Deviant nucleosomes: the functional specialization of chromatin

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B i o p h y s i c i s t s and biochemists have often focussed on chromatin structure simply as an engineering problem. The highly conserved histones are proposed to wrap DNA around them to assemble monotonous arrays of nucleosomes, which, in turn, self-associate to form semi-crystalline higher-order structures. This image has proven very convenient for those scientists who require structurally unilbrm samples in order to interpret their experiments on the physical properties of biological material. However, this homogenous packaging of DNA within the chromosome is not consistent with the experience of those molecular geneticists and cell biologists who investigate chromatin function. These investigators find the organization of DNA within the chromosome to be highly variant, reflecting the functional differentiation of the chromosome into distinct domains. Recent observations provide a molecular explanation that potentially reconciles the dual requirements of packaging DNA into chromatin via nucleosomal structures and the functional differentiation of the chromosome into distinct domains. This review illustrates how the introduction of variants of the histones themselves and the inclusion of specific trans.acting factors, with striking similarities to the histones, can retain some of the architectural features of canonical nucleosomes yet provide the highly differentiated chromatin structures necessary to facilitate events, such as transcription and chromosomal segregation.

Deviant nucleosomes: the functional specialization of chromatin ALAN P. WOLFFEAND DM1TRYPRUSS ~egulatory prote/ns exist w/tb strong sequence and

structural ~ r ~ t e s to the bistone proteins. Molecular £exettc and cea b/o/og/ca/analyses suggest that these proteins are localized at particular sites within the chromosome. Their assembly iato ucleosomal structures cotters slwcializedf u n a i o ~ to ttulividual chromosomal domain~ that covalendy modify lysine and serine residues maintained at conserved sites6. Both the phosphorylation of serine and the acetylation of lysine residues within the N-terminal-tail domains are associated with the modulation of transcriptional activityT,8. Ubiquitination of the C-terminal tail of H2A is also correlated with transcriptional activation9. These modifications can be targeted through unknown mechanisms to particular chromosomal domains. Several core histone variants exist that have specific changes from normal histones both in the N-terminal tails and in the DNA-binding surfaces of the C-terminal histone-fold domains (Fig. 2). For example, differences in amino acid sequence from the normal somatic H2A are conserved in a particular H2A variant (H2A.Z) from ciliate protozoa (Tetrahymena) to humans, suggesting that the H2A.Z protein is biologically important. In fact, the histone H2A.Z variant in Drosophila has been shown to be essential for early development 1°. Until recently, core histone variants were believed to contain the most extreme changes from a normal histone architecture that might be incorporated into a nucleosome. However, these variants are now recognized as only one example of a family of proteins that share the histone-foid structure (Fig. la) and that might be incorporated into a nucleosomal architecture, ahhough these proteins contain wide deviations from normal histone sequences 11. The core histories appgar to have evolved from a DNA-hinding protein that contained only the three tx-helices of the histone-fold domain and lacked any tail domainslt. The archaebacterial protein HMf consists of only the histone-fold domain and has the capacity to wrap DNA around itself within nucleosome-like stnmtures t2. The eukaryotic core histones retained this property, but added the capacity of the assembled nucleoprotein complex to interact with other proteins outside the nucleosome through the addition of the tail domains. Other specific regulatory proteins have made use of their histone-fold domains to confer very specialized properties on individual nucleoso~tes through their replacement of normal histones within chromatin t3,t4, These include the deviant histones: CENP-A, which is found at the centromere (discussed later), and rat macro H2A (Figs 1 and 2). The function of macro H2A is not yet known, although an extended C-terminal

Core htstones and relaLod regulatory proteins Arents, Moudrianakis and colleagues discovered that each of the four core histones (H2A, H2B, H3 and H4) has a very similar.C-terminal-domain structure that directs the formation of specific heterodimers between the histones and that also determines the path of DNA in the nucleosome 1. Each C-terminal domain contains at least three t~-helices spatially arranged in a 'histone fold' (Fig. la). Within this structure, a long central helix is bordered on each side tW a loop segment and a shorter helix. The long central helix acts as a heterodimerization interface (Fig. lb). Dinaerization creates three DNA-binding surfaces through the interaction of loop segments at each end of the long central helix, and through the juxtaposition of the two short a-helices flanking the amino (N) terminal ends of the long central helix 2 (Fig. 113). The C-terminal histone-fold domains of the core histones might be expected to be conserved because of the extensive protein-protein and proteinDNA interactions implicit to their central structural role in the nucleosome. However, the N-terminal-tail domains of the core histones H3 and H4 are as invariant through eukaryotic evolution as the histone-fold domains3. This conservation of N-terminal protein sequence reflects the essential role for the histone tails in gene regulation'~. The N-terminal tails of all of the core histones and the C-terminal tail of histone H2A protrude on the outside of the nucleosome, where they can potentially make contacts with other nucleoprotein complexes. The interaction of the histone tails with non-histone proteins can either activate or repress transcription in a promoter-specific m~:nnet4. 5. The N-terminal-tail domains are also the targets of specific enzymatic activities

