- Email: [email protected]
Structure and dynamic behavior of nucleosomes
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Structure and dynamic behavior of nucleosomes Karolin Luger Genetic studies have identified residues in the structured regions of the histones that are critically involved in the formation of heterochromatin. Any investigation of the events that regulate access to the chromatin substrate must take into account the dynamic nature of the nucleosome, and the regulated inter-conversion between various levels of chromatin higher-order structure. Addresses Department for Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:127–135 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by John Tamkun and David Stillman 0959-437X/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(03)00026-1
Abbreviations bp base pair lrs loss of rDNA silencing NCP nucleosome core particle RNAi RNA interference SHL Superhelix location SIN SWI/SNF-independent
Introduction The DNA of a single eukaryotic cell is compacted in the cell nucleus in a hierarchical scheme of folding into chromatin. At the first level of organization, two tight superhelical turns of DNA are wrapped around an octamer of two copies each of the four histone proteins: H2A, H2B, H3, and H4. This unit, the nucleosome core particle (NCP), represents the basic repeating structure in chromatin (Figure 1). Hundreds of thousands of nucleosomes, connected by 20–80 base pairs (bp) of linker DNA, are compacted further into hierarchically folded higherorder assemblies of unknown architecture (reviewed in [1,2]). Also critically involved in this process are linker histone H1, a variety of non-histone proteins, and divalent metal ions. The nucleosome fulfils different, seemingly conflicting roles in the nucleus. First, DNA is compacted by a factor of five by being forced into a tight superhelix upon interaction with the histone proteins. Second, despite making >120 direct atomic interactions between the histones and the DNA backbone at the 14 ‘superhelix www.current-opinion.com
locations’ (SHLs, numbered 0.5 to 6.5 in Figure 1c), nucleosomes are highly dynamic. This is most apparent in the observed ability of the histone octamer to translocate, or ‘slide’, along the DNA over significant distances (first observed in [3]; Figure 2). The considerable energy barrier for nucleosome sliding in vivo is likely alleviated by the action of ATP-dependent chromatin remodeling factors [4,5]. Third, the inter-conversion of fluid hierarchy chromatin structures that prevail in the interphase nucleus [6] from transcriptionally blocked to transcriptionally active states, as well as the formation of the highly ordered assembly that prevails in metaphase chromosomes are likely to be tightly regulated by reversible modification of histones, of other associated proteins, and of DNA (reviewed in [7–9]). The recently established link between the machinery that is responsible for the RNAi (RNA interference) phenomenon, and the formation of heterochromatin ([10], and reviewed in [11]) represents only a glimpse of many strange and wonderful things that remain to be understood in these regulatory pathways. The organization of eukaryotic DNA in nucleosomes has profound consequences on its ability to serve as a template for the enzymatic activities within the replication, recombination, repair, and transcription machineries. A wide range of approaches have provided new evidence on how chromatin accommodates all of these activities in a dynamic manner. High-resolution structures of NCPs from different organisms [12,13,14,15], or reconstituted with histone variants [16], and of NCPs in complex with small, site-specific minor groove DNA-binding ligands [17] deliver structural information in stunning detail (Table 1). Careful biophysical analyses show that the introduction of mutations at the protein–DNA interface, or the incorporation of variant histones result in profound changes in the types of higher-order structure formed by nucleosomal arrays [18,19]. A wealth of evidence from genetic screens suggests that this structured surface of the histone octamer is also involved in regulating the transition from transcriptionally active euchromatin to highly compacted and transcriptionally inactive heterochromatin [20,21,22,23,24]. Here, I review data that demonstrate the dynamic behavior of chromatin, and consider recent genetic data in light of these structural results.
Nucleosome structure Progress in our understanding of nucleosome and chromatin structure has been made with the determination of a variety of nucleosome three-dimensional structures (summarized in Table 1). First, technical advances have Current Opinion in Genetics & Development 2003, 13:127–135
128 Chromosomes and expression mechanisms
Figure 1
(a)
(b)
H3
H3
H3
H3
H4
H4
H4
H2B H2B
H2A
H2A
H2A (c)
H2B
H2A
(d)
0.5 SIN
6.5
1.5
5.5 lrs
2.5
4.5 3.5
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Overall structure of the nucleosome core particle. (a) Front view of the NCP (protein database 1KX5; [14]), viewed down the superhelical axis. The histone fold domains of H2A, H2B, H3, and H4 are colored in yellow, red, blue, and green respectively, histone tails and extensions are shown in white, DNA is shown in light blue. H3 residues that are responsible for selective replication-coupled assembly are shown in magenta [30]. The red star indicates the site of ubiquitination in yeast [31]. (b) Side view of the same structure, obtained by a 908 rotation around the vertical axis. (c) Partial NCP structure, viewed as in (a), but showing only 73 base pairs and associated proteins. SHLs are indicated, as are the location of the SIN and lrs mutants on H3 and H4. The DNA from an NCP reconstituted with a different DNA sequence (1KX4) was superimposed based on the histone octamer main chain atoms, and is shown in pale yellow. (d) Side view as in (b), but with parts of the DNA removed to reveal the location of the H3 residues identified in [30].
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Structure and dynamic behavior of nucleosomes Luger 129
Figure 2
structure of nucleosomal DNA than was available from the previous structures [14]. Second, the first structure of an NCP containing a histone variant (H2A.Z; [16]) and the structures of chicken [12] and yeast [13] NCPs were also made available. Most recently, NCPs in complex with small, site-specific minor groove DNA-binding ligands were investigated [17]. All of these structures were crystallized at close to physiological salt concentrations (25 mM MnCl2 and 25 mM KCl, pH 6.0).
Incubation at 37°C
The Xenopus laevis nucleosome core particle at near-atomic resolution Current Opinion in Genetics & Development
Shifting behavior of X. laevis NCPs. The histone octamer is asymmetrically positioned with respect to the DNA after salt gradient reconstitution (at 48C) onto a 146 bp DNA fragment [25]. These positions are converted to the thermodynamically stable, symmetrically positioned species upon incubation at elevated temperatures. The temperature and time required for complete shifting is a function of the number of histone–DNA contacts and of the DNA sequence.
