and function

Histone structure M. Mitchell University

of Virginia,

Smith

Charlottesville,

Virginia,

USA

The past year has seen major advances in our understanding of histone and nucleosome structure and function. Direct DNA mapping and thermodynamic experiments have finally provided conclusive evidence that the histones impose an altered helical pitch on the DNA as it is wrapped on the surface of the core histone octamer. Further, it is now clear that lysine acetylation in the amino-terminal domains of histones H3 and H4 can alter the topology of the DNA in chromatin and probably influence its higher-order folding. Genetic experiments reported in the past year have provided a wealth of new information on histone structure and function, including the identification of the peptide domain of histone H4 that is necessary for permanent gene repression, the confirmation that nucleosome structure is critical for centromere function, and evidence that histone acetylation plays a significant role in chromosome dynamics.

Current

Opinion

in Cell

Biology

1991,

3:42+437

Introduction

Histone

The histone proteins, and the core octameric complexes that they constitute, form the fundamental organizational units of the eukaryotic chromosomes. Research interest in the structure and function of histones and chromatin has been strong for over 25 years, beginning with the purification of nuclear chromatin as a more or less de6ned material, through the discovery of the basic composition of the nucleosome. Recent advances in our understanding of the histone proteins at one extreme, and the higher-order folded chromosome at the other, continues to sustain that intense interest, and with good reason. All of the vital processes of nuclear physiology, transcription, replication, repair, recombination, and segregation must deal with the remarkable topological complexity of the chromosome, imposed at the most fundamental level by the histone proteins, Thus, advances in our understanding of histone and nucleosome structure and function are likely to impinge on a wide variety of basic issues in molecular cell biology and genetics.

A schematic diagram illustrating our current view of the nucleosome is shown in Fig. 1. At the center of the nucleosome core particle is an octamer complex of histone proteins, two each of histones H2A, H2B, H3, and H4. The octamer contains a central tetramer, composed of two molecules of histone H3 and two molecules of histone H4, flanked on each side by a dimer, composed of one molecule of histone H2A and one molecule of histone H2B. The octarner is thought to be roughly cyfindrical, with a diameter of approximately 70 A and a thickness of 55-57A The octamer is capable of binding and organizing approximately 146 bp of DNA into 1.75 lefthanded turns around the outside of the protein complex with a pitch of roughly 28& The dimensions of the resulting nucleosome core particle approximate a slightly wedge-shaped disk, about 1lOA in diameter and 55A thick.

This review covers the results of experiments reported during the last year that have clarified and refined our picture of the structure of the nucleosome, that have identified the functional significance of specific histone peptide sequences, and that have provided evidence for the participation of the histones in chromosome segregation and nuclear division. For a review of recent experiments that address the role of chromatin structure in DNA repair, see the raiew by Smerdon elsewhere in this issue (pp 422428).

Both the nucleosome core particle [ 1] and the histone octamer [2) were crystallized several years ago for Xray analysis; however, the structures derived from the two studies are incompatible. Subsequently, preliminary neutron-scattering measurements [3] and circular dichroism (CD) studies [4] were interpreted as showing that the shape and volume of the octamer changed dramaticaUy on going from high concentrations of sodium chloride to high concentrations of ammonium sulfate, conditions approximating those used for ctystallization of

Octamer

and nucleosome

structure

structure

Abbreviation CD-circular

@ Current

Biology

dichroism.

Ltd ISSN 0955-0674

429

430

Nucleus

(a)

and gene expression

(b)

(c)

it is more likely that there have been technical difficulties in solving one or both of the structures. These results are encouraging because they suggest that further analysis and refinement should resolve the ditferences between the two structures and provide a uniform picture of the architecture of the nucleosome.

The path of the DNA

(d)

(e)

Fig. 1. Schematic illustration of the nucleosome, showing some key structural features. The drawings are a subjective attempt to summarize selected data from [1,15,16,17**1 and references cited in those papers. The four core histones are distinguished throughout the panels by their particular shading. Their structures, folding, and interactions are highly over-simplified. (a) The core histone octamer is seen directly down the dyad axis of symmetry through the center of the two histone H3 molecules. The structure is represented as roughly cylindrical with a diameter of 70A and a width of %A, drawn with the edge of the cylinder shown. fb) The nucleosome core particle shown as in panel A, but now with 146 bp of DNA assembled into a left-hand superhelix of 1.75 turns around the octamer. The pitch of the superhelix is about 28A. fc) A side view of the nucleosome core particle with the structure in fb) rotated W’around the vertical axis, showing the diameter face of the nucleosome. fd) Speculation on the locations of the flexible amino-terminal domains of the core histones using the structure and view shown in fb) but extending the DNA to complete two turns on the octamer. The rough locations of amino-terminal domains are indicated by the additional ‘random coils’ shown. The binding sites of the H2B amino-terminal domains are hidden in this perspective. fe) A side view of the nucleosome with the histone amino-terminal domains. The structure in fd) is rotated Waround the vertical axis to produce the same perspective as in fc).

the octamer. This interpretation apparently reconciled the two diRerent crystallographic structures [5]. However, this issue has recently been reevaluated, using optical rotary dispersion and CD spectropolarimetry measurements [6*,7-l. The results of these new studies suggest that the interpretations of the previous data were in error because of scatter caused by histone aggregation in high ammonium sulfate concentrations. When the samples were clarified by ultracentrifugation and the protein concentration and composition redetermined just before taking the measurements, no signiIicant ditferences in secondary structure were detected. Therefore, it is unlikely that both the low-resolution nucleosome structure and the preliminary octamer structure are correct. Rather,

