Sergei G. Bavykin, Sergei I. Usachenkol, Aila I. Lishanskaya2, Valentin ...

Volume 13 Number 10 1985

Nucleic Acids Research

Primary organization of nucleosomal core particles is invariable in repressed and active nuclei from animal, plant and yeast cells

Sergei G.Bavykin, Sergei I.Usachenkol, Aila I.Lishanskaya2, Valentin V.Shick, Alexander V.Belyavsky, Igor M.Undritsov, Alexei A.Strokov, Irina A.Zalenskaya3 and Andrei D.Mirzabekov* Institute of Molecular Biology, USSR Academy of Sciences, Moscow 117984, lInstitute of Bioorganic Chemistry, Academy of Sciences of the Uzbek SSR, Tashkent 700000, 2Institute of Nuclear Physics, USSR Academy of Sciences, Leningrad 188350, and 3Institute of Cytology, USSR Academy of Sciences, Leningrad 194064, USSR Received 12 March 1985; Accepted 20 April 1985 ABSTRACT A refined map for the linear arrangement of histones along DNA in nucleosomal core particles has been determined by DNA-protein crosslinking. On one strand of 145-bp core DNA, histones are aligned in the following order: (5') H2B25,35-H45e,&5-H37u5..u,5/H4mm-H2Baos,lls-H2Aiiu'H313s,1i4/H2Al45 (3') (the subscripts give approximate distance in nucleotides of the main histone contacts from the 5'-end). Hence, the histone tetramer (H3,H4)2 and two dimers (H2A-H2B) are arranged on double-stranded core DNA in a symmetrical and rather autonomous way: H2A/H3-(H2A-H2B)-(H3,H4)2-(H2B-H2A)-H3/H2A. The primary organization was found to be very similar in core particles isolated from repressed nuclei of sea urchin sperm and chicken erythrocytes, from active in replication and transcription nuclei of Drosophila embryos and yeast and from somatic cells of lily. These data show that (i) the core structure is highly conserved in evolution and (ii) the overall inactivation of chromatin does not affect the arrangement of histones along DNA and thus does not seem to be regulated on this level of the core structure. INTRODUCTION Nucleosomal core particle is the basic unit of chromatin structure. It consists of about 145 base pairs (bp) of DNA and a histone octamer containing two molecules each of histones H2A, H2B, H3 and H4. Together with histone HI and spacer DNA of variable length the core particles form nucleosomes (1, 2). The X-ray and electron microscopy studies of core crystals and ordered aggregates of histone octamers performed by Klug's group have already attained 7 A resolution and should hopefully reach 5 h (3-6). In our laboratory we used specific DNA-protein crosslinking combined with two-dimensional gel electrophoresis of the crosslinked material to determine the sequential arrangement of histones along DNA (primary organization) in core particles and Hl-containing nucleosomes (7-9). Recent improvements in this technique and the use of cores from different sources have enabled us in this paper to refine our previous map for the primary organization of nucleosomal core particles (7, 10). Nucleosomal cores, although invariable in all eukaryotes, represent a highly heterogeneous population of particles that differ in histone modifi© I RL Press Limited, Oxford, England.

