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The language of covalent histone modifications
Author: Brian Strahl
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"� 2000 Macmillan Magazines Ltd NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com 41 progress The language of covalent histone modifications Brian D. Strahl & C. David Allis Department of Biochemistry and Molecular Genetics, University of Virginia Health Science Center, Charlottesville, Virginia 22908, USA ............................................................................................................................................................................................................................................................................ Histone proteins and the nucleosomes they form with DNA are the fundamental building blocks of eukaryotic chromatin. A diverse array of post-translational modifications that often occur on tail domains of these proteins has been well documented. Although the function of these highly conserved modifications has remained elusive, converging biochemical and genetic evidence suggests functions in several chromatin-based processes. We propose that distinct histone modifications, on one or more tails, act sequentially or in combination to form a ?histone code? that is, read by other proteins to bring about distinct downstream events. How eukaryotic genomes are manipulated within a chromatin environment is a fundamental issue in biology. At the heart of chromatin structure are highly conserved histone proteins (H3, H4, H2A, H2B and H1) that function as building blocks to package eukaryotic DNA into repeating nucleosomal units that are folded into higher-order chromatin fibres 1,2 (Fig. 1). Once thought of as static, non-participating structural elements, it is now clear that histones are integral and dynamic components of the machinery responsible for regulating gene transcription. The same is probably true for other DNA-templated processes such as replication, repair, recombination and chromosome segregation. An extensive literature documents an elaborate collection of post- translational modifications including acetylation, phosphorylation, methylation, ubiquitination and ADP-ribosylation 3 that take place on the ?tail? domains of histones. These tails, which protrude from the surface of the chromatin polymer and are protease sensitive, comprise ,25?30% of the mass of individual histones 3,4 , thus providing an exposed surface for potential interactions with other proteins (for example, Sir3/4 and Tup1 proteins in yeast 5,6 ). Because of the inherent disordered nature of histone tails, their precise location in higher-order fibres and the atomic details of their structure are not known 1,7 . Long-standing models have suggested that histone modifications may alter chromatin structure by influencing histone?DNA and histone?histone contacts 4,8 . However, growing awareness of the remarkable diversity and biological specificity associated with dis- tinct patterns of covalent histone marks has caused us and others 9?13 to favour the view that a histone ?language? may be encoded on these tail domains that is read by other proteins or protein modules. We refer to this language as the ?histone code? and present evidence supporting the existence of this language and discuss some potential ramifications. To illustrate the potential complexity of covalent marks decorating a single histone tail, we have chosen to focus most of our discussion on a short stretch of core histone H3. However, many of the concepts presented here are likely to apply to all of the histone termini and, in particular, to that of histone H4 10,14 . Lysine acetylation sets the stage Of the modifications listed above, histone acetylation has been the most studied and appreciated 14 . Fuelled, in part, by the discovery of enzymes responsible for bringing about the steady-state balance of this modification?histone acetyltransferases (HATs) and histone deacetyl- ase (HDACs)?compelling evidence has recently been provided that acetylation of specific lysine residues in the amino termini of the core histones plays a fundamental role in transcriptional regulation 2,15 . In H3 from most species, the main acetylation sites include lysines 9, 14, 18 and 23 (refs 3, 16), and, as is the case with the functionally redundant tail of its nucleosomal partner, H4 14 , selected lysines become acetylated during specific cellular processes (Figs 1b and 2). Transcription-linked acetylation, catalysed by the GCN5 family of HATs, shows a preference for lysine 14 of H3 in vitro 17 although an expanded set of lysine residues is likely to be used in vivo 18,19 .Howis this acetylation site specificity in H3 brought about? Solution and crystal structure data of various members of the GCN5 HAT family, including co-crystals of the enzyme with H3 tail peptides 20 , have begun to yield important insights into the enzy- matic mechanisms underlying the site specificity of these HATs 21?25 . One important concept to emerge from these studies is that residues outside the preferred lysine 14 acetylation site in H3 are important for histone-binding specificity. For example, glycine 13 and proline 16 have a critical