allowed the diffraction limit and quality of the electron density map of the original NCP structure (protein database code 1AOI [available in the Protein Data Bank repository: http://www.rcsb.org/pdb/]) [25,26] to be improved in a newer structure (protein database code 1KX3) [14]. A further significant improvement was subsequently achieved by adjusting the length of the DNA from 146 to 147 bp. This resolved crystallizationinduced structural heterogeneity in the DNA, and resulted in a much more accurate representation of the
What have we learned from these efforts? The amount of structural information available from the 1.9 A˚ structure of the NCP (protein database code 1KX5, reconstituted from recombinant Xenopus laevis histones [27] and a ‘symmetric’ 147 bp DNA fragment derived from human a-satellite DNA) [14,15] is unprecedented. A table in [14] lists each and every protein–DNA interaction in the structure. Given the extreme degree of sequence conservation between histones of different species [28], this will be a valuable reference for interpreting biochemical and genetic data. This structure is almost at the resolution where individual atoms can be directly resolved, and consequently >3000 water molecules have been identified. Among these, a large number of watermediated protein–DNA interactions, roughly equal in number to the 120 direct protein–DNA contacts, could potentially contribute significantly to the interaction energy between protein and DNA. However, as pointed out in [14], quantifying their relative importance to nucleosome stability and dynamics is not straightforward
Table 1 List of publicly available nucleosome and histone octamer structures, listed in chronological order. PDB code
Histone source
DNA source
Resol. Description
References
1AOI 1EQZ
X. laevis (recombinant) Gallus gallus (native, salt-extracted) X. laevis H2B, H3, H4; M. musculus H2A.Z, (all recombinant) S. cerevisiae (recombinant) X. laevis (recombinant)
146 bp palindromic, derived from human a-satellite 146 bp palindromic, derived from human a-satellite
2.8 2.5
Original NCP structure Chicken
[25] [12]
146 bp palindromic, derived from human a-satellite
2.6
Histone H2A.Z variant
[16]
146 bp palindromic, derived from human a-satellite
3.1
Yeast
[13]
146 bp palindromic, derived from human a-satellite
2.0
[14]
X. laevis (recombinant) X. laevis (recombinant) X. laevis (recombinant) X. laevis (recombinant) X. laevis (recombinant) G. gallus (native, salt-extracted) G. gallus (native, salt-extracted)
146 bp 147 bp 146 bp 146 bp 146 bp None
Components identical to 1AOI, improved resol. Different DNA sequence Highest resolution NCP to date NCP- ligand co-crystal structure 1 NCP- ligand co-crystal structure 1 NCP- ligand co-crystal structure 1 Original histone octamer structure –
–
1F66
1ID3 1KX3 1KX4 1KX5 1M18 1M19 1M1A 2HIO 1HQ3
None
palindromic, palindromic, palindromic, palindromic, palindromic,
derived derived derived derived derived
from from from from from
human human human human human
a-satellite B 2.6 a-satellite 1.9 a-satellite 2.45 a-satellite 2.3 a-satellite 2.65 3.1 2.15
[14] [14] [17] [17] [17] [55]
Structures of histone octamer alone are also listed at bottom. The code in the first column identifies the structure in the Protein Data Bank (PDB) repository (http://www.rcsb.org/pdb/). Resol, resolution (A˚ ).
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Current Opinion in Genetics & Development 2003, 13:127–135
130 Chromosomes and expression mechanisms
although the structure provides an unprecedented dataset for analysis. These solvent-mediated interactions are thought to help accommodate structural variations in the DNA that are a consequence of sequence variability found throughout the genome [14]. This observation suggests that solvent molecules could play a role in facilitating nucleosome mobility. Variations on the theme: DNA sequence
An argument against the general applicability of the published NCP structures has been the fact that all structures have been obtained from NCPs reconstituted with virtually identical DNA sequences. Of particular interest then is the finding that the overall structure of the NCP reconstituted with a significantly different DNA sequence is highly similar (protein database code 1KX4; [14]). Minor changes in the structure of the DNA (Figure 1c) are a reflection of the flexibility of nucleosomal DNA (also see below), since the full complement of histone–DNA interactions is also observed in this second structure. The differences in binding energy between different DNA sequences that have been observed experimentally [29] are likely to be a result of differences in the energy required for DNA distortion. Variations on the theme: histones
Given the high degree of evolutionary conservation of the histone proteins, it is not surprising that the 2.5 A˚ structure of an NCP reconstituted from chicken histones and a similar 146 bp DNA sequence (protein database code 1EQZ; [12]) is not much different from the earlier structures with Xenopus histones [14,25]. Protein and DNA structure, as well as crystal packing, is highly similar between Xenopus and chicken NCPs. By contrast, yeast histones are relatively divergent from those of higher eukaryotes [28]. Intriguingly, H2A and H3 in yeast appear to be evolutionary derivatives of the replacement histones H3.3 and H2A.X [30]. The yeast NCP structure (protein database code 1ID3) shows that the amino acid differences between H3.3 and replicationdependent H3 that are responsible for excluding the latter from replication-independent assembly in higher eukaryotes [30] have no impact on nucleosome structure, and are shielded from the solvent by the DNA supercoil ([13]; Figure 1b,d). One of the more surprising outcomes from the structure determination of the yeast NCP is the change in internucleosomal contacts in the crystal packing, which is a result of minor protein sequence differences between yeast and higher eukaryotes [13]. Although one has to be cautious of interpreting in vivo higher-order structure from in vitro crystal packing, these comparisons are likely to be significant considering that both types of interaction require energetically favorable interactions between highly charged nucleosome surfaces. Thus, it is Current Opinion in Genetics & Development 2003, 13:127–135
tempting to speculate that post-translational modifications of histones (either in the structured regions or in the histone tails), or the localized introduction of histone variants into chromatin may change the equilibrium between different types of nucleosome–nucleosome interactions. In this context, the identification of an ubiquitination site in a region of H2B that is critically involved in the formation of crystal contacts is particularly intriguing ([31]; Figure 1). One first step towards testing this hypothesis would be to use established in vitro systems [18,19,32,33] to study the ability of yeast histones to assemble into defined higher-order arrays, and to investigate the effect of post-translational modifications on histone tails and on the structured domains of metazoan histones. Histone variants within any species are highly conserved specialized histones that co-exist with the major histone types in the nucleus and have the potential to locally alter chromatin structure (reviewed in [34,35]). The structure of the essential histone variant H2A.Z (protein database code 1F66) supports the notion that this may take place not only at the level of the nucleosome but in the context of higher-order structure. Subtle changes in nucleosomal surface are the major hallmark of NCPs containing the essential histone variant H2A.Z [16]. These surface differences, which are located in regions of H2A.Z that are essential for viability [36], change the equilibrium between different states of chromatin higher-order structure that can be studied in vitro by analytical ultracentrifugation [19]. The presence of H2A.Z in nucleosomal arrays facilitates intranucleosomal interactions, as evident in a higher degree of folding to a compact structure in response to increasing Mg2þ, whereas internucleosomal interactions (fiber–fiber interactions) are disfavored.
Mutational and genetic studies Sin mutations: subtle changes, major effects?
Could a similar consequence of structural changes altering the balance of dynamic interactions also be responsible for the observed phenotype of histone mutants that affect transcription? SWI/SNF-independent (SIN) histone mutants in yeast partially relieve the requirement of some genes for the chromatin remodeling factor SWI/ SNF [37,38]. Differences occur between the relative effect of the mutants at different genes [38], implying a role for DNA sequence or local chromatin environment. The mutants found in genetic screens are located at the protein–DNA binding interface formed by the L1 and L2 loops of histones H3 and H4 (Figure 1c; [39]). It was further shown that the R45H SIN mutant of H4 — although competent to assemble chromatin and to sustain viability if present as the sole source of H4 in yeast — leads to increased accessibility of nucleosomal DNA in vivo, and impairs the ability of nucleosomes to supercoil DNA [40]. Similar data exist for the H3 SIN mutants [41]. Intriguingly, another SIN mutant, H4 R45C [37], www.current-opinion.com
Structure and dynamic behavior of nucleosomes Luger 131
Figure 3
bonds between the histone main chain and the DNA backbone at the site of mutation are disrupted in a subtle yet complex manner (U Muthurajan, K Luger, unpublished data). Given the relatively moderate structural effects of the H4 R45 C mutation (Figure 3), it is intriguing to speculate that the formation of regular higherorder structure is incompatible with highly mobile nucleosomes. It remains to be seen whether the other original SIN mutations [42] and other mutations in histones that give a SIN phenotype — but are located at the interface between the (H3–H4)2 tetramer and the (H2A–H2B) dimer [42] — have a similar effect on nucleosome sliding and higher-order structure formation.