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(a)

tail has been added to the histonefold domain of H2A (Ref. 15). This tail contains the leucine zipper, H2A, _ ~ , = ~ v ne 367C which is a dimerization motif often MacroH 2 A ' N = - ' ~ @ ~ found in transcription factors. This leucine zipper might interact with CBF.cN i C a sequence specific DNA-binding protein to tether a specific nucleospinal structure at a defined site. H2BN 38 5S 93 106 C More extreme variations from normal histone sequence are CBF-Ak ~ " = ' ~ C found in transcriptional regulatow proteins, These regulatory proN 45 64 87 121 C teins maintain the histone-fold H3====RK=APRR=GeFI ==: domain and make use of it both CENP-A--N--==RK=APRR=G:R=== C to direct specific protein-protein interactions and to bind to DNA TAF1140 ~ ¢ ~ q ~ ' ~ ~ ~1 C (Refs 16, 17), Regulatory proteins with these properties include the important components of the H4=N=== I__ 31 50 84 ~ " = / ' % 5 9 3 basal transcription factor TFIID, known as TATA-binding-protein TAF1160N C associated factors (TAF),60 and -40, and the related CCAAT-box(b) binding proteins, NF-Y (CBF) and HAP2, -3 and -5 (Fig. 1). TAFu40 and TAFu60 exist as heterodimers NH2B H2AC in TFIID, TF,40 resembles H3, and TAFIÁ60 resembles H4, Both proteins have extended C-terminal NH2A° tails that interact with other components of TFIID and transcriptional activators. It has been proposed that TAF.40 and TAFu60 participate in the assembly of nucleosome-like structures, excluding normal histones from the TATA l~6uae 1. Histones and regulatory proteins comaining the histone fold. (a) Each core box, yet maintaining DNA in a histone is ~hown in a linear representation with the approximai:: regions of 0t-helix semi-compacted state competent shown as tvlinders. Numbers indicate the first amino acid relative to the N-terminusof for transcriptionl6. Metazoan NF-Y the histone protein for each helix, The related proteins are shown below each histone, (CBF) and Saccbaromyces cemvLciae Proteins related to histone H2A are in green, to hisrone H2B in blue, to historic H3 in yellow and to histone H4 in red. Note that the entire N4emlinal tail of the core histones is HAP2, -3 and -5 are highly related not shown to scale. (b) The heterodimerization of histones H3 and H4 and of histones trimeric proteins. The evolutionarily H2A and H2B, The C-temlinalstructured domain of each core histone is shown conserved peptide sequences of [color coded as in (a)] in die hislone fold, "Ii~einteraction between the hlstone-fold two of the subunits (CBF-A and domains is described as a 'handshake 't, The sites of interaction with DNA within the CBF-C, or HAP3 and.. HAP5) heterodimer are indicated by the arrows. The positions of the N-tennlnal tails of the core resemble the histone-fold domains histones are shown as dashed lines, of histones H2B and H2A, respectively (Fig. 1). These domains are essential for DNA binding in the presence of the third protein (CBF-B or HAP2) that condlat bends DNA, but that also fulfills additional functions fers sequence specificity 17. The NF-Y (CBF) and HAP2, by virtue of interaction with other transcription factors, Thus, it might be important to retain DNA in a relatively -3 and -5 proteins are transcriptional activators. It appears advantageous for a large number of compact structure within a nucleosome-like architeceukaryotic DNA-binding proteins to retain their histonetune, but also useful for the historic-like protein to like character (see later). This requirement might be assume other more specialized functions, similar to the architectural role of the HMG box in I)lfferentlatin8 chromattn at the centromcre the assembly of large regulatory nucleoprotein complexes tS. In certain instances, it is possible to faciliThe centromene provides an excellent example of a specialized chromosomal domain. The two sister cbromatate nucleoprotein-complex assembly through the use of a generic HMG-box protein, such as HMGI, which tids are held together at the centromere until mitosis, alters or stabilizes a bend in the double helix. In more then tile attachment of the centromere to the mitotic specialized cases, a specific transcription factor has spindle at the kinetochore mediates chromosome segreevolved that incorporates a structure related to HMG1 gation, The human centromere contains repetitive