3439

Nucleic Acids Research cation, in the presence of histone variants and nonhistone proteins (for review see 1, 2, 11) and in some physical properties (12-14). It appears that this heterogeneity may be both the cause and a result of many functional states of chromatin. The cores from transcribed chromatin regions must have some peculiarities in structure since they are preferentially digested by DNAase 1 (15, 16), can be enriched in HM6 14 and 17 proteins (17) and ubiquitin conjugated to histone H2A (18), show an increased affinity for HMG 14 and 17 proteins (19) and for RNA polymerase II (20) and contain hyperacetylated histone species (reviewed in 21). The cores isolated from transcribed ribosomal chromatin of Physarum polycephalum show exposed H3 cystein residues, and in electron micrographs they look like partly unfolded bipartite particles (22). Replication and transcription of DNA in chromatin should apparently affect the DNA-histone contacts. To cite a few extreme examples, it was shown that histones got removed from actively transcribed ribosomal genes (23), heat shock genes (24) and from the 5'-regulatory, DNAase I hypersensitive gene regions (24, 25). It is clear therefore that a comparison of the arrangement of DNA-histone contacts in nucleosomes of functionally different chromatin regions could be of essential interest. The "protein image' hybridization technique developed in this laboratory (24) enables one to compare the primary organization of nucleosomes in various genes and within different regions of the same gene, and such experiments are now in progress. In this paper, we have made use of another approach and compared the core primary organization in repressed nuclei of sea urchin sperm and chicken erythrocytes and in highly active in replication and transcription nuclei of Drosophila embryos, lily buds and yeast. Our results show that the primary organization of core particles is nearly identical in all three higher eukaryote kingdoms. The core organization is also very similar in both active and repressed nuclei which suggests that the overall repression of chromatin is unlikely to be regulated on the level of the arrangement of histones along core DNA.

MATERIALS AND METHODS Isolation of nuclei Nuclei from sea urchin sperm (Strongilocentrotus intermedius) were prepared as in (26). Yeast nuclei were isolated according to the method (27) with some modifications. In particular, spheroplasts were made from exponentially growing

3440

Nucleic Acids Research culture of Saccharomyces cerevisiae strain DC5 using snail gut enzymes instead of Zymolyase. After centrifugation in Percoll gradient, the nuclei were washed three times in 0.5-. NP-40, 50 mM Na-phosphate (6.8) by centrifugation at 17,500 g for 15 min, three times in the same buffer containing 2 mM EDTA without detergent and three times in 0.35 M NaCl, 2 mM EDTA, 10 mM Na-phosphate (pH 6.8). Isolation of nuclei and chromatin from Drosophila embryos and chicken erythrocytes was performed as described in (7) and (28). Nuclei of lily (Lilium candida, L.) were prepared as will be described elsewhere (Bavykin et al., in preparation). Sepals from buds (60 g) were powdered with dry ice. The ice was evaporated at -200C, the homogenate was stirred in 1.5 1 of buffer 1 containing 2/. Triton X-100, 0.3 M sucrose, 0.5 mM diisopropyl fluorophosphate, 17. dimethylsulphoxide, 50 mM Tris-Cl (pH 7.6) for 12 hr at 40C, filtered through several layers of cheesecloth and centrifuged for 10 min at 300 g. The pellet was washed twice in 350 ml of the same buffer containing 1% Triton X-100, 0.4 M sucrose, centrifuged for 10 min at 500 g, suspended in 100 ml of buffer 1 containing 0.25h Triton X-100, 0.25 M sucrose, layered over three volumes of the same buffer containing 0.25/ Triton X-100, 0.5 M sucrose and centrifuged for 20 min at 1000 g. The nuclei were washed once with 50 mM Tris-Cl (pH 7.6), centrifuged for 10 min at 500 g and finally washed three times in 0.35 M NaCl, 2 mM EDTA, 10 mM Na-phosphate (pH 6.8) by centrifugation at 5000 g for 10 min. Histone-DNA crosslinking in sea urchin sperm, yeast and lily nuclei DNA-histone crosslinking was conducted as described in (7,29). Nuclei (2000 A2&0 units) were suspended in 50 ml of 0.5 mM EDTA, 0.5 mM diisopropyl fluorophosphate, 17. dimethylsulphoxide, 50 mM Na-cacodylate (pH 7.0) and dimethylsulfate was added to a final concentration of 4.3 mM under vigorous stirring. The DNA was methylated at 40C for 18 hr. The methylated nuclei were washed twice with 80 mM NaCl, 5 mM EDTA, 20 mM Na-phosphate (pH 6.8). Depurination of methylated bases in DNA was performed by incubation of nuclei at 450C for 8 hr in 50 ml of the above buffer containing 0.5 mM diisopropyl fluorophosphate added in dimethylsulphoxide. The nuclei were washed twice in 100 mM Na-phosphate (pH 6.8) and suspended in the same solution where the histone-DNA aldimine bonds formed were reduced by adding 1/10 volume of freshly prepared 250 mM Na-borohydride and incubating at 40C for 30 min in the dark. Finally, the nuclei were washed successively with 100 mM, 50 mM and 20 mM Na-phosphate (pH 6.8).