role in leading to a restricted GCN5?H3 peptide recognition site, G-K14*-X-P (ref. 20). Thus, as is the case with protein kinases and phosphatases, short preferred consensus motifs are likely to exist for individual HATs and HDACs 26 which help to establish the final histone code. Acetylation of specific lysine residues in H3 is also associated with biological processes apart from transcription (Fig. 2). During DNA replication, for example, new histones are rapidly synthesized and assembled onto the replicated DNA. H3 and H4 are brought to replicating chromatin in a pre-acetylated state that becomes erased after replication is completed and the newly assembled chromatin matures 27,28 . Whereas the sites of deposition-related H4 acetylation are highly conserved 29,30 (for example, lysines 5 and 12; see Fig. 2), the situation with H3 is less clear. However, lysine 9 in H3 appears to have a more dominant role in histone deposition and chromatin assembly in some organisms 17,27,30 . The finding that a chromatin assembly complex in Drosophila, called RCAF (for replication- coupling assembly factor), contains H4 specifically acetylated at lysines 5 and 12 suggests that these acetylation sites play an important role in chromatin assembly 31 . Does this acetylation pattern represent a code that has been deciphered by a component of a histone chaperone complex? Finally, we note that the spacing between acetylatable lysines (Fig. 1b) is strikingly regular in the amino termini of many histones (for example, lysines at 9, 14, 18 and 23 in H3; and 5, 8, 12 and 16 in H4), and, curiously, this spacing periodicity is reminiscent of that of an a-helix (that is, 3.6 residues). To our knowledge, no group has systematically attempted to expand or contract the characteristic three-to-four residue spacing between many known acetylation sites. Along this line, the alternating, and seemingly, invariant pattern of deposition-related acetylation wherein lysines 5 and 12, but not lysines at 8 and 16, are acetylated in newly synthesized H4 is particularly intriguing 29,30 (Fig. 2). Beyond histone acetylation Phosphorylation, particularly that of histones H1 and H3, has long been implicated in chromosome condensation during mitosis 32,33 . However, converging evidence suggests that H3 phosphorylation � 2000 Macmillan Magazines Ltd (specifically serine 10; see Fig. 1b) is also directly correlated with the induction of immediate-early genes such as c-jun,c-fos and c-myc 34?36 . Mutations in Rsk-2, recently shown to be an H3 kinase in vitro, are associated with Coffin?Lowry syndrome in humans and result in a loss of epidermal growth factor-stimulated H3 phosphorylation in vivo 9,37 . Transcriptional activation in response to mitogenic and other stimuli are altered in Coffin?Lowry cells 38 , suggesting a potential direct role for H3 phosphorylation in regulating gene transcription through a remodelling step that is most consistent with chromatin decondensation, a result seemingly at odds with the use of this mark in chromosome condensation. The potential importance of the serine 10 phosphorylation mark in H3 is strengthened by the finding that MSK1, a kinase activated by growth factor and stress stimuli, also phosphorylates H3 in vitro 39 . Interestingly, another H3 kinase has recently been identified that is associated with dosage compensation in flies 40 .InDrosophila, equalization of transcription from the sex chromosomes is achieved by a twofold upregulation of transcription from the male X chromosome 41 that is associated with acetylation of H4 at lysine 16 42 . Thus, H4 acetylation on lysine 16, possibly in concert with H3 phosphorylation at serine 10, may establish a combinatorial mark that leads to enhanced transcription from the male X chromosome. What about other histone modifications? Just as histone acetylation and phosphorylation have become topics of renewed interest, we expect other known histone modifications will soon be back in the limelight. Methylation of lysine and/or arginine residues, for example, are among the least understood post-translational modifications affecting histones. This is partly because the responsible enzymes are not known, immunological reagents selective to detect histone methylation do not exist, and, unlike histone acetylation and phosphorylation, the charge on the individual lysine and arginine residues is not greatly affected, making electrophoretic resolution of methylated histones difficult. H3 and H4 are the predominant histones modified by methylation, and sequencing studies from many organisms indicate that multiple lysines in H3 (4, 9 and 27) are the preferred sites of methylation 3,43,44 . Moreover, lysine residues can be mono- di- or trimethylated, adding yet another layer of complexity to this histone mark. The recent discovery of a nuclear receptor co-activator-interacting protein, called CARM1, which possesses arginine-specific, histone H3-selective methyltransferase (HMT) activity 45 provides evidence to support the notion that histone methylation contributes to transcriptional activation. CARM1 HMT activity is required for ligand-dependent transcriptional activation, and the finding that CARM1 functions through association with co-activators contain- ing