinity than those located near the nucleosomal dyad (e.g. Li et al. 1994). This has been explained by the phenomenon of transient exposure of binding sites by partial dissociation of terminal regions of DNA from the histone octamer (see above). It has also been suggested that partial dissociation of the histone octamer (for example, dissociation of a H2A/H2B dimer) would have to occur before a transcription factor would be able to bind to nucleosomal DNA (Figure 3; Spangenberg et al. 1998), a process which may be facilitated by ATPdependent chromatin-remodelling factors (e.g. Bruno et al. 2003) or histone chaperones (Chen et al. 1994, Ito et al. 2000, Bao et al. 2004). Finally, the flexible histone tails are likely to be involved in the regulation of nucleosomal DNA accessibility (for example, Vitolo et al. 2000, and see above). Thus, transcription factor binding to nucleosomes can have an effect on nucleosome sliding and positioning (e.g. McPherson et al. 1993), DNA breathing by capturing the DNA in the unbound state (White & Luger 2004), and chromatin remodelling, the latter most likely through the recruitment of ATP-dependent chromatin remodelling factors (see, for example, Tsukiyama et al. 1994, Cirillo et al. 2002).

Conclusions and outlook It has become clear that the many cellular activities that impinge upon chromatin structure operate via multiple and complex mechanisms. Mounting evidence demonstrates that ATP-dependent chromatin remodelling factors, histone-chaperones, histone modifying enzymes, and nucleosome-binding proteins affect different levels of chromatin organization in a highly orchestrated and concerted manner. It is highly unlikely that, for example, every promoter is made accessible via a unified order of events; rather, each and every incidence of regulated DNA accessibility will have to be studied independently to identify the important players and order of events that are necessary for the regulation of DNA accessibility. An additional uncertainty is the structure of the actual substrate itself. For a complete understanding of regulated DNA accessibility at any given site, we need to understand, at molecular detail, the structure of the underlying chromatin. Acknowledgments I thank Jayanth V. Chodaparambil and Srinivas Chakravarthy for help with figures, Rajeswari Edayathumangalam and Young-Jun Park for unpublished data, and all members of the Luger Laboratory for helpful discussion. References Abbott DW, Ivanova VS, Wang X, Bonner WM, Ausio J (2001) Characterization of the stability and folding of H2A.Z chromatin particles: implications for transcriptional activation. J Biol Chem 276: 41945Y41949. Agresti A, Scaffidi P, Riva A, Caiolfa VR, Bianchi ME (2005) GR and HMGB1 interact only within chromatin and influence each other’s residence time. Mol Cell 18: 109Y121. Ahmad K, Henikoff S (2002) The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol Cell 9: 1191Y1200. Akey CW, Luger K (2003) Histone chaperones and nucleosome assembly. Curr Opin Struct Biol 13: 6Y14. Anderson JD, Widom J (2000) Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites. J Mol Biol 296: 979Y987. Angelov D, Charra M, Seve M, Cote J, Khochbin S, Dimitrov S (2000) Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex binding. J Mol Biol 302: 315Y326.

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