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Nucleic Acids Research Isolation of core particles from crosslinked nuclei of sea urchin sperm, lily and yeast Crosslinked nuclei from sea urchin sperm or lily buds were suspended, respectively, in 0.4 and 1.5 mM CaCl2, 10 mM Tris-Cl (pH 8.0) at a concentration of 150 A240 units/ml and digested with 85 units/ml micrococcal nuclease (Worthington) for 20 min at 370C. The reaction was terminated by adding EDTA to 5 mM. Highly purified core particles were isolated from the digest by electrophoresis in a polyacrylamide gel (7, 30). Crosslinked yeast nuclei (20 A2&0 units/ml) were incubated with micrococcal nuclease at a concentration of 15 units/ml in 0.5 mM CaCl2, 20 mM Na-phosphate (pH 6.8) for 20 min at 370C, and the nucleosome core particles were purified by sucrose gradient centrifugation in 1 mM EDTA, 20 mM Na-phosphate (pH 6.8) for 35 hr at 75,000 g. Isolation and crosslinking of cores from chicken erythrocyte and Drosophila embryo chromatin The core particles from uncrosslinked chromatin of chicken erythrocytes and Drosophila embryos were prepared according to (7). First, histone Hi was removed from chromatin on Dowex AG-50-X2 (31), then the chromatin was treated with micrococcal nuclease and the core particles were purified by sucrose gradient centrifugation. Histones were crosslinked to DNA in isolated core particles as recommended in (7). Enrichment and labeling of crosslinked DNA-histone complexes lost free histones were removed from the crosslinked material before its labeling with 1251 or 32p by precipitating DNA together with crosslinked histones as cetyl trimethylammonium (Cetavion) salts (7). Then the crosslinked complexes were labeled at tyrosine residues of histones with 125I as described (32), taking 0.5 mCi of Na'25I for labeling 1 A240 units of the complex (the amount used for one two-dimensional electrophoresis). DNA in the crosslinked complex was labeled at the 5' end with 32P according to (33). The complex (1 A2&o units) in 0.03 ml of 10 mM

MgCl2, 10 mM dithiothreitol, 0.1 mM spermidine, 40 mM BICIN (pH 9.0) was incubated at 370C for 1.5 hr with 2 units of polynucleotide kinase and 0.5 mCi of ('-32P)ATP. This amount was sufficient for 1 to 4 twodimensional electrophoreses, depending on the gel size. Next, most of the free DNA was removed from the crosslinked complex by hydroxylapatite chromatography of SDS-protein complexes (34) as described in (24) with some modifications. 32P-labeled crosslinked material (1 A240 units) was dissolved in 0.03 ml of 1% SDS, 10 mM dithiothreitol, 5 mM Na-phosphate(pH 6.4) and incubated at 100°C for 1 min to denature DNA. Then 0.27 ml of 0.1% SDS, 5 mM