Although the DNA in the nucleosome is wrapped about 1.75 times around the outside of the histone protein octamer, removal of the histone from the DNA generates only about 1.1 negative superhelical turns per nucleosome in the DNA The solution to this ‘linking number paradox’ has been the subject of intense study and considerable controversy since it was first framed by Finch et al [8]. They pointed out that the paradox could be solved if the helical pitch of the DNA on the nucleosome was overwound relative to that of DNA in solution. This proposal was supported by an analysis of DNase I cutting sites on the DNA [ 91 and periodicities in the sequences of nucleosomal DNA fragments [ 101, among other studies. However, an analysis of the linking number changes seen in small circular DNA rings reconstituted with a single nucleosome suggested that the helical pitch of the DNA in the nucleosome remained the same as B-DNA in solution, about 10.5-10.65 bp per turn [ 111. The question is of obvious importance; a change in the helical pitch of the DNA could potentially affect many functions, including the recognition of DNA-binding sites by proteins and chromosome topology. Two reports appeared in the last year that provide fresh evidence for an altered helical pitch of the DNA on the nucleosome. Using two different approaches, both measurements suggest an average pitch of approximately 10.1 bp per turn for the 146 bp of DNA in the nucleosome. Hayes et al. [12*] used hydroxyl radical footprinting to measure directly the helical repeat of a DNA segment from the Xenopus borealis 5s RNA gene when bound to either a nucleosome or a calcium phosphate surface. On the calcium phosphate surface, the peaks of hydroxyl radical cleavage had a periodicity of 10.49 bp per turn, in good agreement with the value of B-form DNA in solution. However, the DNA on the nucleosome had an average pitch of 10.18 bp per turn; over most of the nucleosome the result was actually closer to 10.05 bp per turn, but near the dyad axis the periodicity increased locally to 10.7 bp per turn. Shrader and Crothers [13-l used a series of short syn thetic DNA sequences with different anisotropic bending properties to measure the effects of the phasing of these flexible segments on the thermodynamics of nucleosome formation. The segments were combined as pentamers or hexamers at the center of a 161 bp DNA fragment so that the flexible sequences were repeated at defined intervals every 9.5-11.0 bp. The optimum phasing for nucleosome formation was approximately 10.1 bp per turn. Unlike the 5s RNA gene studies, no evidence for underwound DNA at the dyad axis was observed.

Histone

Both of these reports indicate that the DNA on the nucleosome is overwound by about 0.25-0.35 bp per turn. Depending on the assumptions made, these results may not completely resolve the linking number paradox, but they show conclusively that the helical pitch of the DNA is altered during its binding to the core histone octamer.

The flexible

amino-terminal

domains

Each of the core histones has at least two distinct structural domains, a flexible amino-terminal domain rich in basic ammo acids, accounting for 20-30% of the protein, and a more structured globular domain. Histones H2A and H3 also have short unstructured carboxy-terminal domains. In assembled chromatin these domains are sensitive to proteases. A recent comparison of the native and trypsinized histone octamer further emphasizes this structural specialization [14*]. All of the sharp resonances found in the octamer by nuclear magnetic resonance measurements, that is, all of the flexible protein segments, were contributed by the trypsin sensitive amino-terminal and carboxy-terminal domains. Recent experiments have begun to provide clues about the location of the amino-terminal domains within the nucleosome core particle. Ebralidse et al [ 151 were able to crosslink His18 in the amino-terminal domain of histone H4 at positions on the DNA about 1.5 helical turns from the nucleosome dyad axis. Ausio et al. [l6] purified dimer and tetramer histones, selectively trypsinized to remove the amino-terminal domains, and used them to reconstitute nucleosomes in d-0. The isolated histones were mixed and matched to create specific particles lacking the H2A-H2B domains only, the H3H4 domains only, or all of the protease-sensitive domains. The results of thermal melting measurements of these reconstituted nucleosomes are consistent with a model in which the amino-terminal domains of H2A-H2B stabilize the 10 bp DNA segments about 20 bp in from each end of the nucleosomal DNA, and the H3H4 domains stabilize the central 90 bp. Hill and Thomas [17**] used a three-stage radiolabelling protocol to identify amino-terminal domain lysine residues that interact with the DNA in sea urchin sperm chromatin. This protocol has been used previously to map the locations of the stronger lysineDNA interactions on the globular surface of the octamer [ 181. The lysines in the ammo-terminal domain of histone H2A were found to be inert to radiolabelling in both chromatin and core particles, suggesting that this histone is not bound to DNA in either case and may play a role in the higher order structures of compacted chromatin. The amino-terminal domains of sea urchin sperm H2B are unusual; the chromatin contains three sperm-specific variants of H2B with 10-20 ammo acid extensions composed of a five-amino-acid repeat motif. The methyfation experiments show that these H2B domains bind to the linker DNA between nucleosomes. However, two of the variants also have peptides that are bound to DNA in the nucleosome core particle. The amino-terminal domain of hisB