HAT activity is particularly intriguing given that Rsk-2 interacts with the transcriptional co-activator CREB-binding protein (CBP) 46 . These data suggest that large multisubunit enzyme com- plexes containing multiple histone- and non-histone-modifying activities 9,47 work in concert with other chromatin remodelling machines 48,49 to regulate gene transcription (Fig. 3, left panel). The ?histone code? hypothesis Considering only the electrostatic requirements for folding the chromatin polymer 4,8,50 , histone acetylation, through the neutralization of positive charge, and histone phosphorylation, through the addition of negative charge, would probably cause decondensation of the chromatin fibre 51 . Thus, the use of multiple marks on histone tails (that is, combining acetylation and phosphorylation) could serve to amplify the readout of upstream signalling pathways causing greater changes in the overall charge density of tails that progress 42 NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com Nucleosome core particle H3N ? Higher-order folding 10-nm filament H4 H2B H2A H3 Chromatin fibre On Off H3 Globular domain N-terminal 'tail' N ? a b Unmodified histone H3 Modified histone H3 H3 H4 4 9 10 14 18 23 27 28 N-ARTKQTARKSTGGKAPRKQLATKAARKSAP... 1 5 8 12 16 20 N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT... ? * CENP-A N-MGPRRRSRKPEAPRRRSPSPTPTPGPSRRG... ? ? ? 7 9 17 27 Figure 1 Chromatin organization and the tail of histone H3. a, General chromatin organization. Like other histone ?tails?, the N terminus of H3 (red) represents a highly conserved domain that is likely to be exposed or extend outwards from the chromatin fibre. A number of distinct post-translational modifications are known to occur at the N terminus of H3 including acetylation (green flag), phosphorylation (grey circle) and methylation (yellow hexagon). Other modifications are known and may also occur in the globular domain. b, The N terminus of human H3 is shown in single-letter amino-acid code. For comparison, the N termini of human CENP-A, a centromere-specific H3 variant, and human H4, the nucleosomal partner to H3, are shown. Note the regular spacing of acetylatable lysines (red), and potential phosphorylation (blue) and methylation (purple) sites. The asterisk indicates the lysine residue in H3 that is known to be targeted for acetylation as well as for methylation; lysine 9 in CENP-A (bold) may also be chemically modified (see text). The above depictions of chromatin structure and H3 are schematic; no attempt has been made to accurately portray these structures. Acetylated Histone deposition? ? Higher-order combinations N N N N ? Phos/acetyl Transcription Phosphorylated Mitosis/meiosis Methylated Transcription? SMC/ Condensins? ? ? ? ? N N H3 Residue: 1 4 9 10 14 18 23 28 Acetylated Bromodomain Transcription Modification Associated Function state protein/module Unmodified Sir3/Sir4/Tup1 Silencing N termini N H4 N 5 12 Acetylated Histone deposition RCAF? CENP-A N Phosphorylated Mitosis? ? ? 7 17 27 N Acetylated Transcription 8 16 ? Figure 2 The ?histone code? hypothesis. Histone modifications occur at selected residues and some of the patterns shown have been closely linked to a biological event (for example, acetylation and transcription). Emerging evidence suggests that distinct H3 (red) and H4 (black) tail modifications act sequentially or in combination to regulate unique biological outcomes. How this hierarchy of multiple modifications extends (depicted as ?higher-order combinations?) or how distinct combinatorial sets are established or maintained in localized regions of the chromatin fibre is not known. Relevant proteins or protein domains that are known to interact or associate with distinct modifications are indicated. The CENP-A tail domain (blue) might also be subjected to mitosis-related marks such as phosphorylation; the yellow bracket depicts a motif in which serines and threonines alternate with proline residues. � 2000 Macmillan Magazines Ltd lead to greater changes in the chromatin structure of target genes. Indeed, evidence supports synergism between histone acetylation and phosphorylation in the induction of immediate-early genes after mitogenic stimulation 52 , and it seems likely that these signalling pathways may converge at other loci as well 35 . H3 phosphorylation at serine 10, possibly in conjunction with phosphorylation at serine 28 (Figs 1b and 2; and see below), is also required for proper segregation and condensation of chromosomes during mitosis and meiosis 53,54 . If the function of H3 phosphoryla- tion is to ?open? chromatin, how then can H3 phosphorylation at the same site also be involved in chromosome condensation? This question seems to beg a simple answer: perhaps a single histone modification does not function alone. We will refer to the hypoth- esis?that multiple histone modifications, acting in a combinator- ial or sequential fashion on one or multiple histone tails, specify unique downstream functions?as the histone code hypothesis. What is the evidence in support of this hypothesis? Serine 28 in H3 is embedded in surrounding sequences similar to serine 10 (that is, both are R-K-S*), and data has shown that serine 28 is phosphorylated during chromosome