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Nucleic Acids Research Na-phosphate was added, and the solution was loaded onto a thermostated at 300C column containing 0.5 .1 of hydroxylapatite synthesized in silicagel (35); 30 min later, the column was washed with 5 ml of 0.1% SDS, 5 mM Naphosphate (pH 6.4); the free DNA was removed by 0.1% SDS, 0.15 M Na-phosphate (pH 6.4), and the crosslinked DNA-protein complex was eluted with 0.1% SDS, 0.5 M Na-phosphate. The elution was monitored by radioactivity measurements. The material was dialysed at 40C for 12 hr against 0.1% SDS (changed twice), DNA fragments from DNAase I digest of rat liver nuclei (36) were added to the 0.1 mg/ml concentration, and the crosslinked DNA was precipitated with 3 volumes of ethanol from 0.2 M NaCl. Two-dimensional electrophoresis of crosslinked DNA-histone complexes All electrophoretic separations were performed as described earlier (7) with the modifications specified below. The Laemmli buffer system (37) was used, 7 M urea was present when indicated. Electrophoresis in the first direction was conducted in thinner gels (0.6 mm) which enabled us to shorten significantly the further treatment of gel strips and thus improve resolution in the second direction. A gel strip containing 125I-histones was washed successively in 66% formic acid, 2% diphenylamine in 66% formic acid, then incubated in the latter solution for 15 min at 700C, washed four times in 66% formic acid, several times in water until neutral pH, and finally two times in 0.1% SDS, 0.125 M Tris-HCl (pH 6.8). All washes were done on a magnetic stirrer for 10 min each. Histones in a first-dimension gel strip containing 32P-labeled crosslinked material were hydrolysed in the gel in the second direction with Pronase (38) as described (7) except that there was no need to switch off the current during hydrolysis. Gel dimensions and other electrophoretic parameters are given in the figure legends. RESULTS Chromatin Chromatin of sea urchin sperm and chicken erythrocytes is almost completely repressed and participates neither in replication nor in transcription. On the other hand, chromatin from Drosophila embryos, lily buds and especially from yeast is effectively involved in these processes, with all nucleosomes undergoing replication in each cycle of cell division and at least partly participating in transcription. However, it is difficult to estimate quantitatively what portion of nucleosomes is involved in transcription. In the exponentially growing yeast used in this study, DNA is transcribed by at least 40% (39), and all chromatin shows the "active" pro3443


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d Figure 1. Electrophoresis of histones from sea urchin sperm (a), chicken erythrocyte (b), yeast (c) and lily bud (d and e -short and long exposure) core particles in 15. polyacrylamide gel in the presence of 0.1X SDS (37) perties upon digestion with DNAase I and fractionation (40); we conclude therefore that at least a considerable part of the yeast cores originate from transcriptionally active chromatin. Another aim of this study was to compare the primary organization of care nucleosomes isolated from different sources spanning all three higher eukaryote kingdoms. Sea urchin sperm and lily buds seemed particularly suited for the purpose of establishing a possible correlation between the primary organization of nucleosomes and core histones structure. The striking feature of sea urchin spermal chromatin is the presence of several variants of histone H2B which molecules contain a considerably extended Nterminal part with an additional cluster of 4-5 lysine and several arginine residues (41). These spermal H28 histones have a decreased mobility and are better resolved from other histones by electrophoresis in SDS gels. We have taken advantage of this fact to try and achieve a higher resolution in mapping histone contacts along core DNA. In lily, as well as in other plants, H2A and H2B histones (except one H2A subfraction in lily) are usually longer and migrate slower in SDS gels than the same histones from animal cells (Figure 1). The positioning of lily histones was determined when the SDS electrophoretic system was used in the second direction after the histoneshad been electrophoresed in a Triton-acetic acid-urea gel (42) in the first direction (not shown). 3444


4

6

Figure 2.Two-dimensional sequencing gel electrophoresis of single-stranded, 32P-labeled DNA crosslinked to histones in sea urchin spermal cores. Electrophoresis in the first direction was carried out in 7 M urea under DNA denaturing conditions in 17% polyacrylamide slab gel (165x365x0.6 mm) at a constant current of 3 mA and 7 mA for the concentrating and separating gel, respectively, for 30 min until bromphenol blue ran about 1.5 length of the separating gel. After digestion of histones with Pronase in the gel (38), electrophoresis was continued in the second direction in 15% polyacrylamide slab gel (300x400xl mm) containing 7 M urea at a constant current of 10 MA and 20 mA for the concentrating and separating gel, respectively, until bromphenol blue reached the bottom of the gel. The broken lines show position in the gel and the figures give the length of ethidium-bromide-stained DNA fragments from DNAase I digests of rat liver nuclei. Their precise values are 20, 31, 41, 52, 63, 73, 83, 93, 103, 113, 124, 133 Ind 142 bases (46, 47). The positions of 32P-labeled DNA fragments crosslinked to different histones and arranged on separate diagonals were revealed by autoradiography and are indicated by solid lines. The extreme right diagonal contains uncrosslinked DNA fragments. At the left is the autoradiogram of the top part of the gel after a shorter exposure.