(a) SHL 0.5
The lrs mutants: the other side of the coin?
H3 T118
H4 R45
(b) SHL 0.5
H3 T118
H4 R45C
Current Opinion in Genetics & Development
Structure of an NCP containing the SIN mutant H4 R45C. (a) Detailed view of H4 R45 and H3 T118 at SHL þ0.5 in wild-type NCP. (b) Detail from the H4 R45C mutant NCP structure, shown in green in the same orientation. The DNA from wild-type NCP has been superimposed in white.
when reconstituted into nucleosomal arrays, eliminates Mg2þ-dependent intramolecular folding of chromatin fibers [18]. Experiments similar to the one shown in Figure 2 were used to demonstrate that nucleosomes containing the H4 R45 SIN mutations (and all other SIN mutations) slide significantly faster or reposition at lower temperatures (A Flaus, U Muthurajan, K Luger, unpublished data). This can be rationalized on inspection of the 11 mutant nucleosome crystal structures that show that www.current-opinion.com
The histone folds of H3 and H4, and of H2A and H2B, pair together to form tightly interlocked, crescent shaped, quasi-symmetric heterodimers ([25]; Figure 4). Because of this quasi-symmetry, each crescent-shaped heterodimer can be structurally superimposed onto itself by a rotation of 1808 around this symmetry axis. Thus, the two ‘ends’ of each (H3–H4) ‘crescent’ (and the residues in H3 and H4 that are involved in DNA binding) are structurally equivalent, yet organize different regions of the DNA [39]. The same holds true for the (H2A–H2B) dimer (Figure 4). The SIN-mutants of H3 and H4 are all clustered at the one end of the (H3–H4) heterodimer (Figure 4) that organizes the central turn of nucleosomal DNA (SHL 0.5; Figures 1c and 4). Would the structurally equivalent protein residues at SHL 2.5, if mutated, have similar effects, or are these residues functionally different from those at SHL 0.5? Some answers to these questions have emerged from an unexpected corner. A genetic approach has identified a localized region on the nucleosome that is crucially involved in a process termed ‘chromatin silencing’ in yeast [20]. All of the affected residues (loss of rDNA silencing [lrs] mutants) are located within close proximity at the opposite end of the crescent-shaped H3–H4 heterodimer (Figure 4). They cluster around (and include) H3 K79, which has recently been shown to be the substrate for a specific H3 methyl transferase. This modification has the potential role to mark open (‘euchromatic’) chromatin regions ([22,43,44]; reviewed in [45]), possibly by changing the association between nucleosomes, or by preventing the binding of protein factors that are necessary for silencing in yeast. Within the identified region, different subregions appear to be necessary for the three different types of silencing that are known to exist in yeast because individual mutations affect them to varying extents. This is consistent with the notion that subtly different mechanisms are responsible for the three types of silencing. Intriguingly, five of the mutations identified in this genetic screen are the exact structural equivalents of Current Opinion in Genetics & Development 2003, 13:127–135
132 Chromosomes and expression mechanisms
Figure 4
Structural relationship of SIN and lrs mutants. The H3–H4 (top) and H2A–H2B (bottom) histone-fold dimers (colored as in Figure 1) are shown in the exact same orientation, based on a superposition of Ca atoms in the histone fold. Residues affected by SIN and lrs mutations in H3–H4, and structurally equivalent residues in H2A–H2B are shown. The inset to the left shows the lrs region of H3–H4 in a view generated by the rotation of H3–H4 by 1808 around the indicated axis, to emphasize the structural and functional similarity of SIN and lrs mutants.
the five original SIN mutants (Table 2). Thus, mutation of equivalent amino acid residues that fulfil the same function at different SHLs in wild-type nucleosomes appear to prohibit the formation of transcriptionally inactive chromatin. Biochemical characterization of these mutants is not yet available, but it will be of interest to see whether the effects on nucleosome structure, sliding, and ability to form higher-order assemblies in vitro are similar to those observed with the SIN mutant histones. These results also beg the question whether similar
consequences are to be expected upon mutation of the equivalent set of residues in the (H2A–H2B) dimer at SHLs 3.5 and 5.5 (Figures 1c and 4; Table 2).
Nucleosome dynamics and accessibility Site-specific recognition of DNA must occur in a highly compacted chromatin state. Whereas the co-existence of site-specific transcription factors (or enzymes) and histones on the same DNA in vitro ([46,47] and references therein) and in vivo [48,49] has been well documented,
Table 2 Structural correlation of SIN and lrs histone mutations. SIN mutant SHL 0.5
lrs mutant SHL 2.5
(H2A-H2B) SHL 3.5
(H2A-H2B) SHL 5.5
Function in wild-type NCP
H4 H4 H3 H3 H3 H4
[H3 L82 S] H3 R83 A H4 T80 A H4 R78 G H4 R67 H3 K79
[H2A Y39] H2A R42 H2B T85 H2B R83 H2B G72 –
[H2B L42] [H2A R77] H2A T76 [H2A K75] H2A E64 H2B H46
Stabilization of L1L2 loops Side chain reaches into minor groove H-bond with arginine that reaches into the minor groove Intra-chain salt bridge with D (not observed in H2B R83) Surface-exposed Surface-exposed, methylated by Dot1p (see text)
V43 I R45 H, C T118 I R116 H E105K G42
Mutations shown in bold have been identified by the respective genetic screens [20,37]. Residues in H2A and H2B that are equivalent to the SIN mutants (column 3) or to the lrs mutants (column 4) are also shown. Mutations shown in brackets are not the precise structural equivalent to those listed in the first and second column, but fulfill similar functions. See Figure 4 for details on structure.
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Structure and dynamic behavior of nucleosomes Luger 133
the structural implications of such interactions on both binding partners remain elusive. The crystal structures of three NCPs in complex with small polyamide molecules (protein database codes 1M18, 1M19, 1M1A [50]) that bind the minor groove of nucleosomal DNA in a sitespecific manner with nanomolar affinity represent a first step towards answering this question [17]. These studies not only confirmed earlier findings that nucleosomal DNA indeed is recognized with high affinity and in a sequence-specific manner [51] but showed that the nucleosome is much more dynamic and adjustable than previously assumed. By extension, this suggests that nucleosomal DNA may adapt in a similarly plastic manner in the presence of transcription factors that require access to only one face of the DNA. Similarly, subtle structural differences imposed by DNA sequence are easily accommodated (Figure 1c).
tion, recombination, and repair resemble very much those of a man who has lost his car keys in a dark alleyway, yet chooses to look for them under the street lamp.