structure and function

Smith

tone H3 was also found to interact with linker DNA, and probably with the 10 bp immediately &mking the 146 bp of DNA on the nucleosome core particle. The amino-terminal domain of histone H4 was bound exclusively to the DNA within the nucleosome. At first glance, the results of Hill and Thomas [ 17**] appear to be in con!lict with those of Ausio et al [ 161. However, the reconstitution experiments always removed pairs of amino-terminal domains, either H2A and H2B, or H3 and H4. If the binding sites of the ammo-terminal domains of H2A and H3 are largely outside the nucleosome core particle, as suggested by the labeling experiments, then the results of the two studies can be reconciled. Taken together, they suggest a model in which the amino-terminal domain of histone H3 binds to the DNA at its entry and exit from the core particle, the amino-terminal domain of histone H2B binds to the DNA shortly after it has begun to wrap around the octamer, the amino-terminal domain of histone H4 binds to sites on the internal 90 bp of nucleosomal DNA, and the amino-terminal domain of histone H2A serves to stabilize the higher-order chromatin structure (Fig. 1). These results are provocative because they suggest that the four amino-terminal domains of the core histones may have distinct functions. Genetic evidence supporting different roles for the histone H3 and H4 amino-terminal domains is provided by the phenotypes of viable amino-terminal deletion mutants in yeast [ 191. Deletion of either the H3 or the H4 amino-terminal domain caused a delay in the cell cycle during nuclear division. However, the H4 amino-terminal deletion mutant had additional phenotypes that were not shared by the H3 aminoterminal deletion mutant, such as temperature-sensitive growth and loss of gene silencing. Simultaneous deletion of both ammo-terminal domains produced synthetic lethality, a result similar to that reported previously for the amino t&mini of H2A and H2B [ 201. Significantly, loss of growth of the double mutant did not occur at nuclear division, the phenotype common to the two deletion mutants, but randomly throughout the division cycle. These results suggest that the two domains play different roles, with synthetic lethality in the double mutant caused by the synergistic loss of multiple functions.

linker

histones

Histone Hl, and the erythrocyte-specik histone H5, appear to bind to the linker DNA outside the core particle, further constmining the DNA in two full turns around the octamer [21]. The effects of histone Hl on DNA wrapping in the nucleosome have been extensively studied. Zivanovic et al [22*] have recently extended their nucleosome reconstitution experiments on small circular DNA rings to examine the effects of adding histone H5. Single monosomes were reconstituted onto 359 bp and 368 bp closed ckcular DNA fragments derived from pBR322, with and without histone H5. The reconstituted complexes were then relaxed with topoisomerase I and ex-

431

432

Nucleus

and gene expression

amined by gel electrophoresis and electron’microscopy. In these experiments the addition of histone H5 to the nucleosome resulted in a large increase in supercoiling of the DNA; the linking number change increased from approximately - 1.1 to - 1.7 turns. Electron micrographs of the histone H5 particles showed the presence of crossed DNA loops at the exit of the nucleosome. This large increase in supercoiling was not seen in previously reported experiments where histone H5 was added to polynucleosomes reconstituted on circular minichromosomes [23,24]. One explanation for the ditference may be that interactions between the nucleosomes at the higher packing density prevent their reorientation when histone H5 is added, although Morse and Cantor [24] were unable to find any evidence for nucleosome interaction in their experiments. Recently, both Shrader and Crothers [13**] and Yao et al [ 25.1 have obtained convincing evidence of weak interactions between dinucleosomes. The proteins of the histone Hl family have long been recognized as having a major role in higher order structures and chromatin condensation [26]. Recent covalent crosslinking studies by Mirzabekov et al. [27-] have focused on three residues in histone H5 that are in contact with the DNA, Thrl, I-&25, and His62. The site of His25 crosslinking was found in the linker DNA proximal to the nucleosome and the reaction at this site was independent of the degree of chromatin compaction. In contrast, I-I&62-DNA crosslinking occurred further away from the nucleosome and was strongly favored in condensed chromatin; it was the predominant crosslinked product in whole nuclei and in isolated chromatin condensed with Mg*+ ions. In chromatin fragments, crosslinking at His62 required chains of at least three nucleosomes and probably more. The Thrl crosslink product was only seen in soluble chromatin at a low ionic strength, suggesting that it may be primarily involved in protein-protein structure in native chromatin. Thus, there is an interesting correlation between the state of compaction of the chromatin, and changes in the interaction of histone H5 with the DNA. The central globular domain of histone H5, approximately amino acids 22-100, is sufficient to bind to the nucleosome and stabilize two turns of the DNA [ 2 11. A gene fragment containing this globular domain has recently been engineered for expression in EL&e&&z coli [28*]. The protein produced is a fusion of the first three residues of histone H5 with amino acids 23106. Crystals of the protein that defract beyond 2.5A resolution have been obtained. The solution of the crystal structure of this part of histone H5 should provide valuable insights Into the binding of linker histones to chromatin.

Histone

and nucleosome

function

The amino acid sequences of the histones have been highly consewed among eukaryotes and, of course, so

has the structure of the nucleosome. Some of the functional consequences of histone and nucleosome structure are now becoming clear from recent biochemical and genetic experiments. The first sequence-speciiic function of a histone protein, gene silencing by histone H4, has recently been identilied in the yeast Saccha romps

Silencing:

cereukzhe.