Except spermal H2B and lily H2A and H2B histones, all other core histones are highly conservative in their primary structure (43) and have similar mobilities in SDS gels (Figure 1). In relation to other properties of the chromatin used in this study, we can mention that in highly transcriptionally active yeast chromatin histone HI is likely to be absent (44). This is consistent with an earlier observation that even a moderate transcription leads to the removal of Hi from chromatin (24). The repeat length of chromatin depends on its activity (being longer in repressed nuclei) and is about 240, 210, 180 and 165 bp in chromatin from sea urchin sperm, chicken eryth-

3445

Nucleic Acids Research rocytes, Drosophila embryos and yeast, respectively (1).

Experimental approach To study the nucleosome structure as it exists in nuclei, histone-DNA contacts were fixed by crosslinking directly in nuclei of sea urchin sperm, yeast and lily. Then the nuclei were digested with micrococcal nuclease and the cores were purified either by gel electrophoresis (3(0) for sea urchin sperm and lily buds or by sucrose gradient centrifugation for yeast. On the other hand, the chicken erythrocyte and Drosophila embryo cores were crosslinked after their isolation in order to compare our results with previous studies. The method of locating protein contacts on DNA was described in full detail elsewhere when used to study the primary organization of nucleosomes (7) and the RNA polymerase-promoter complex (45). The method consists of crosslinking protein NH2-groups to DNA partially depurinated under mild conditions (29). The crosslinking causes the DNA to split in such a manner that only the 5-terminal DNA fragment becomes attached to protein molecules. Thus, the length of a crosslinked DNA fragment precisely shows the distance of a protein crosslinking site from the DNA 5 -end. This length can be assessed by using two systems of two-dimensional diagonal gel electrophoresis. Electrophoresis in the first direction is the same for the two systems, with fractionation of crosslinked material depending on the size of both DNA and proteins. Then in the first system of the two-dimensional gel electrophoresis, which serves to identify the crosslinked DNA (DNA-identifying system), histones are digested directly in the gel by Pronase (38) and the released 32P-labeled DNA fragments are separated in the second direction according to their length in the presence of unlabeled size markers (DNA fragments from DNAase I digest of rat liver nuclei, see ref. 36). The crosslinked proteins decrease the DNA mobility in the first direction proportionally to the protein size. As a result, the DNA fragments attached to different histones fall on different diagonals in the gel of the second direction. All core histones from sea urchin sperm are particularly well resolved in the first direction in gels containing and not-containing urea (see Figure la) and therefore the DNA fragments crosslinked to them are also completely separated into four distinct diagonals in the second direction. Thus, location of binding sites for all four core histones along spermal core DNA can be precisely (± 3-5 nucleotides) read from positions of radioactive spots on these diagonals (Figures 2-3). To attain a better resolution and identify the crosslinked spermal DNA fragments of about 60-140, 40-80 and 20-40 nucleotides long, we used 3 twodimensional gels of different length and concentration (Figures 2-3). For 3446


Figure 3. Two-dimensional gel electrophoresis of 32P-labeled DNA crosslinked to histones in spermal cores used to resolve intermediate and short DNA fragments. Electrophoresis in the first direction was carried out (A) in 15X polyacrylamide slab gel (165x365xO.6 mm) and (B) in 18X polyacrylamide slab gel (165x165x0.6 mm) at a constant current of 3 mA and 7 mA for the concentrating and separating gel, respectively. Electrophoresis in the second direction was performed (A) in 15X polyacrylamide slab gel (200x400xl mm) and (B) in 30X polyacrylamide slab gel (200x x200xl mm) at 7 mA and 14 mA for the concentrating and separating gel, respectively. For other details see Figure 2.