Experimental evidence for a high degree of accessibility of nucleosomal DNA also comes from the study of DNA repair in a mononucleosome [46,52–54]. The activities studied here are only suppressed two- to eight-fold on a nucleosome, and their action does not require the unraveling or unfolding of nucleosomes. However, repair may be facilitated by the removal of histone tails [46] or by the presence of SWI/SNF in the reaction mixture [54].
Conclusions and outlook Histones are structurally and functionally bipartite. The structured histone-fold domains are responsible for DNA binding, whereas the flexible histone tails function as signaling and docking platforms for a diverse range of protein factors, and appear to be either directly or indirectly involved in the formation of higher-order structure. All of these histone-tail functions are regulated by a fiendishly complex set of post-translational modifications at specific amino acid side chains within the tails. As posttranslational modifications were thought to occur exclusively on the histone tails, the prevailing view on the role of the nucleosome in transcription regulation and chromatin compaction has been very ‘tail-centric’. This view is changing with the recent identification of enzymes that modify residues in the structured regions of the histones. It has become clear that the biological effects of posttranslational modifications, histone mutations, and also DNA-sequence variations have to be investigated with regard to their effect on nucleosome mobility and chromatin higher-order structure. This has proven to be particularly difficult for several reasons. First, the available nucleosome structures provide only a static snapshot of a highly dynamic and malleable structure. Second, structural information on any level of higher-order chromatin organization is virtually absent. And third, in vitro models for higher-order structure are difficult to obtain and to handle experimentally. Until these gaps are filled, our efforts to understand the impact of chromatin structure on the vital processes of DNA transcription, replicawww.current-opinion.com
Acknowledgements I thank U Muthurajan for the preparation of Figures 3 and 4, R Suto for Figure 2, and A Flaus and all present members of the Luger Laboratory for critical discussion.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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10. Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA: Regulation of heterochromatic silencing and histone H3 lysine9 methylation by RNAi. Science 2002, 297:1833-1837. Deletion of some genes that are part of the machinery responsible for RNAi results in the aberrant accumulation of complementary transcripts from centromeric heterochromatic repeats. This suggests a functional link between RNAi and the establishment and maintenance of heterochromatin. 11. van Leeuwen F, Gottschling DE: Genome-wide histone modifications: gaining specificity by preventing promiscuity. Curr Opin Cell Biol 2002, 14:756-762. 12. Harp JM, Hanson BL, Timm DE, Bunick GJ: Asymmetries in the nucleosome core particle at 2.5 A resolution. Acta Crystallogr D Biol Crystallogr 2000, 56:1513-1534. 13. White CL, Suto RK, Luger K: Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions. EMBO J 2001, 20:5207-5218. Sequence differences in a region that is ubiquitinated by Rad6 [31] lead to changes in crystal packing, suggesting that there are several ways to pack nucleosomes in a thermodynamically favorable way. 14. Davey CA, Sargent DF, Luger K, Maeder AW, Richmond TJ: Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution. J Mol Biol 2002, 319:1097-1113. The highest-resolution structure of any protein–DNA complex of this size. The manuscript focuses on the role of structured water at the protein– DNA interface. Water molecules not only contribute significantly to the stability of DNA-binding but also take part in adapting the histone surface to conformational variation in the DNA. It is suggested that bridging water molecules may play a principal role in facilitating nucleosome mobility. 15. Davey CA, Richmond TJ: DNA-dependent divalent cation binding in the nucleosome core particle. Proc Natl Acad Sci USA 2002, 99:11169-11174. Current Opinion in Genetics & Development 2003, 13:127–135
134 Chromosomes and expression mechanisms
16. Suto RK, Clarkson MJ, Tremethick DJ, Luger K: Crystal structure of a nucleosome core particle containing the variant histone H2A.Z. Nat Struct Biol 2000, 7:1121-1124. 17. Suto RK, Edayathumangalam RS, White CL, Melander C, Gottesfeld JM, Dervan PB, Luger K: Crystal structures of nucleosome core particles in complex with minor groove binding ligands. J Mol Biol 2003, 326:371-380. Three NCP–polyamide co-crystal structures show that ligand binding leads to local and global changes in DNA conformation, while maintaining a full complement of histone–DNA interactions. Experimental evidence is provided to support the hypothesis that twist defects can jump into nucleosomal DNA and diffuse along the DNA length, because it shows that twist defects are actively stabilized at multiple nucleosomal locations. 18. Horn PJ, Crowley KA, Carruthers LM, Hansen JC, Peterson CL: The SIN domain of the histone octamer is essential for intramolecular folding of nucleosomal arrays. Nat Struct Biol 2002, 9:167-171. Defined nucleosomal arrays were analyzed by analytical ultracentrifugation. H4 R45C substitution eliminates Mg2þ-dependent, intramolecular folding of the nucleosomal arrays, suggesting that Sin-versions of histones may alleviate the need for SWI/SNF in vivo by disrupting higherorder chromatin folding. 19. Fan JY, Gordon F, Luger K, Hansen JC, Tremethick DJ: The essential histone variant H2A.Z regulates the equilibrium between different chromatin conformational states. Nat Struct Biol 2002, 19:172-176. Surface changes in the H2A.Z histone variant NCP structure [16] facilitate the intramolecular folding of nucleosomal arrays while simultaneously inhibiting the formation of highly condensed structures that result from intermolecular association. 20. Park JH, Cosgrove MS, Youngman E, Wolberger C, Boeke JD: A core nucleosome surface crucial for transcriptional silencing. Nat Genet 2002, 32:273-279. The residues identified by a genetic screen cluster around (and include) H3 K79. Some are the structural and functional equivalents of the SIN mutants [37]. The surface may interact with specific silencing proteins, or it may be directly involved in fostering nucleosome–nucleosome contacts. 21. Dlakic M: Chromatin silencing protein and pachytene checkpoint regulator Dot1p has a methyltransferase fold. Trends Biochem Sci 2001, 26:405-407. 22. van Leeuwen F, Gafken PR, Gottschling DE: Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell 2002, 109:745-756. 23. Ng HH, Xu RM, Zhang Y, Struhl K: Ubiquitination of histone H2B by Rad6 is required for efficient Dot1- mediated methylation of histone H3-lysine 79. J Biol Chem 2002, 277:34655-34657. Rad6-mediated ubiquitination of H2B lysine 123 [31] is important for efficient methylation of lysine 79 of histone H3, suggesting that Rad6 affects telomeric silencing, at least in part, by influencing methylation of histone H3. 24. Feng Q, Wang H, Ng HH, Erdjument-Bromage H, Tempst P, Struhl K, Zhang Y: Methylation of H3-Lysine 79 is mediated by a new family of HMTases without a SET domain. Curr Biol 2002, 12:1052-1058. 25. Luger K, Maeder AW, Richmond RK, Sargent DF, Richmond TJ: X-ray structure of the nucleosome core particle at 2.8 A˚ resolution. Nature 1997, 389:251-259. 26. Luger K, Maeder AW, Sargent DF, Richmond TJ: The atomic structure of the nucleosome core particle. J Biomol Struct Dyn 2000, 11:185-189.