a site-specific

function

Permanent repression of the yeast silent-mating type loci is complex and depends on several cis and tramacting factors, including speciiic DNA binding sites for the products of ABFl and R4PI, a replication origin DNA sequence consensus, the amino-terminal acetylase genes NATl/ARDZ perhaps acting through histone H2B, and the products of four regulatory genes, SIRI-4. Finally, the amino-terminal domain of histone H4 is also necessary for silencing [ 291. Three reports in the last year have shown that this silencer activity maps to a specific region within the amino-terminus of histone H4 [30**-32**]. Single-point mutations in amino acids 16-19 all resulted in the complete or partial loss of silencing as assayed by northern blot analysis or the decreased mating ability of mutant cells (Table 1). Although deletion of the aminoterminal domain caused de-repression at both the HML and the h!MR silent loci, the point mutations largely affected the a-mating sequences at HML [31-l; currently the reasons for this difference are not known. An important clue to the functional role of the H4 silencer site has been provided by a reversion analysis of H4 point mutant.5 [ 32.01. Among the extragenic suppressors that restored silencing to H4 mutants, one complementation group mapped to the SI&I gene. These sir3 suppressors did not restore silencing to the H4 aminoterminal deletion mutant, showing that the suppressors did not bypass the need for the H4 domain but rather improved the function of a defective domain. These results suggest that the silencer function of histone H4 involves protein-protein interactions. However, suppression by the sir3 mutations did not show any obvious allele specificity for the various histone H4 point mutations, and therefore the interaction between H4 and SIX? may be indirect. The exact nature of the interaction awaits further genetic and biochemical experiments. There is now excellent biochemical evidence for a distinct chromatin structure at the yeast silent loci. Nasmyth [33] first showed that the patterns of DNase I hypersensitive sites at the silent-mating type loci were dependent on SIR gene function and differed between silent and derepressed strains. Recently, Chen et al [34**] used nucleosome fractionation by mercurya5nity chromatography to show that the chromatin structure at the silent HMRa locus is in a different conformation from that of transcribed genes. Yeast nucleosomes from regions of the chromatin containing gene sequences, including sequences from the expressed MA% locus, were retained on the column via a cysteine residue at position 110 in histone H3. (Although CysllO is the natural amino acid in metazoan histone H3 proteins, the allele had to be engi-

Histone

Table 1. Mating

eficiency

of MATa

histone

H4 mutants.

Relative mating \mino-terminal

sequence

efficiency

SGRGKGGKGLGKGGAKRHRKI _ --R--.-e--.--.---._ -

1.0 1.0

--R--R----.---..---

_ _

1.0

-R------R-.---..--

0.9

_ _ .-R.-R-e-R.-...w... __.-

0.5-1.0

__..._-.

-R

_.___.

0.04

.---R.-R-.-R-.-R--.--

O.WO9 m--R

__.__

__.-__.___..__-

0.24-0.5

.K-.-.

__.._..-

.___.__.

__..

__.-

._

0.17

-R-e. _.___

0.38

w-K.-

0.70

-s--R-

1.14

-.-,A..A---A--..--.--

0.2

_ _ .-G.-,.--G.-------.

0.5

_ -.Q.-Q-..Q...Q...--

O.OcQl

_ -.A.-,,..A-..A..---

o.m2 __

__..__

.

.

..-

_.

.A.

_.

_.

0.00004

.-G--e-.

< o.ooo1

--_..._._..___

.(J.---

.-__...-___.__

..y..-.

--...____.____._

< O.oaol 0.06

..G

_...

< O.cGal

.G-..

< 0.0@31

-e-,--e

0.006

---GCompiled )f the first

from

:ode) of yeast ubstitutions

references

21 amino histone

acids

[30**-32.01. (given

H4 is shown

in the mutant

The wild-type

in the single-letter

0.25 sequence amino

acid

in the top line. The amino

alleles are listed below

acid

the wild type.

neered by oligonucleotide-directed mutagenesis in yeast; cells carrying the allele were wild type for all properties tested.) The gene-region nucleosomes were retained on the column whether transcription was active or not, which is consistent with the higher basal level of histone acetylation in yeast and supports previous evidence that most of the yeast genome is in an accessible chromatin conformation. However, in a major exception to this rule, nucleosomes from the silent h%!Ru locus were not retamed on the affinity column, indicating that the H3 cysteines were inaccessible. Thus, the silent-mating type loci may be the yeast equivalent of heterochromatin in meta zoan cells.

Histone

acetylation

The amino-terminal domains of the core histones are the site of many post-translational mod&cations, of which the reversible acetyiation of the ~-amino group of lysines is among the most extensiveiy studied. Acetylation neutralizes the positive charge of the lysine side chains and is expected to weaken the interaction of the amino-termi-

structure

and function

Smith

nal domains with the DNA Previous biochemical studies have shown that histone hyperacetylation has minor but distinct effects on nucleosome structure in vifro, for example causing slower mobility in polyacryiamide nucleoprotein gels [35*]. Recently, Oliva et al [36*] have used electron spectroscopic imaging to show that hyperacetylation destabilizes nucleosome particles to the electrostatic stress imposed by spreading on a carbon film grid. Increases in histone H3 and H4 acetylation have long been correlated with changes in transcriptional activity. For example, when nucleosomes are affinity purified using antibodies specific for acetylated histones, the associated DNA is enriched for sequences from actively transcribed genes [371. Conversely, when nucleosomes from active chromatin are affinity-purified by mercuryagarose column chromatography, the histones are hyperacetylated (references cited in [34=*]). This correlation was greatly strengthened by the results of recent experiments reported by Tazi and Bird [38**]. Regions of GCrich DNA, ‘CpG islands’, occur at the 5’ domains of many mammalian housekeeping genes. Because of their unique base composition and lack of DNA methylation, these regions may be isolated as chromatin by using appropriate restriction endonucleases. Analysis of this purified ‘active’ chromatin revealed nucleosome-free regions of DNA, a lack of the linker histone Hl, and hyperacetylation of histones H3 and H4. Results reminiscent of these mammalian studies have also been reported with the use of a different mapping approach in flies. Immunotluorescent staining of polytene chromosomes with antibodies specific for acetylated species of histone H4 showed islands of acetylated histones that were localized to the flanking region of transcribed puffs [ 39.1. Recent genetic results in yeast now strongly support the importance of histone H3 and H4 acetyiation in cell physiology, and provide clues to the functions of this post-translational modification. Histone H4 contains four lysines in its amino-terminal domain that are subject to reversible acetylation. Megee et al. [30**] engineered a set of point mutations within the ammo-terminal domain of yeast histone H4 designed to mimic different states of acetyiation. Lysines were replaced with arginines to simulate the unacetylated state, and with glutamines to simulate the hyperacetylated state. When the four lysines were replaced by arginines, preventing any histone H4 acetylation, the resulting mutant cells were inviable by several tests. Subsequently, Park and Szostak [31**] obtained cells that were alive with this mutated histone H4, although the cells grew very poorly. We now know that this discrepancy is due to differences in the yeast strain backgrounds; although the complete arginine allele is indeed lethal in the original strain tested, the same plasmid construct will support minimal growth in another laboratory strain (PC Megee and MM Smith, unpublished data). In any case, it is clear that blocking histone H4 acetylation is very harmful to cell growth. Many lines of evidence show that the lysines in the ammoterminal domains are not acetyiated at random but rather in a specific order (references cited in [39@]>. For ex-