NH.~ H3c

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FIcAmE2. Sequence alignments between histones and related proteins. (a) Comparison between histone H2A, human, Drosophilaand Tetrabymena H2A.Zvariants, and macro H2A. identities are indicated by dashes. Gaps to maximize alignment are shown by the dots. The zig-zag lines ha the H2A sequences represe_nt the divergent C-terminal tail sequences. Numbers of amino acids are indicated where the sequences are very different (e.g. N-terminal tails). Helical regions are indicated (helix 1, 2 and 3 from the N-terminus). (b) Comparison between histone H3 and CENP-A;alignments as in (a). ~x-satellite DNA. Each repeating unit of 171bp direcLs both the positioning of a single nucleosome and the sequence-selective binding of the HMGI or HMGY proteins19, 20. Human centromeric, hromatin contains a highly deviant histone called CENP-A. This has significant identity over the histone-fold domain to histone H3, yet has a very different N-terminal tail (Figs 1 and 2). CENP-A is found within nucleosomes and presumably heterodimerizes with H4 (Refs 13, 21). importantly, Sullivan and colleagues discovered that the histonefold domain of CENP-A targeted the protein to the centromere 21. This result implies that other histone variants, such as H2A.Z, might be targeted to particular DNA sequences, because the DNA-binding surfaces of the histone-foid domains show a similar number of sequence differences compared to the normal somatic histone H2A as seen between histone H3 and CENP-A (Fig. 2). It is possible that CENP-A has arisen through the necessity of having a specialized nucleosomal structure at the centromere where the N-terminal-tail domain makes highly selective contacts with other centromeric components, such as the large hydrophilic DNA-binding proteins CENP-B and CENP-C (Fig. 3). Experimental support for this hypothesis comes from genetic experiments in S. cemvisiae where a CENP-A homolog, CSE4 is essential for correct sister chromatid segregation 14. This suggests that nucleosomes that include CENP-A will assemble a specialized differentiated chromosomal domain that might serve to facilitate attachment of the

kinetochore and the function of the centromere in the segregation of chromosomes. Deviant nucleosomes - the 'winged helix' connection The assembly of a differentiated domain at the centromere establishes an interesting precedent for the targeting of core histone variants to particular sites within chromatin. A very similar targeting of linker histones and isomorphous transcription factors to nucleosomes containing defined DNA sequences also occurs, Linker histones, such as histone H1 or HS, contain a structured nucleic-acid-binding domain known as the 'winged helix'. This domain is found in a variety of sequence-specific transcriptional regulators, including the prokaryotic catabolite gene activator protein and the eukaryotic hepatocyte nuclear factor 3 protein (HNF3) z2,z3. The winged helix consists of a bundle of three ¢~-helices attached to a three-stranded anti-parallel iS-sheet. HNF3 binds across the major groove of DNA via one of the u-helices suggesting that the structured domain of the linker histone will contact nucleosomal DNA in the same way, The linker histone contains additional basic N- and C-terminal domains that influence the path of linker DNA between adjacent nucleosomes 24. Linker histones, such as histone HI, have been shown to direct the exact positioning of nucleosomes with respect to DNA sequence 25. This positioning relies on the sequence and structure selective recognition of DNA by the linker histone, and protein-protein

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(b)