29. Widom J: Role of DNA sequence in nucleosome stability and dynamics. Q Rev Biophys 2001, 34:269-324. 30. Ahmad K, Henikoff S: The histone variant H3.3 marks active chromatin by replication- independent nucleosome assembly. Mol Cell 2002, 9:1191-1200. The histone variant H3.3 is the exclusive substrate for ‘replication-independent’ deposition. Residues on ‘major’, replication-dependent H3 that are responsible for excluding it from this pathway have been identified (see also Figure 1). 31. Robzyk K, Recht J, Osley MA: Rad6-dependent ubiquitination of histone H2B in yeast. Science 2000, 287:501-504. 32. Simpson RT, Thoma F, Brubaker JM: Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure. Cell 1985, 42:799-808. 33. Hansen JC, Ausio J, Stanik VH, van Holde KE: Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1. Biochemistry 1989, 28:9129-9136. 34. Redon C, Pilch D, Rogakou E, Sedelnikova O, Newrock K, Bonner W: Histone H2A variants H2AX and H2AZ. Curr Opin Genet Dev 2002, 12:162-169. 35. Wolffe AP, Pruss D: Deviant nucleosomes: the functional specialization of chromatin. Trends Genet 1996, 12:58-62. 36. Clarkson MJ, Wells JR, Gibson F, Saint R, Tremethick DJ: Regions of variant histone His2AvD required for Drosophila development. Nature 1999, 399:694-697. 37. Kruger W, Peterson CL, Sil A, Coburn C, Arents G, Moudrianakis EN, Herskowitz I: Amino acid substitutions in the structured domains of histones H3 and H4 partially relieve the requirement of the yeast SWI/SNF complex for transcription. Genes Dev 1995, 9:2770-2779. 38. Fleming AB, Pennings S: Antagonistic remodelling by Swi-Snf and Tup1-Ssn6 of an extensive chromatin region forms the background for FLO1 gene regulation. EMBO J 2001, 20:5219-5231. Differences occur between the relative effect of the mutants at different genes, implying a role for DNA sequence or local chromatin environment. 39. Luger K, Richmond TJ: DNA binding within the nucleosome core. Curr Opin Struct Biol 1998, 8:33-40. 40. Wechser MA, Kladde MP, Alfieri JA, Peterson CL: Effects of Sinversions of histone H4 on yeast chromatin structure and function. EMBO J 1997, 16:2086-2095. 41. Kurumizaka H, Wolffe AP: Sin mutations of histone H3: influence on nucleosome core structure and function. Mol Cell Biol 1997, 17:6953-6969. 42. Recht J, Osley MA: Mutations in both the structured domain and N-terminus of histone H2B bypass the requirement for Swi-Snf in yeast. EMBO J 1999, 18:229-240. 43. Ng HH, Feng Q, Wang H, Erdjument-Bromage H, Tempst P, Zhang Y, Struhl K: Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. Genes Dev 2002, 16:1518-1527. 44. Lacoste N, Utley RT, Hunter JM, Poirier GG, Cote J: Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase. J Biol Chem 2002, 277:30421-30424. 45. Varga-Weisz PD, Dalgaard JZ: A mark in the core: silence no more! Mol Cell 2002, 9:1154-1156.
27. Luger K, Rechsteiner TJ, Richmond TJ: Preparation of nucleosome core particle from recombinant histones. Methods Enzymol 1999, 304:3-19.
46. Chafin DR, Vitolo JM, Henricksen LA, Bambara RA, Hayes JJ: Human DNA ligase I efficiently seals nicks in nucleosomes. EMBO J 2000, 19:5492-5501.
28. Sullivan S, Sink DW, Trout KL, Makalowska I, Taylor PM, Baxevanis AD, Landsman D: The histone database. Nucleic Acids Res 2002, 30:341-342. Annotated alignments of full-length non-redundant sets of sequences are now available on the web in both HTML and PDF formats. The database also provides summaries of information on solved histone-fold structures, post-translational modifications of histones, and the human histone gene complement.
47. Angelov D, Charra M, Seve M, Cote J, Khochbin S, Dimitrov S: Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex binding. J Mol Biol 2000, 302:315-326.
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48. Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, Zaret KS: Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell 2002, 9:279-289. www.current-opinion.com
Structure and dynamic behavior of nucleosomes Luger 135
49. Zhu Z, Thiele DJ: A specialized nucleosome modulates transcription factor access to a C. glabrata metal responsive promoter. Cell 1996, 87:459-470.
53. Kosmoski JV, Ackerman EJ, Smerdon MJ: DNA repair of a single UV photoproduct in a designed nucleosome. Proc Natl Acad Sci USA 2001, 98:10113-10118.
50. Dervan PB: Molecular recognition of DNA by small molecules. Bioorg Med Chem 2001, 9:2215-2235.
54. Hara R, Sancar A: The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle. Mol Cell Biol 2002, 22:6779-6787.
51. Gottesfeld JM, Melander C, Suto RK, Raviol H, Luger K, Dervan PB: Sequence-specific recognition of DNA in the nucleosome by pyrrole- imidazole polyamides. J Mol Biol 2001, 309:625-639. 52. Nilsen H, Lindahl T, Verreault A: DNA base excision repair of uracil residues in reconstituted nucleosome core particles. EMBO J 2002, 21:5943-5952.
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55. Arents G, Burlingame RW, Wang BC, Love WE, Moudrianakis EN: The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix. Proc Natl Acad Sci USA 1991, 88:10148-10152.
Current Opinion in Genetics & Development 2003, 13:127–135
Structure and dynamic behavior of nucleosomes Karolin Luger Genetic studies have identified residues in the structured regions of the histones that are critically involved in the formation of heterochromatin. Any investigation of the events that regulate access to the chromatin substrate must take into account the dynamic nature of the nucleosome, and the regulated inter-conversion between various levels of chromatin higher-order structure. Addresses Department for Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:127–135 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by John Tamkun and David Stillman 0959-437X/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(03)00026-1
Abbreviations bp base pair lrs loss of rDNA silencing NCP nucleosome core particle RNAi RNA interference SHL Superhelix location SIN SWI/SNF-independent
Introduction The DNA of a single eukaryotic cell is compacted in the cell nucleus in a hierarchical scheme of folding into chromatin. At the first level of organization, two tight superhelical turns of DNA are wrapped around an octamer of two copies each of the four histone proteins: H2A, H2B, H3, and H4. This unit, the nucleosome core particle (NCP), represents the basic repeating structure in chromatin (Figure 1). Hundreds of thousands of nucleosomes, connected by 20–80 base pairs (bp) of linker DNA, are compacted further into hierarchically folded higherorder assemblies of unknown architecture (reviewed in [1,2]). Also critically involved in this process are linker histone H1, a variety of non-histone proteins, and divalent metal ions. The nucleosome fulfils different, seemingly conflicting roles in the nucleus. First, DNA is compacted by a factor of five by being forced into a tight superhelix upon interaction with the histone proteins. Second, despite making >120 direct atomic interactions between the histones and the DNA backbone at the 14 ‘superhelix www.current-opinion.com
locations’ (SHLs, numbered 0.5 to 6.5 in Figure 1c), nucleosomes are highly dynamic. This is most apparent in the observed ability of the histone octamer to translocate, or ‘slide’, along the DNA over significant distances (first observed in [3]; Figure 2). The considerable energy barrier for nucleosome sliding in vivo is likely alleviated by the action of ATP-dependent chromatin remodeling factors [4,5]. Third, the inter-conversion of fluid hierarchy chromatin structures that prevail in the interphase nucleus [6] from transcriptionally blocked to transcriptionally active states, as well as the formation of the highly ordered assembly that prevails in metaphase chromosomes are likely to be tightly regulated by reversible modification of histones, of other associated proteins, and of DNA (reviewed in [7–9]). The recently established link between the machinery that is responsible for the RNAi (RNA interference) phenomenon, and the formation of heterochromatin ([10], and reviewed in [11]) represents only a glimpse of many strange and wonderful things that remain to be understood in these regulatory pathways. The organization of eukaryotic DNA in nucleosomes has profound consequences on its ability to serve as a template for the enzymatic activities within the replication, recombination, repair, and transcription machineries. A wide range of approaches have provided new evidence on how chromatin accommodates all of these activities in a dynamic manner. High-resolution structures of NCPs from different organisms [12,13,14,15], or reconstituted with histone variants [16], and of NCPs in complex with small, site-specific minor groove DNA-binding ligands [17] deliver structural information in stunning detail (Table 1). Careful biophysical analyses show that the introduction of mutations at the protein–DNA interface, or the incorporation of variant histones result in profound changes in the types of higher-order structure formed by nucleosomal arrays [18,19]. A wealth of evidence from genetic screens suggests that this structured surface of the histone octamer is also involved in regulating the transition from transcriptionally active euchromatin to highly compacted and transcriptionally inactive heterochromatin [20,21,22,23,24]. Here, I review data that demonstrate the dynamic behavior of chromatin, and consider recent genetic data in light of these structural results.