433

434

Nucleus

and gene expression

ample, histone I-I3 is preferentially modified’at Lysl4 and Lys23, followed by Lysl8 [35*,40=]. In mammalian cells monoacetylated histone H4 is mod&d almost exclusively at Lysl6 [40*]. It is thus tempting to speculate that this speciIicity has critical functional significance. The genetic results are in partial support of this view. Glutamine, alanine, and glycine substitutions at ~ysl6 of yeast histone H4 specifically de-repress transcription at HMLar [30**-32**] (Table 1). Furthermore, as described earlier, it is likely that this de-repression occurs because of disruption of site-specific interactions rather than non-speciiic charge neutralization per se. However, a variety of single and double arginine substitutions failed to manifest major phenotypic defects, showing that no single lysine site is essential for function [30**]. On the contrary, the underlying mechanism for several phenotypes, such as sporulation [31**], temperature sensitivity, and delayed nuclear division, is more consistent with a generalized reduction of charge in the amino-terminal domain (PC Megee, BA Morgan and MM Smith, unpublished data) [30-l. Fii, the genetic results strongly suggest that histone I-I3 and H4 acetylation has a role in nuclear division. Three different classes of mutants produce a major delay during the G2 + M portions of the division cycle, deletions of the amino-terminal domain of H3, deletion of the amino-terminal domain of H4, and the complete glutamine replacement mutant of H4 [30**] (BA Morgan, BA Mittman and MM Smith, unpublished data). These results predict that one or more lysine residues must be deacety lated, and thus charged, for efficient completion of nuclear division.

Histones

and the centromere

Panially defective centromere function is one potential mechanism that may account for a delay in nuclear clvision in the yeast histone mutants. The structure of the chromatin at yeast centromeres has been carefully studied by Bloom and colleagues (references in [ 41**] ), and consists of a phased array of nucleosomes surrounding a 220 bp segment that is highly resistant to nuclease digestion. This 220 bp centromere ‘core’ element in eludes the three required centromere c&acting DNA sequence motifs, CDE I, CDE II, and CDE III. The importance of the histone proteins for centromere function has been tested by blocking expression of a single histone protein, either H2B or H4 [41**]. Abrupt repression of histone expression in yeast results in nucleosome depletion and a lirst-cycle arrest at nuclear division. When the structure of the centromere of chromosomeIIIwase xamined following the block on histone gene expression, new nuclease-sensitive sites were exposed within the CDE II element of the centromere core. This increased access to CDE II was coniirmed by assaying the ability of the restriction enzyme D~UI to cut the chromatin DNA at three closely spaced sites within CDE IL In control cells, less than 5% of the CDE II elements were cut with the enzyme, but after nucleosome depletion approximately 40% of the elements were cut.

This disruption of centromere structure required DNA replication in the absence of histone gene expression, precisely the conditions required for nucleosome depletion. These changes in the structure of the centromere core could be indirect effects of the depletion of the nucleosomes in the arrays Banking the centromere. An intriguing possibility, however, is that the histone octamer may be a structural component of the 220 bp centromere core. In either case, histone hyperacetylation mutations might be expected to impair the efficiency of centromere function, resulting in a delay at nuclear division. Supercoiling

and higher

order

structure

Inefficient chromosome condensation before mitosis provides a second attractive model for the nuclear division defect seen in the yeast mutants. Recent results show that histone acetylation may alter DNA topology in the chromatin by directly affecting the path of the DNA Experiments by Bradbury and colleagues showed that high levels of histone acetylation reduced the linking number change per nucleosome in covalently closed circular minichromosomes reconstituted in vitro [42]. In the last year, Norton et al. [43**] have shown that this change in topology can be entirely explained by changes in histone H3 and H4 acetylation. In nucleosomes reconstituted with hyperacetylated H3 and H4, the linking number was reduced to only -0.81 turns per nucleosome, compared to - 1.04 obtained for control nucleosomes. There are two likely mechanisms for this linking number change. An alteration in the path of the DNA entering and leaving the nucleosome would be sufficient to produce the altered DNA topology. Alternatively, a relatively small increase in the effective radius of the octamer as a result of amino-terminal acetylation would also be sufficient to change the linking number by a few tenths of a turn per nucleosome [ 43**]. Histone acetylation may also affect chromatin compaction indirectly through histone Hl. It has been known for some time that removal of the amino-terminal domains of the core histones interferes with chromatin condensation. When chromatin stripped of histone Hl was trypsinizd to remove the amino-terminal domains, it remained unfolded when histone Hl was added back, even under conditions that favored compaction of control chromatin [44]. Recently, Ridsdale et al. [45*] have shown a correlation between the state of acetylation of the core histones and the ability of histone Hl to promote the salt-dependent aggregation of the chromatin. Sodium butyrate induces hyperacetylation of active chromatin by inhibiting histone deacetylation. When chromatin from control cells and cells treated with sodium butyrate were compared, fragments containing the hy peracetylated active genes were preferentially resistant to precipitation by histone Hl. A number of years ago the salt-dependent, Hl-dependent folding of chromatin was shown to be qualitatively consistent with an electrostatic mechanism treating the DNA and histones as polyelectrolytes [ 461. Clark and Kirnura [47*] have extended this analysis to predict quantitative