condensation in mam- malian cells 55 (Fig. 1b). Whether serine 28 is phosphorylated during interphase or during immediate-early gene induction is not yet known. Thus, the formal possibility remains that multiple phos- phorylation events on the same histone tail, or on several tails, may be required for efficient chromosome condensation during mitosis and meiosis (see below). Even if the H3 tail is doubly marked by serine 10 and serine 28 phosphorylation during mitosis, it seems likely that this is not the complete story regarding chromosome condensation. H3 phos- phorylation at serine 10 initiates in the pericentric heterochromatin, an A/T-rich region of satellite DNA closely associated with centro- meric DNA 53 . Centromeric DNA itself is packaged with specialized proteins, one of which is a specialized H3 ?variant? found in both yeast and humans, CENP-A 56 . CENP-A differs from H3 primarily in its unique N-terminal tail (Fig. 1b). Apart from lysine 9, arginine residues comprise all other positively charged side chains. Thus, the role of acetylation at lysine 9 (if it occurs) probably differs from that of acetylation of canonical H3 in association with transcription. Moreover, the CENP-A tail contains many serine and threonine residues, which raises the possibility that this specialized H3, like the main H3, becomes phosphorylated during mitosis. An alternating S/T/G-P motif repeats five times in this tail generating an 11-amino- acid stretch flanked on either side by arginine residues (Fig. 1b). This motif suggests that CENP-A may be phosphorylated during mitosis, and it will be interesting to determine whether the CENP-A and H3 proteins are substrates for the same or unique sets of kinases and phosphatases. It would also be of interest to determine whether the SMC/condensing proteins, which have a central role in mitotic chromosome condensation, bind to these tails and contain histone- modifying activities 57 (Fig. 2). The enzymology of multiple histone modifications The existence of multiple modifications within a short stretch of the same histone tail (Fig. 1b), begs the question: how is a complex, multimark code established and maintained in the first place? One attractive hypothesis is that covalent modification of a histone tail by one enzyme influences the rate or efficiency with which a second enzyme follows using the now-modified histone tail as substrate. Does site-specific phosphorylation or methylation influence the ability of a HAT to recognize and bind to the tail? Alternatively, does a histone kinase or methylase care whether a histone tail is acetylated at a specific lysine residue? To that end, we point out that many modifications are close enough to each other on the histone tail (Fig. 1b) to influence, positively or negatively, the ability of enzymes to further modify these residues. Along this line, modifications on one histone tail might influence the outcome of other enzymatic activities acting on other histone tails. The explosion of recent discoveries of histone-modifying enzymes, many available in recombinant form, paves the way for future experimental tests of some of these questions. Structural studies with modified histone substrates will be necessary to determine which residues, if any, are used to stabilize or promote interactions with modified substrates. Site-directed mutagenesis of these residues, followed by in vivo and in vitro assays, will help to progress NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com 43 Transcription Mitosis Cellular signals CondensedDecondensed Nucleus Cell membrane HMT KINASE KINASE HMT? H3 HDAC? HAT? ? HDAC? Act HAT H3 CENP-A ? NuRC BD ?BD ? H3 ?BD ? NuRC BD ?? H3 CENP-A ?? Docking with downstream complexes Figure 3 Coordinated recruitment of histone-modifying activities. Recent discoveries suggest that distinct histone-modifying activities interact to form multisubunit complexes that probably work in concert with nucleosome remodelling complexes (NuRCs; for example, Swi/Snf, RSC, NURF) to remodel chromatin. Interactions demonstrated thus far include CARM1, a histone methyltransferase (HMT), with histone acetyltransferase (HAT)- containing coactivators that interact with nuclear receptors (Act) 45 and Rsk kinase with the CBP/p300 HAT 46 . Not depicted is the possibility of non-histone substrates being bona fide targets of these activities. In addition to remodelling nucleosomes (indicated by zigzag DNA), NuRCs may chemically modify and/or bind histone tails. Binding of a NURC or HAT complex (depicted as single ovals) to histone tails may be mediated by the bromodomain (BD). Although many of the complexes identified to date are implicated in events leading to transcription (left panel; green nucleosomes), we speculate that similar, but unique, complexes may exist that modify and direct chromatin condensation (right panel; red nucleosome). This ?code? of modifications may dictate the biological outcome through changes caused in higher-order chromatin structure or may direct the downstream biological effect by recruiting and interacting with docking proteins or complexes that remain to be identified. Although H3 and CENP-A are the only histones depicted here, it is likely that all other histones are subjected to