Nucleosome structure Progress in our understanding of nucleosome and chromatin structure has been made with the determination of a variety of nucleosome three-dimensional structures (summarized in Table 1). First, technical advances have Current Opinion in Genetics & Development 2003, 13:127–135
128 Chromosomes and expression mechanisms
Figure 1
(a)
(b)
H3
H3
H3
H3
H4
H4
H4
H2B H2B
H2A
H2A
H2A (c)
H2B
H2A
(d)
0.5 SIN
6.5
1.5
5.5 lrs
2.5
4.5 3.5
Current Opinion in Genetics & Development
Overall structure of the nucleosome core particle. (a) Front view of the NCP (protein database 1KX5; [14]), viewed down the superhelical axis. The histone fold domains of H2A, H2B, H3, and H4 are colored in yellow, red, blue, and green respectively, histone tails and extensions are shown in white, DNA is shown in light blue. H3 residues that are responsible for selective replication-coupled assembly are shown in magenta [30]. The red star indicates the site of ubiquitination in yeast [31]. (b) Side view of the same structure, obtained by a 908 rotation around the vertical axis. (c) Partial NCP structure, viewed as in (a), but showing only 73 base pairs and associated proteins. SHLs are indicated, as are the location of the SIN and lrs mutants on H3 and H4. The DNA from an NCP reconstituted with a different DNA sequence (1KX4) was superimposed based on the histone octamer main chain atoms, and is shown in pale yellow. (d) Side view as in (b), but with parts of the DNA removed to reveal the location of the H3 residues identified in [30].
Current Opinion in Genetics & Development 2003, 13:127–135
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Structure and dynamic behavior of nucleosomes Luger 129
Figure 2
structure of nucleosomal DNA than was available from the previous structures [14]. Second, the first structure of an NCP containing a histone variant (H2A.Z; [16]) and the structures of chicken [12] and yeast [13] NCPs were also made available. Most recently, NCPs in complex with small, site-specific minor groove DNA-binding ligands were investigated [17]. All of these structures were crystallized at close to physiological salt concentrations (25 mM MnCl2 and 25 mM KCl, pH 6.0).
Incubation at 37°C
The Xenopus laevis nucleosome core particle at near-atomic resolution Current Opinion in Genetics & Development
Shifting behavior of X. laevis NCPs. The histone octamer is asymmetrically positioned with respect to the DNA after salt gradient reconstitution (at 48C) onto a 146 bp DNA fragment [25]. These positions are converted to the thermodynamically stable, symmetrically positioned species upon incubation at elevated temperatures. The temperature and time required for complete shifting is a function of the number of histone–DNA contacts and of the DNA sequence.
allowed the diffraction limit and quality of the electron density map of the original NCP structure (protein database code 1AOI [available in the Protein Data Bank repository: http://www.rcsb.org/pdb/]) [25,26] to be improved in a newer structure (protein database code 1KX3) [14]. A further significant improvement was subsequently achieved by adjusting the length of the DNA from 146 to 147 bp. This resolved crystallizationinduced structural heterogeneity in the DNA, and resulted in a much more accurate representation of the
What have we learned from these efforts? The amount of structural information available from the 1.9 A˚ structure of the NCP (protein database code 1KX5, reconstituted from recombinant Xenopus laevis histones [27] and a ‘symmetric’ 147 bp DNA fragment derived from human a-satellite DNA) [14,15] is unprecedented. A table in [14] lists each and every protein–DNA interaction in the structure. Given the extreme degree of sequence conservation between histones of different species [28], this will be a valuable reference for interpreting biochemical and genetic data. This structure is almost at the resolution where individual atoms can be directly resolved, and consequently >3000 water molecules have been identified. Among these, a large number of watermediated protein–DNA interactions, roughly equal in number to the 120 direct protein–DNA contacts, could potentially contribute significantly to the interaction energy between protein and DNA. However, as pointed out in [14], quantifying their relative importance to nucleosome stability and dynamics is not straightforward
Table 1 List of publicly available nucleosome and histone octamer structures, listed in chronological order. PDB code
Histone source
DNA source
Resol. Description
References
1AOI 1EQZ
X. laevis (recombinant) Gallus gallus (native, salt-extracted) X. laevis H2B, H3, H4; M. musculus H2A.Z, (all recombinant) S. cerevisiae (recombinant) X. laevis (recombinant)
146 bp palindromic, derived from human a-satellite 146 bp palindromic, derived from human a-satellite
2.8 2.5
Original NCP structure Chicken
[25] [12]
146 bp palindromic, derived from human a-satellite
2.6
Histone H2A.Z variant
[16]
146 bp palindromic, derived from human a-satellite
3.1
Yeast
[13]
146 bp palindromic, derived from human a-satellite
2.0
[14]
X. laevis (recombinant) X. laevis (recombinant) X. laevis (recombinant) X. laevis (recombinant) X. laevis (recombinant) G. gallus (native, salt-extracted) G. gallus (native, salt-extracted)
146 bp 147 bp 146 bp 146 bp 146 bp None
Components identical to 1AOI, improved resol. Different DNA sequence Highest resolution NCP to date NCP- ligand co-crystal structure 1 NCP- ligand co-crystal structure 1 NCP- ligand co-crystal structure 1 Original histone octamer structure –
–
1F66
1ID3 1KX3 1KX4 1KX5 1M18 1M19 1M1A 2HIO 1HQ3
None
palindromic, palindromic, palindromic, palindromic, palindromic,
derived derived derived derived derived
from from from from from
human human human human human
a-satellite B 2.6 a-satellite 1.9 a-satellite 2.45 a-satellite 2.3 a-satellite 2.65 3.1 2.15
[14] [14] [17] [17] [17] [55]
Structures of histone octamer alone are also listed at bottom. The code in the first column identifies the structure in the Protein Data Bank (PDB) repository (http://www.rcsb.org/pdb/). Resol, resolution (A˚ ).