Fmt~'ltt 3, The putative arrangement of CEN'P-Ain the nucleosome. (a) The C-terminal histone-foM domains of the core histones are shown. The core hi.stones are shown in blue except for CEN'P-A. which is sho~ax in ~,'ellow. (b) Putative clustering of CENP-A N-terminal tails in a nucleosomal array. These tails might have protein--protein interactions with ~xher components of the centmn,m~, such as CENP-B ancL'or CENP-C. contacts m a d e bet~'een the w i n g e d helix d o m a i n of the linker histone and the histone-fold domains o f the core histones 26. During Xenopus laevis development, early embryonic variants o f linker histones are progressively replaced with normal somatic histone H1. The inclusion o f histone HI into nucleosomes assembled on the tandemly reiterated oocyte-t3'pe 55 rilx)somal RNA e n c o d i n g genes directs the positioning of histone-DNA contacts over key promoter elements ar.d represses transcription 27. Similar to the reiterated u-satellite DNA o f the h u m a n centromere, the repetitive r,~ature o f 5S rRNA e n c o d i n g genes has e n a b l e d a m o r e detailed analysis o f chromatin structure than has b ~ . n possible for single, unique nucleosomes. Nevertheless, evidence also exists for the assembly of specialized nucleosomes on regulatory DNA. The m o u s e serum albumin e n h a n c e r esists in the active state within an array o f precisely positioned nucleosome-like particles 28. Specific enhanc,er-binding factors, including HNF3, are part o f the n u d e o s o m e like particles a n d HNF3 can actively direct their positioning with respect to DNA sequence. These observations lead to the hypothesis that HNF3 repla.ces linker histones within chromatin containing the serum albumin enhancer 28, thereby eztablishing a precise regulatory nucleoprotein architecture (Fig. 4). The replacement o f histone H1 b y HNF3 w o u l d be analogou.'.~ to the replacement o f histone H3 b y CENP-A at the centromere. In both instances, a sequence-selective histonelike regulatory protein w o u l d direct the assembly of a differentiated chromatin domain. This might e n c o m p a s s a single variant nucleosome, as at the serum albumin enhancer, or long art'ays o f variant nucleosomes, as at the mammalian centromere,

~ture prospects The application o f molecular genetics, and cell biology, to chromosomal organization and function has led

to s o m e surprising conclusions. A w i d e variety o f histone-like proteins can b e a s s e m b l e d into nucleosomal structures. In many instances these deviant nucleosomes include specific DNA sequences. Core a n d linker

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Fmum~ 4. A specialized nucleosome on the mouse serum albumin enhancer. Two nueleosomes (N1 and N2; see Ret*,27) ate shown positioned on the enhancer of the mouse serum albumin gene (numbers are relative to the 5' end of the albumin enhance0, The boundaries of micrococcal nuclease digestion are indicated by the buckets. The positions of uans~'ription-fa~.'~orbinding sites are shown as is the potemiai site of HNF3 or linker histone H1 interaction with the nucleosomal structures. The helix that interacts with DNA is shaded red.

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histone variants, proteins of the histone-fold and winged-helix families, can all contribute to the local differentiation of functional chromosomal domains. What are the advantages for regulatory proteins of maintaining a histone-like structure within a nucleosomal architecture? One immediate advantage is the stability of the nucleosome during the cell cycle. Histones H3 and H4 do not exchange out of chromatin outside of S-phase, moreover, molecular mechanisms exist to reassemble nucleosomes efficiently on newly replicated DNA (Red, 29). Thus, CENP-A could maintain a very stable association with the centromere even through the replication process. Histones H2A and H2B do exchange out of chromatin, but do so predominantly during transcription 29. It is very difficult to disrupt core histone interactions within a nucleosome in vivo. Regulatory proteins that assemble in association with the core histones will be resistant to displacement from DNA by competing protein-DNA interactions. Linker histories have a much less stable association with nucleosomal DNA. Relatively weak interactions offer the potential for a role in regulatory events where transcription needs to be reversibly activated. For example HNF3 might replace linker histones on the serum albumin enhancer to prevent their repressive influence on transcription (Fig. 4). A second advantage of transcription factors resembling the histones lies in the utilization of nucleosomal architecture (see also our earlier discussion of HMG proteins). As the physical analysis of chromatin deafly demonstrates, nucleosomal arrays do efficiently self-assemble into higher-order chromatin structures. The absence of a nucleosome or the presence of a large non-nucleosomal nucleoprotein complex will probably interfere with this self-assembly process30. Thus, the req~2;.rement for the assembly of higher order chromatin structures might impose constraints on the properties of nucleoprotein complexes in particular chromosomal regions. The easiest way to maintain higher order structure, yet have functional specialization, would be to modify the proteins within a nucleosome, while maintaining the basic function of DNA compaction. A distinct feature of histone interactions with nucleosomal DNA is the exposure of DNA on the surface of the nucleosome. One side of DNA is occluded on the histone surface, but the other is exposed and potentially accessible to other regulatory proteins. Thus, histonelike interactions with DNA could facilitate the assembly of muiticomponent complexe#L There are only a limited number of ways through which proteins can recognize the double helical DNA molecule. The core histones and the linker histones provide two examples of protein structures able to have specific interactions with DNA. The cell has made use of proteins with histone-like structures to carry out particular functions. Eukaryotic regulatory proteins have evolved to operate in a chromatin environment. A feature of chromatin is the assembly of precise nucleoprotein structures, in which regulatory proteins are accommodated into nucleosomal architectures. The future will increasingly emphasize not the monotony of chromatin structure but the capacity of nucleosomes to adopt a fascinating variety of form and function.