Histone

aspects of chromatin folding. The results of their calculations are in agreement with several experimental studies and suggest that neutralization of the negative phosphate charges in the linker DNA is the major factor controlling higher order folding. We have seen that the phenotypes of some of the amino-terminal domain mutations, such as delayed nuclear division, may be correlated with the overall positive charge density of the lysines in the domain. It is thus attractive to speculate that the molecular basis for some of these mutant phenotypes may be the result of alterations in the dynamics of chromatin folding.

struchwe and function

Smith

A, LUTTER LC: The Helical Periodicity of DNA Nucleosome. Nucleic Aci& Res 1981, 94267-4283.

on the

9.

KLJJG

10.

SATCHVEU SC, Drtew HR, TRAvEtts AA: Sequence ties in Chicken Nucleosome Core DNA J Md 191:65%75.

11.

ZIVANOVIC Y, GOULET I, REVET B, LE BRET M, PRUNEU. A: Chromatin Reconstitution on Small DNA Rings: II. DNA Supertolling on the Nucleosome. / Md Bid 1988, 200~267-290.

Periodic& Bid 1986,

12.

HAYES JJ, Tuurus TD, WOLFFE AP: The St~cture of DNA in a Nucleosome. Proc Nat1 Acad Sci US4 1990, 87:740>7409. ~dirtXt assay Of DNA StNCNfe within the nUCieOSQIne core partiCk USing hydroxyi radical mapping shows that the helical pitch of the DNA on the nucleosome is overwound with respect to B-form DNA in solution. SHRAJIER TE, CROIHERS DM: EfFects of DNA Sequence and Histone-histone Interactions on Nucleosome Placement. / Mol Biol 1990, 216:69+34. Further evidence for an altered helical pitch of the DNA on the nucleosome, artificial sequences with Bexible segments are combined to investigate the effect of their arrangement on the positioning of the nucleosome. The optimal period between segments is approximately 10.1 bp. Also reported are the nature of the tlexible sequences, the position of adenine tracts, and weak phasing interactions between nucleosomes. 13. ..

Acknowledgements Work from the author’s laboratory cited above is supported by NM grant GM 28920. 1 thank all my colleagues in the field and paniculariy members of the Smith laboratory for helpful discussions and comments.

14. .

References

and recommended

Papers of special interest, published have been highlighted as: . of interest .. of outstanding interest

within

S~HR~TH GP, YAU P, WAI BS, GATEWOOD JM, A NMR Study of Mobility in the Histone Leti 1990, 268:117-120. A brief report of the high-frequency nuclear magnetic tra of native and trypsinized histone octamers. Au the trypsin-sensitive domains.

reading the annual

period

of review,

1.

RICHMOND TJ, FINCH JT, RUSHTON B, RHODES D, KLL~G AI Structure of the Nucleosome Core Particle at 7A Resolution. Nature 1984, 311:532-537.

2.

BURUNGM~E RW, LOVE WE, WANG B, HAMLW R, XUONG N. MOU~UN~WS EN: Crystallographic Structure of the Octameric Histone Core of the Nucleosome at a Resolution of 3.3 Angstrom. Science 1985, 228:546-553.

3.

UBERBACHER EC, HARP JM, WIUUNSON-SING~EY E, BLINICK GJ: Shape Analysis of the Histone Octamer in Solution. Science 1986, 232:1247-1249.

4.

PARK K, F&MAN GD: The Histone Octamer, ally Flexible Structure. Biocbemisfstr)~ 1987,

5.

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Cell 1988,

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EM: FE&S

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15.

EBRAUD~E KK, GRACHEV SA MIRUBEKOV AD: A Highly Basic Histone H4 Domain Bound to the Sharply Bent Region of Nucleosomal DNA Nature 1988, 331:365-367.

16.

AUSIO J, D~NC F, VAN HOLDE KE: Trypsinized Nucleosome Core Particles of the Histone Tails’ in the Stabilization J Mol Biol 1989, 206:451463.

Use of Selectively to Analyze the Role of the Nucleosome.

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HIU CS, THOMAS JO: Core H&tone-DNA Interactions in Urchin Sperm Chromatin. The Amino-terminal Tail of H2B Interacts with Linker DNA Eur J B&hem 1990, 187:145-153. Chemical labeling of tysines is used to identify sites in the amino-terminal domain of the histones that bind to linker DNA The sea urchinspennspecific H2B terminal domains show strong biding to the linker DNA Specific binding patterns of the other amino-terminal domains are also reported. 18.

LMBERT SF, THOMAS JO: Lysine-containing DNA-biding Regions on the Surface of the Histone Octamer in the Nucleosome Core Particle. Eur J Biochem 1986, 160191-201.

19.

MORGAN Bq M~TMAN BA, SW MM: The Highly Conserved N-terminal Domains of Histones H3 and H4 are Required for Normal Cell Cycle Progression. Md Celf Biol 1991, in press.

20.

SCHUSTER T, HAN M, GRUNSTEIN M: Yeast H2B Amino Termini Have Interchangeable 1986, 45445451.