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Current Opinion in Genetics & Development 2003, 13:127–135
130 Chromosomes and expression mechanisms
although the structure provides an unprecedented dataset for analysis. These solvent-mediated interactions are thought to help accommodate structural variations in the DNA that are a consequence of sequence variability found throughout the genome [14]. This observation suggests that solvent molecules could play a role in facilitating nucleosome mobility. Variations on the theme: DNA sequence
An argument against the general applicability of the published NCP structures has been the fact that all structures have been obtained from NCPs reconstituted with virtually identical DNA sequences. Of particular interest then is the finding that the overall structure of the NCP reconstituted with a significantly different DNA sequence is highly similar (protein database code 1KX4; [14]). Minor changes in the structure of the DNA (Figure 1c) are a reflection of the flexibility of nucleosomal DNA (also see below), since the full complement of histone–DNA interactions is also observed in this second structure. The differences in binding energy between different DNA sequences that have been observed experimentally [29] are likely to be a result of differences in the energy required for DNA distortion. Variations on the theme: histones
Given the high degree of evolutionary conservation of the histone proteins, it is not surprising that the 2.5 A˚ structure of an NCP reconstituted from chicken histones and a similar 146 bp DNA sequence (protein database code 1EQZ; [12]) is not much different from the earlier structures with Xenopus histones [14,25]. Protein and DNA structure, as well as crystal packing, is highly similar between Xenopus and chicken NCPs. By contrast, yeast histones are relatively divergent from those of higher eukaryotes [28]. Intriguingly, H2A and H3 in yeast appear to be evolutionary derivatives of the replacement histones H3.3 and H2A.X [30]. The yeast NCP structure (protein database code 1ID3) shows that the amino acid differences between H3.3 and replicationdependent H3 that are responsible for excluding the latter from replication-independent assembly in higher eukaryotes [30] have no impact on nucleosome structure, and are shielded from the solvent by the DNA supercoil ([13]; Figure 1b,d). One of the more surprising outcomes from the structure determination of the yeast NCP is the change in internucleosomal contacts in the crystal packing, which is a result of minor protein sequence differences between yeast and higher eukaryotes [13]. Although one has to be cautious of interpreting in vivo higher-order structure from in vitro crystal packing, these comparisons are likely to be significant considering that both types of interaction require energetically favorable interactions between highly charged nucleosome surfaces. Thus, it is Current Opinion in Genetics & Development 2003, 13:127–135
tempting to speculate that post-translational modifications of histones (either in the structured regions or in the histone tails), or the localized introduction of histone variants into chromatin may change the equilibrium between different types of nucleosome–nucleosome interactions. In this context, the identification of an ubiquitination site in a region of H2B that is critically involved in the formation of crystal contacts is particularly intriguing ([31]; Figure 1). One first step towards testing this hypothesis would be to use established in vitro systems [18,19,32,33] to study the ability of yeast histones to assemble into defined higher-order arrays, and to investigate the effect of post-translational modifications on histone tails and on the structured domains of metazoan histones. Histone variants within any species are highly conserved specialized histones that co-exist with the major histone types in the nucleus and have the potential to locally alter chromatin structure (reviewed in [34,35]). The structure of the essential histone variant H2A.Z (protein database code 1F66) supports the notion that this may take place not only at the level of the nucleosome but in the context of higher-order structure. Subtle changes in nucleosomal surface are the major hallmark of NCPs containing the essential histone variant H2A.Z [16]. These surface differences, which are located in regions of H2A.Z that are essential for viability [36], change the equilibrium between different states of chromatin higher-order structure that can be studied in vitro by analytical ultracentrifugation [19]. The presence of H2A.Z in nucleosomal arrays facilitates intranucleosomal interactions, as evident in a higher degree of folding to a compact structure in response to increasing Mg2þ, whereas internucleosomal interactions (fiber–fiber interactions) are disfavored.
Mutational and genetic studies Sin mutations: subtle changes, major effects?
Could a similar consequence of structural changes altering the balance of dynamic interactions also be responsible for the observed phenotype of histone mutants that affect transcription? SWI/SNF-independent (SIN) histone mutants in yeast partially relieve the requirement of some genes for the chromatin remodeling factor SWI/ SNF [37,38]. Differences occur between the relative effect of the mutants at different genes [38], implying a role for DNA sequence or local chromatin environment. The mutants found in genetic screens are located at the protein–DNA binding interface formed by the L1 and L2 loops of histones H3 and H4 (Figure 1c; [39]). It was further shown that the R45H SIN mutant of H4 — although competent to assemble chromatin and to sustain viability if present as the sole source of H4 in yeast — leads to increased accessibility of nucleosomal DNA in vivo, and impairs the ability of nucleosomes to supercoil DNA [40]. Similar data exist for the H3 SIN mutants [41]. Intriguingly, another SIN mutant, H4 R45C [37], www.current-opinion.com
Structure and dynamic behavior of nucleosomes Luger 131
Figure 3
bonds between the histone main chain and the DNA backbone at the site of mutation are disrupted in a subtle yet complex manner (U Muthurajan, K Luger, unpublished data). Given the relatively moderate structural effects of the H4 R45 C mutation (Figure 3), it is intriguing to speculate that the formation of regular higherorder structure is incompatible with highly mobile nucleosomes. It remains to be seen whether the other original SIN mutations [42] and other mutations in histones that give a SIN phenotype — but are located at the interface between the (H3–H4)2 tetramer and the (H2A–H2B) dimer [42] — have a similar effect on nucleosome sliding and higher-order structure formation.
(a) SHL 0.5
The lrs mutants: the other side of the coin?
H3 T118
H4 R45
(b) SHL 0.5
H3 T118
H4 R45C
Current Opinion in Genetics & Development
Structure of an NCP containing the SIN mutant H4 R45C. (a) Detailed view of H4 R45 and H3 T118 at SHL þ0.5 in wild-type NCP. (b) Detail from the H4 R45C mutant NCP structure, shown in green in the same orientation. The DNA from wild-type NCP has been superimposed in white.