Ar.lmowledSetmats I thank Ms Thuyro for prepa,-ation of the manuscript, and David Landsman and Van Mouddanakis for usefui discussions.

References 1 Arents, G. etaL (1991) Proc. NatlAcad. Scl. USA88, 10148-10152 2 Arents, G. and Moudrianakis, E.N. (1993) Proc. NaaAcad. Set. USA 90, 10489-10493 3 De Lange, RJ., Fambtough, D.M., Smith, E.L and Bonner, J. (1969)./. Biol. Chem. 244, 5669--5679 4 Grunstein, M. etal. (1992) in TranscrtptfonalRegulatton (McKnight, $. and Yamamoto, K., eds), pp. 1295-1315, Cold Spring Harbor Laboratory Press 5 Hecht, A. etal. (1995) CellSO, 583-592 6 Turner, B.M., Birtey, A.J. and Lavender,J. (1992) Cell69, 375-384 7 Mahadevan, L.C., Willis, A.C. and Barrah, M.J. (1991) Cell 65, 77%783 8 Hebbes, T.R., Clayton, A.L.,Thome, A.W. and Crane-Robinson, C. (1994) EMBOJ. 13, 1823--1830 9 Levinger, L. and Varshavsky, A. (1982) Cell28, 375-385 1 0 Van Daal, A. and Elgin, S.C.R. (1992) Mol. Biol. Cell. 3, 593-6O2 I ! Baxevanis, A.D., Arents, G., Moudrianakis, E.N. and Landsman, D. (1995) Nn¢lefc Acids Res. 23, 2685-2691 12 Sandman, K. et ai. (1990) Proc. Naa Acad. Set. USA 87, 5788-5791 13 Sullivan, K.F., Hechenberger, M. and Masri, K. (1994) J. Cell Blol. 127, 581-592 14 Stoler, S., Keith, K.C., Kumick, K.E. and FitzGerald-Hayes, M (1995) Genes Dev. 9, 573-586 15 P e ~ n , J . R . and Fried, V.A. (1992) Sc~-nce257. 1396-1400 16 Kokubo, T. et aL (1993) Nature367, 484--48"7 17 Sinha, S., MaRy, S.N., Lu,J. and de Crombrugghe, B. (1995) Pro¢. NatlAcad. Sci. USA 92, 1624-1628 18 Grosschedl, R., Giese, K. and PageI,J. (1994) Trends Genet. 10, 94-100 19 Zhang, X-Y., Fittler, F. and Horz, W. (1983) Nucleic Acids Res. 11, 4287-4307 2 0 Strauss, F. and Varshavsky, A. (1984) Cell37, 889--901 21 Palmer, D.K. et al. (1987)./. CelIBiol. 104, 805-815 22 Ramakrishnan, V, et aL (1993) Nature362, 2It.b-224 23 Clark, K.L.. Halay, E.D.. Lai, E. and Burley, S.K+(1993) Nature364, 412--420 24 Pruss, D, Hayes,JJ, and Wolffe, A.P. (1994) BioFAsavs 17, 161-170 25 Meersseman, G., Pennings, S. and Bmdbu~, E.M. (1991) J. Mol. Biol 220, 89-100 26 Ura. K.. Hayes, J.J. and Wolffe, A.P. (1995) FatCBOJ, 14, 3752-3765 27 Bouvet, P., Dimitrov, S. and Wolffe, A.P. (1994) Genes Dev. 8, 1147-1159 28 McPherson, C.E., Shim, E.Y., Friedman, D.S. and Zaret, K.S. (1993) Cell75, 387-398 29 Jackson, V. (1990) Biochemistry 29, 719--731 30 Hansen, J.C. and Ausio,J. (1992) Trends Biochem. Sci. 17, 187-191 31 Truss, M. etaL (1995) EMBOJ. 14, 1737-1751

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