lily nucleosomal cores, this system did not separate the diagonals of DNA fragments attached to histone H3 and to the main H2A fraction. In this case, identification of the H2A-bound DNA fragments was carried out using a very weak H2A-subfraction diagonal which was detected between the H3 and H4 diagonals indicated in Figure 4. For the cores from any other source, the diagonals for H2A- and H2B-crosslinked DNA fragments were separated rather poorly in the DNA-identifying twodimensional system. Then we used a second system of two-dimensional gel electrophoresis which identified the crosslinked proteins (protein-identifying 3447


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Figure 4. Two-dimensional gel of 32P-labeled DNA from lily bud crosslinked cores. Experimental parameters were the same as in Figure 2.

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system) and provided an improved resolution for the crosslinked histones from Drosophila, yeast and chicken erythrocytes: following fractionation of crosslinked material in the first direction, the DNA was hydrolysed directly in the gel, and the released 125I1-abeled histones were then separated in the second direction. In this gel (Figure 5), the spots of crosslinked histones are shifted to the left from the spots of uncrosslinked ones (the extreme right part of the autoradiogram). By comparing the proteinand DNA-identifying gels (Figures 5-6), one can accurately determine the DNA length. Here we have compared such gels for chicken erythrocyte cores only; the comparison for Drosophila cores was done earlier (7) and the same for yeast and lily buds is to be published elsewhere. A better resolution of two-dimensional gels here as compared with previously published results (7) has been attained owing to the removal of most uncrosslinked DNA from the crosslinked material on a hydroxylapatite column prior to DNA-identifying two-dimensional gel electrophoresis, and also because we used thinner and longer gels, gel concentrations and current were optimized, etc. Arrangement of histones on DNA The data on the arrangement of histones on DNA in nucleosomal cores summarizing 3-5 experiments, such as presented in Figures 2-6, are shown for chicken erythrocytes, Drosophila embryos and yeast particles in Figure 7A, for spermal cores in Figure 7B and for lily particles in Figure 7C. Also given are relative intensities of histone crosslinking to different DNA seg3448

Nucleic Acids Research ments. Here we have discriminated for the first time the binding sites between histones H2A and H2B in the area of 110-130 nucleotides and between H2A and H3 within 130-145 nucleotides from the 5' DNA termini. The previously published data (7) are supplemented with newly discovered weak crosslinking sites for all histones. We have also found a direct correlation between the appearance of the crosslinking sites H445,,7 and the presence of particles containing about 135 bp long DNA in the core preparations (in pure nucleosomal cores these crosslinking sites were not observed). We do not know the reason for the horizontal twinning of the H3,5.95 spots in the DNA gels of spermal cores (Figure 2). This may have resulted from partial modification or proteolysis of H3. The vertical doubling of the H4sa,&S spots in the gels of spermal cores on the same figure seems to be due to an incomplete digestion of this histone by Pronase before electrophoresis in the second direction. From Figures 7 and 8 it is apparent that all the cores studied have very similar arrangement of histone crosslinking sites along DNA, even though the relative intensity of crosslinking can differ at a particular site. Only the strong crosslinking site H2M and the weak site H3105 show some specificity. These sites were found only in the cores from sea urchin sperm and lily buds. It seems that the additional cluster of 4 to 5 lysine residues in the extended N-terminal part of spermal H2B (41) is involved in the H2B.. contact. We have to admit here that the H2B crosslinking in the H2B22-52 area significantly varied in intensity and relative sharpness of spots in two-dimensional gels, even when the experiments were done with the same core preparations. This probably reflects a high sensitivity of crosslinking to the experimental conditions owing to an oscillation of H2B between the complementary DNA strands across the DNA grooves (7) and between the adjacent turns of superhelical core DNA (see Figure 9) which may account for the H2B crosslinking in this area. Less pronounced, though still noticeable, variations in intensity were also observed for crosslinking of other histones. Another dissimilarity, already recorded earlier (8), is the fact that the H2A,5 site is found only in the cores crosslinked after their isolation, e. g. in chicken and Drosophila particles. This is probably an indication that the isolation procedure affects the core structure. DISCUSSION Structural implications We have not found any essential differences in the primary organization of nucleosomal core particles originating from all three higher eukaryote kingdoms (animals, plants and fungi)(51), namely from sea urchin sperm (Fi3449