when reconstituted into nucleosomal arrays, eliminates Mg2þ-dependent intramolecular folding of chromatin fibers [18]. Experiments similar to the one shown in Figure 2 were used to demonstrate that nucleosomes containing the H4 R45 SIN mutations (and all other SIN mutations) slide significantly faster or reposition at lower temperatures (A Flaus, U Muthurajan, K Luger, unpublished data). This can be rationalized on inspection of the 11 mutant nucleosome crystal structures that show that www.current-opinion.com
The histone folds of H3 and H4, and of H2A and H2B, pair together to form tightly interlocked, crescent shaped, quasi-symmetric heterodimers ([25]; Figure 4). Because of this quasi-symmetry, each crescent-shaped heterodimer can be structurally superimposed onto itself by a rotation of 1808 around this symmetry axis. Thus, the two ‘ends’ of each (H3–H4) ‘crescent’ (and the residues in H3 and H4 that are involved in DNA binding) are structurally equivalent, yet organize different regions of the DNA [39]. The same holds true for the (H2A–H2B) dimer (Figure 4). The SIN-mutants of H3 and H4 are all clustered at the one end of the (H3–H4) heterodimer (Figure 4) that organizes the central turn of nucleosomal DNA (SHL 0.5; Figures 1c and 4). Would the structurally equivalent protein residues at SHL 2.5, if mutated, have similar effects, or are these residues functionally different from those at SHL 0.5? Some answers to these questions have emerged from an unexpected corner. A genetic approach has identified a localized region on the nucleosome that is crucially involved in a process termed ‘chromatin silencing’ in yeast [20]. All of the affected residues (loss of rDNA silencing [lrs] mutants) are located within close proximity at the opposite end of the crescent-shaped H3–H4 heterodimer (Figure 4). They cluster around (and include) H3 K79, which has recently been shown to be the substrate for a specific H3 methyl transferase. This modification has the potential role to mark open (‘euchromatic’) chromatin regions ([22,43,44]; reviewed in [45]), possibly by changing the association between nucleosomes, or by preventing the binding of protein factors that are necessary for silencing in yeast. Within the identified region, different subregions appear to be necessary for the three different types of silencing that are known to exist in yeast because individual mutations affect them to varying extents. This is consistent with the notion that subtly different mechanisms are responsible for the three types of silencing. Intriguingly, five of the mutations identified in this genetic screen are the exact structural equivalents of Current Opinion in Genetics & Development 2003, 13:127–135
132 Chromosomes and expression mechanisms
Figure 4
Structural relationship of SIN and lrs mutants. The H3–H4 (top) and H2A–H2B (bottom) histone-fold dimers (colored as in Figure 1) are shown in the exact same orientation, based on a superposition of Ca atoms in the histone fold. Residues affected by SIN and lrs mutations in H3–H4, and structurally equivalent residues in H2A–H2B are shown. The inset to the left shows the lrs region of H3–H4 in a view generated by the rotation of H3–H4 by 1808 around the indicated axis, to emphasize the structural and functional similarity of SIN and lrs mutants.
the five original SIN mutants (Table 2). Thus, mutation of equivalent amino acid residues that fulfil the same function at different SHLs in wild-type nucleosomes appear to prohibit the formation of transcriptionally inactive chromatin. Biochemical characterization of these mutants is not yet available, but it will be of interest to see whether the effects on nucleosome structure, sliding, and ability to form higher-order assemblies in vitro are similar to those observed with the SIN mutant histones. These results also beg the question whether similar
consequences are to be expected upon mutation of the equivalent set of residues in the (H2A–H2B) dimer at SHLs 3.5 and 5.5 (Figures 1c and 4; Table 2).
Nucleosome dynamics and accessibility Site-specific recognition of DNA must occur in a highly compacted chromatin state. Whereas the co-existence of site-specific transcription factors (or enzymes) and histones on the same DNA in vitro ([46,47] and references therein) and in vivo [48,49] has been well documented,
Table 2 Structural correlation of SIN and lrs histone mutations. SIN mutant SHL 0.5
lrs mutant SHL 2.5
(H2A-H2B) SHL 3.5
(H2A-H2B) SHL 5.5
Function in wild-type NCP
H4 H4 H3 H3 H3 H4
[H3 L82 S] H3 R83 A H4 T80 A H4 R78 G H4 R67 H3 K79
[H2A Y39] H2A R42 H2B T85 H2B R83 H2B G72 –
[H2B L42] [H2A R77] H2A T76 [H2A K75] H2A E64 H2B H46
Stabilization of L1L2 loops Side chain reaches into minor groove H-bond with arginine that reaches into the minor groove Intra-chain salt bridge with D (not observed in H2B R83) Surface-exposed Surface-exposed, methylated by Dot1p (see text)
V43 I R45 H, C T118 I R116 H E105K G42
Mutations shown in bold have been identified by the respective genetic screens [20,37]. Residues in H2A and H2B that are equivalent to the SIN mutants (column 3) or to the lrs mutants (column 4) are also shown. Mutations shown in brackets are not the precise structural equivalent to those listed in the first and second column, but fulfill similar functions. See Figure 4 for details on structure.
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Structure and dynamic behavior of nucleosomes Luger 133
the structural implications of such interactions on both binding partners remain elusive. The crystal structures of three NCPs in complex with small polyamide molecules (protein database codes 1M18, 1M19, 1M1A [50]) that bind the minor groove of nucleosomal DNA in a sitespecific manner with nanomolar affinity represent a first step towards answering this question [17]. These studies not only confirmed earlier findings that nucleosomal DNA indeed is recognized with high affinity and in a sequence-specific manner [51] but showed that the nucleosome is much more dynamic and adjustable than previously assumed. By extension, this suggests that nucleosomal DNA may adapt in a similarly plastic manner in the presence of transcription factors that require access to only one face of the DNA. Similarly, subtle structural differences imposed by DNA sequence are easily accommodated (Figure 1c).
tion, recombination, and repair resemble very much those of a man who has lost his car keys in a dark alleyway, yet chooses to look for them under the street lamp.
Experimental evidence for a high degree of accessibility of nucleosomal DNA also comes from the study of DNA repair in a mononucleosome [46,52–54]. The activities studied here are only suppressed two- to eight-fold on a nucleosome, and their action does not require the unraveling or unfolding of nucleosomes. However, repair may be facilitated by the removal of histone tails [46] or by the presence of SWI/SNF in the reaction mixture [54].
Conclusions and outlook Histones are structurally and functionally bipartite. The structured histone-fold domains are responsible for DNA binding, whereas the flexible histone tails function as signaling and docking platforms for a diverse range of protein factors, and appear to be either directly or indirectly involved in the formation of higher-order structure. All of these histone-tail functions are regulated by a fiendishly complex set of post-translational modifications at specific amino acid side chains within the tails. As posttranslational modifications were thought to occur exclusively on the histone tails, the prevailing view on the role of the nucleosome in transcription regulation and chromatin compaction has been very ‘tail-centric’. This view is changing with the recent identification of enzymes that modify residues in the structured regions of the histones. It has become clear that the biological effects of posttranslational modifications, histone mutations, and also DNA-sequence variations have to be investigated with regard to their effect on nucleosome mobility and chromatin higher-order structure. This has proven to be particularly difficult for several reasons. First, the available nucleosome structures provide only a static snapshot of a highly dynamic and malleable structure. Second, structural information on any level of higher-order chromatin organization is virtually absent. And third, in vitro models for higher-order structure are difficult to obtain and to handle experimentally. Until these gaps are filled, our efforts to understand the impact of chromatin structure on the vital processes of DNA transcription, replicawww.current-opinion.com
Acknowledgements I thank U Muthurajan for the preparation of Figures 3 and 4, R Suto for Figure 2, and A Flaus and all present members of the Luger Laboratory for critical discussion.
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