21.

AUAN J, HARTMAN PG, CRANE-ROBINSON C. Avtu!s FX: The Structure of Histone HI and its Location in Chromatitx Nature 1980, 288:67%79.

6. .

G~DFF~EY JE, BAXEVAN~S AD, MOUDRMNAKIS EN: Spectropolarimetric Analysis of the Core Histone Octamer and its Subunits. BiocbemiWy 1990, 29965-972. Re.examination of the secondary structure changes in the octamer over a range of salt and temperature conditions. Complex changes in the B-sheet content of the tetramer structure are observed between 0.1 and 2.0 M NaCI.

BWBURY Octamer.

Histone HZ4 Functions.

and Gzfi

BAX!ZWW, AD, GODFREY JE, MOUDRLWAKIS EN, PARK K, FASMAN GD: Effect of Aggregation of Histone Octamers in High-salt Solutions on Circular Dichroism Spectra, Biochemishy 1990, 291973976. A collaboration between two laboratories to re-evaluate the effect of high-sait sohrents on the StNCNre of the octamer. The results show that the apparent change in octamer shape produced in high ammonium sulfate wds an artefact of aggregation.

nv~~ov~c Y, DLIBAND-GOUE-T I, SCHULTZ P, STOFER E, OUDET P, PRUNEU A: Chromatin Reconstitution on SmalJ DNA Rings. III. Histone H5 Dependence of DNA Supercoiling in the Nucleosome. / Md Biol 1990, 214:479-495. Reports experimenls Studying the eftect of adding histone H5 to monosomes ECOnStiNted on smd DNA rings. Very good electron micrcscopic piCNR3 show crossed loops generated by the linker histone.

8.

23.

FINCH JT, LUI-I-ER LC, RJIODF.S D, BROWN RS, RUSHTON B. Uvrrr M, KIK A: Structure of Nucleosome Core Particles of Chromatin. Nature 1977, 269:29-36.

22. .

STEIN A: DNA Wrapping in Number FVoblem Re-examined 8:480?&820.

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MORSE RH, WR CR: E&xc of Trypsh&atidn and Histone H5 Addition on DNA mt and Topology in Reconstituted Minichromosomes. Nuckzic Acti Res 1986. 14:32933310.

25. .

YAO J, L~WARY PT, WLOOM J: Direct Detection of Linker DNA Bending in Defined-length OIigomers of Chromatin. Proc Natl Acud Sci LISA 1990, 87:76037607. Examines the Rexibility of the linker DNA in short oligonucleosomes. Finds that under ionic conditions favoring compaction of extended chromatln, the linker DNA in dinucleosomes bends as the nvo nucleo somes come into contact 26.

THOMA F, KOUER’ TH. KLUG A: Involvement in the Organization of the Nucleosome dependent Superstructures of ChromatIn. 83403-427.

27. ..

MIIUBEKOV

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AD, PRUSS DV, EBRAUDLZ KK: Chromatin Superstructure-dependent CrosslInking with DNA of the Histone H5 Residues Thrl. Hb25, and His62. / Mol Biol 1990, 211:479-491. Investigates changes in linker histone SvUCNre with respect to the folded srate of the chromatin. Shows that the preferred crosslinking sites in histone H5 are reorganized as the packed nuclear chromatin is unfolded.

28.

GRAZLWO V, GERCHMAN SE, WONACOTT AJ, SW-EET RM, WEUS JR, WHITE SW, RAMAKRISHNAN V: Crystallization of the Globular Domain of Hlstone H5. / Mol Biol 1990, 212:253257. Prellmjnxy report of crystals of the central globular domain of histone H5. The protein was obtained from a fragment of the histone H5 gene engineered by recombinant DNA for expression in E coli

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29.

KAYNE PS, 1(1M UJ, HAN M, MUUEN JR, YOSHIZAKI F, GRUNUEIN M: Extremely Conserved Histone H4 Amino-terminus is Diipensable for Growth but Essential for Repressing the Silent Mating Loci In Yeast. cell 1988, 55:27-39.

30. ..

MEGEE

PC, MORGAN I34 MIITMAN 04 SMITH MM: Genetic -is of Hbtone H4: Essential Role of Lysines Subject to Reversible Acetylation. Science 1990, 247:841+345. Reporrs the an&is of point mutations in the amino-terminal domain of histone H4 in yeast. First mapping of the gene-silencing function of histone H4 to a speciiic peptide sequence in the amino-terminal domain. Acetyiacion site mutants have defects in nuclear division and possibly DNA replication. 31. ..

PARK EC, SZOSTAK JW: Point Mutations In the Yeast Histone H4 Gene Prevent Silencing of the Silent Mating Type Locus HML. Mol Cell Biol 1990, 10:49324934. This brief report is important for two reasons: first, it shows that point mutations in the histone H4 amino-terminal domain that abolish gene silencing act preferentially at h?MLa; second, it shows that acetylation site murams have sporularion defects. 32. ..

LM, KAYNE PS, KAHN ES, GRUNSTEU‘I M: Genetic Evidence for an Interaction Between SIR3 and Histone H4 in the Repression of the Silent Mating Loci In Saccharomyces @n?visiae Pm Nat1 Acad Sci USA 1990, 87:62ti290. A reversion analysis of histone H4 point mutants defective in gene si. lenclng. Provides an important clue fo the functional role of the H4 si. lencer site. Extragenic suppressors were found in the SIR.? gene whose product l.s a watzsacting protein known to control silencing. 33.