this type of regulation. � 2000 Macmillan Magazines Ltd dissect meaningful functional relationships. How is the histone code read? Could histone modifications exist simply to regulate chromatin structure? Core histone acetylation alone has been shown to relax higher-order chromatin structure in vitro, and to promote factor- binding to cognate DNA elements 4,8 . Alternatively, histone mod- ifications could also act as specific ?receptors? to recruit unique biological complexes that mediate downstream function (Figs 2 and 3). Phosphorylation of H3 at serine 10 is closely associated with both chromosome condensation during mitosis and immediate- early gene induction following mitogenic stimulation. One possible explanation for this discrepancy is shown in Fig. 3. Here we envisage that a phosphorylation mark alone, or in combination with other marks (such as phosphorylation at serine 28), may recruit a binding factor that, in turn, has a role in mediating chromosome condensa- tion and segregation. In contrast, a distinct mark or set of marks (for example, phosphorylation and acetylation at residues 10 and 14, respectively) may provide a unique binding surface to recruit factors promoting decondensation and transcription (for example, Swi/ Snf; see below). Assuming that phosphorylation of CENP-A occurs during mitosis, this marked tail may provide an attractive binding surface for a kinase that carries out general H3 phosphorylation at serine 10 and/or serine 28. In the specific case of the CENP-A tail (Fig. 1b), clusters of arginine residues are in some cases interrupted by serine residues (for example, R-R-R-S*-R-K) where the marked serine is serine 7. Phosphorylation at these positions may possibly serve to modify interactions with proteins that recognize this basic patch in the CENP-A tail. One appealing feature of the histone code hypothesis is that it offers a possible explanation for ?exceptions? to the general rule that histone acetylation correlates positively with gene activation, whereas histone deacetylation acts to create repressive chromatin. For example, recent studies on the mouse mammary tumour virus (MMTV) promoter suggest that histone acetylation is actually involved in transcriptional repression (T. K. Archer and C. L. Smith, personal communication). Similarly, mutations in the HDAC homologue RPD3 cause enhancement of position effect variegation in flies, not suppression as would first be expected 58 . Along these lines, pericentric heterochromatin in flies 42 and silent loci in yeast 59 are marked by acetylation of lysine 12 of H4. The disparity of having histone acetylation linked to both gene activation and repression is reminiscent of the situation with histone H3 phosphorylation being linked to both chromosome condensation and immediate-gene induction. Part of the solution to this paradox may be in having unique histone codes read by distinct sets of proteins that then bring about different downstream responses. If correct, it may be that mitosis-specific HATs, HDACs and HMTs act during chromosome condensation and that distinct sets of histone-modifying enzymes mark chromatin for deconden- sation during gene activation (Fig. 3). Who reads the code? Direct evidence that H3 and H4 tails can act as specific ?receptors? has been provided for Sir3 and Sir4 5 and Tup1/Ssn6 (ref. 6) proteins involved in transcriptional silencing and repression in yeast. More- over, Tup1 binding is influenced by the acetylation state of the H3 and H4 N-terminal tails: unacetylated or monoacetylated H3 and H4 are more strongly bound by Tup1 compared with hyper- acetylated H3 or H4, suggesting that alterations in tail structure and/or charge due to acetylation can modulate non-histone pro- tein/tail binding interactions. Whether or not other histone marks (such as phosphorylation, methylation) regulate these interactions further remains an important issue for future studies. Recent evidence shows that the bromodomain of human PCAF (P300/CBP-associated factor), a domain of little known function which is shared between many, but not all HATs, binds acetylated lysine in the context of H3 and H4 tail sequences 60 . This result suggests that protein motifs may have evolved to recognize histone modifications 61 (Fig. 3). Precedence for this type of receptor?ligand interaction already exists in nature. Phosphorylated tyrosine, for example, is known to be read in specific contexts by SH2-containing modules that, in turn, have an impact on downstream biological events 62 . Have similar mechanisms evolved for lysine acetylation, as well as other covalent histone modifications 61 ? Relevant to this discussion may be the observation that the spacing of acetylatable lysines in the N termini of many histones is regular. Is this spacing part of the histone recognition motif? To that end, we note that many bromodomain-containing proteins have two adjacent bromodomain modules (for example, TAF II 250). Whether in these proteins each bromodomain functions indepen- dently or synergistically to bind acetylated lysines, in one or more histone tails, is not known, but remains an intriguing possibility. If bromodomains bind acetylated histones, then this suggests that other chromatin-associated polypeptides