JOHNSON

NASMYIH KAz The Regulation of Yeast Mating-type Chromatin Structure by SIR: an Action at a Distance Affecting Both Transcription and Transposition. Cell 1982, 30:567-578.

CHEN T& SOUTH MM, LE S, S’IERNGLWZ R, A~.FREY VG: Nucleasome Fractionation by Mercury-affinity Chromatography. Contrasting Distribution of TranscrlptionaIIy Active DNA Sequences and Acetylated Histones in Nucleosome Fractions of Wild-type Yeast CeIIs and Cells Expressing a Hlstone H3 Gene Altered to Encode a Cysteine-110 Residue. J Btbl them 1991, 266&8%98. Biochemical evidence for a distinct chromatin structure at the yeast silent loci. Extends the mercury-al%lty purllicatlon technique of All.

34 ..

frey’s laboratory to a genetically engineered yeast system. Most of the yeast genes tested were in an accessible chromarin StNCNre whether actively transcribed or not. However, the silent-mating type kxl were packaged into a SVUCNre that blocked access to the histone H3 cysfeine residues. 35. .

MARVIN KW, YAU P, BRADBURY EM: lsolation

and Characterizatlon of Acetylated H&ones H3 and H4 and Their Assembly into Nucleosomes. J Bid C!wm 1990, 265:1983!+19847. Biochemical purification of acetylated histone subtypes. A new lifth site of histone H3 acetylation was found at Lys27. The properties of reconStiNEd nucleosome core particles are reported. OLM R, BAZKI-I’-JONES DP, UXKLUR 1 DIXON GH: HIstone Hyperacetylation Can Induce Unfolding of the Nucleosome Core Particle. Nucleic Acids Res 1990, 18:273%2747. An electron microscopic investigation of histone hyperacetylation and nucleosome srability. Shows that acetylarion destabilized the nucleosome to the electrostatic shear forces imposed by spreading on a charged carbon surface. 36. .

37.

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TAZI J, BIRD A: Alternative Chromatin Structure at CpG Islands. Cell 1990, 60:90’+920. Eeatty strengthens the correlation between increases in histone H3 and H4 acetylation and changes in transcriptional activity. Advantage is taken of restriction enzymes that cut in CpG islands to purify chromatin from the promoter regions of active genes. The purilied chromarin was devoid of histone Hl and contained hyperacetylated histones. 38.

39. .

TURNER BM, FRANCHI L, WALLACE H: Islands

of Acetylated Histone H4 in Polytene Chromosomes and Their Relationship to Chromatin Packaging and Transcriptional Activity. J Cell Sci 1990, 96:335-346. Antibodies to specific species of acetylated histone H4 were used fo localize the modified protein in polytene chromosomes. Distinct bands of acetyiated histone were observed, and analysis of puff sites suggested that they were located in regions flanking actively transcribed genes. 40.

THORNE

.

ROBINSON

AW, K~UCIEK D, MITCHELSON K, SALITIERE P, CRANEC: Patterns of Histone Acetylation. Eur J Bib&em 1990, 193:701-713. investigates the distribution of specific acetylation sites in histones H2B, H3, and H4. A strict order of occupancy was found in histone H3: Lysl4, Lys23, and Lysl8 Histone H4 was preferentially modified at Lyslb, while histone H2B showed less speciIicity.

41. ..

SAUNDERS

MJ,

YEH

E,

GRUNSTEIN

M,

BLOOM

some Depletion Alters the Chromatin Structure charomyces cerwisiae Centromeres. Mol Ceil 10:5721-5727. This work is particularly important because it investigates blocking histone gene expression on centromere structure cleosome depletion resulted in the aberrant exposure of DNA in CDE II, as assayed by nuclease sensitivity. 42.

Nucleoof SacBiol 19%).

K:

the effect of in yeast. Nucentromere

NORTON VG, IMAI BS, YAU P, BRADBURY EM: Histone Acetylation Reduces Nucleosome Core Particle Linking Number Change. Cell 1989, 57:44w57.

43. ..

NORTON VG, w KW, YAU P, BRADBURY EM: Nucleosome Linking Number Change Controlled by Acetylation of Iilstones H3 and H4. J Bid &em 1990, 265:1984%19852. important investigation of relative contributions of histone H3-H4 and histone H2.-H2B acetylation on DNA linking number changes in minichromosomes reCOnStiN@d in oitm High levels of histone H3-H4 acetyiation are sulficient fo effect a change in linking number from - 1.01 fo - 0.81 per nucleosome. 44.

ALAN J, HARBORNE N, RAU DC, GOUID Core Histone Tails’ in the Stabilization Solenoid. J Cell Bid 1982, 932X+297.

45. .

RKXD.U

JA,

HENDZEL

MJ,

DELCWE

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H: F%rticipation of of the Chromatin JR: Histone Histones to ConChromatin. J Bid

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Histone Investigates the relationship between salt-dependent histone HIinduced chtomatin precipitation and histone acetylation. Suggests that histone hypetacetylation decreases the ability of linker histones to bring about the higher-order folding of chromatin. 46.

W~DOMJ: Physicochemical Studies of the Folding of the 100 A Nucleosome Fiient into the 300 A Filament. J Mel Bid 1986, 190:411~24.

47. .

Cwuc DJ, I(IMuRA T: Electrostatic Mechanism of Chromatin Folding. / Mel Bid 1990, 21138~.

struchwe and function

Smith

Theoretical treatment of &dependent Hl-dependent folding of chromath Well written and worth reading even if you are not interested in following the details of the mathematics. i

MM Smith, Depattment of Microbiology, Box 441 Jon-ion Building, School of Medicine, University of Virginia, Chadottesille, Virginia 22908, USA

437