containing this domain may also function through recognition and binding to specific acetylation patterns on histones (Fig. 3). For example, components of the Swi/Snf and RSC (for remodels the structure of chromatin) family of ATP-dependent chromatin remodelling enzymes contain bromodomains 61 . Is it possible that these remodelling complexes function through a combination of factor recruitment 48 and recog- nition of distinct acetylation patterns on the histone tails at promoters regulated by these complexes 49 ? Some support for this is provided by genetic evidence that suggests that Swi/Snf function is partially redundant with the functions of Gcn5-containing HAT complexes at specific promoters 63?65 and by studies that show that ATP-dependent remodelling complexes like NURF (nucleosome remodelling factor) require histone tails to function properly 66 . Additionally, could the interdependency of Swi/Snf remodelling complexes with HATs be due, in part, to the fact that each is capable of leaving different covalent marks on the chromatin fibre as part of its remodelling function? In so doing, are signals laid down on the histone tails that recruit the next remodelling complex? It is known, for example, that Gcn5-containing HAT complexes are recruited to specific promoters in yeast after the recruitment of Swi/Snf 48,49 . Could this recruitment partly be caused by the ability of Swi/Snf to leave a mark for HATs to see? It will be of interest to determine whether any nucleosome remodelling complexes contain enzymatic activities that leave covalent marks on histones. Parallels in nature and conclusions Like chromatin, microtubules are polymers composed of highly conserved subunits, (a- and b-tubulin) that heterodimerize to form the repeating unit of this cytoskeletal fibre. Tubulins also have ?tail? domains that are decorated by a diverse array of post-translational modifications, some of which are in common with histones (acetyl- ation and phosphorylation) 67,68 . Like histone tails, the microtubule tails, which are located in the C terminus, also lack a defined structure at atomic resolution 69 but are known to be recognized by microtubule-associated proteins(MAPs)?polypeptides thought to impart dynamic features to the polymer. We wonder whether unique combinations of post-translational modifications exist on tubulins that modulate their function as in chromatin. The apparent parallels between these two types of cellular polymers are intriguing and suggest that a general theme is used by nature to regulate the dynamics of large polymers. In both cases, the complexity and potential redundant nature of these covalent marks may underlie the general difficulty in obtaining clear phenotypes in mutational analyses of known modification sites. In summary, the large network of post-translational modifica- tions that decorates histone tails appears to represent a mechanism for differential regulation of chromatin activity in several distinct biological settings. The histone code described here is by no means deciphered, and we have begun to consider the staggering possi- progress 44 NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com � 2000 Macmillan Magazines Ltd bility that every amino acid in histone tails has specific meaning and is part of the vocabulary of the overall code. The realization that histone variants, such as the centromere-associated protein CENP- A, exist in special chromosomal locations adds yet another level of variation to the chromatin fibre and the histone code. The recent track record suggests the continued need to: (1) identify and map the sites for the complete dictionary of covalent histone modifica- tions in all histones; (2) mutate and identify phenotypes associated with each of the modifications, singly and in combination; (3) identify and characterize the enzymes systems that add or subtract these modifications; (4) determine how complexes containing these activities are recruited to key genomic targets; and (5) learn how these covalent marks specify interactions with downstream partners or modulate higher-order structures. After a long incubation period, interest in covalent modifications of histones is at an all-time high. Understanding the rules and the consequences of this histone code is likely to impact on many, if not all, DNA-templated process with far-reaching implications for human biology and disease. M 1. Luger, K. & Richmond, T. J. The histone tails of the nucleosome. Curr. Opin. Genet. Dev. 8, 140?146 (1998). 2. 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Cell Biol. 4, 53?57 (1992). 69. Nogales, E., Whittaker, M., Milligan, R. A. & Downing, K. H. High-resolution model of the microtubule. Cell 96, 79?88 (1999). Acknowledgements Research surrounding this topic in our laboratory is supported by grants from the NIH to C.D.A. and B.D.S. We wish to thank current laboratory members, especially C. A. Mizzen, for critical review of this manuscript, and T. K. Archer and C. L. Smith for kindly sharing unpublished data. Correspondence and requests for materials should be addressed to C.D.A. (e-mail: allis@virginia.edu). progress NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com 45 "
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