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Figure 5. Two-dimensional gel electrophoresis of 121I-labeled histones crosslinked to DNA in Drosophila embryo (A), yeast (B) and chicken erythrocyte (C, D) cores. The crosslinked complex was electrophoresed in the first direction in 15% polyacrylamide slab gel (Q20000x0.6 mm) containing 7 M urea at 3 mA and 7 mA for the concentrating and separating gel, respectively. Following hydrolysis of DNA in the gel, electrophoresis in the second direction was performed in 15% polyacrylamide slab gel (200x400x1 mm) at 3 mA and 5 mA for the concentrating and separating gel, respectively. Electrophoresis was stopped when bromphenol blue reached the bottom of the gel. The spots of uncrosslinked histones are seen at the extreme right of the gel. The figure at each spot indicates the size of DNA fragments crosslinked to histones as determined from Figure 6. (D) A longer exposure of the gel shown in (C). Xunidentified spot. gure 7A), lily buds (Figure 7C), Drosophila embryos, Ehrlich ascite mouse cells and rat liver (the latter three were studied earlier in this laboratory, ref. 7), and yeast (Figure 7A). The few exceptions were the strong contact H2Bsm in sea urchin sperm, the relatively weak contact H3oso in somatic cells of lily and the H2A75 contact found only in cores crosslinked after isolation. These data further support the idea that the core nucleosome structure is highly conserved in evolution (1). Figure 8A summarizes data on the arrangement of main histone contacts along one strand of core DNA. Figures 8B and 9 show, respectively, models for the symmetrical arrangement of histones on the double-stranded linear and superhelical DNA. It can be seen that histones are aligned on one core DNA strand in the following order: (S') H2B25,35-H4sa,&o-H375X.ffiffi/H4..-H2B,sol,g-H2A,l.-H3135,14o/H2A,4s (3'). On the double-stranded core DNA the order of histones is as follows: H2A/H3-(H2A-H2B)-(H3,H4) 2- (H2B-H2A)-H3/H2A. The weak contacts of histones with core DNA which are absent in Figures 8,A-B are shown in Figures 7,A-C. These weak contacts might arise as a result of simultaneous interaction of histone molecules and their lysine re-

3451


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3452

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of core DNA (52, 53); the presence of very few histone-DNA ionic bonds in this region (54); the existence of compact dinucleosomes formed by overlapping of the adjacent core particles at their terminal regions (55), and the localization of sites preferentially accessible to DNAase I (Figure BA) at distances of 10, 20, 40, 50, 120 and 130 bases from the 5 -end of core DNA (12, 48-50). It should be pointed out that some of the most accessible sites are situated between the regions of dimer (H2A-H2B) and tetramer (H3,H4)2 crosslinking. The histone octamer of a nucleosomal core particle consists of relatively autonomous, although interacting with each other, specific histone complexes: one tetramer (H3, H4)2 and two dimers (H2A-H28) (1). In core nucleosomes, the tetramer and the two dimers are comparatively independent: the tetramer is located in the center of the core DNA from -2.5 to +2.5 sites (Figures 8B and 9) while the two dimers flank the tetramer on both sides and bind to both ends of the core DNA. This model was supported in a study of the subnucleosome structure (56). On the other hand, within DNA areas accomodating the (H2A-H2B) dimers and, in particular, the (H3,H4)2 tetramer there is a significant overlapping between histones. Simultaneous interacti-

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Figure 8. (A) A map for the main histone contacts located on one DNA strand which summarizes results of the crosslinking studies on cores from chicken erythrocytes, Drosophila embryos, yeast, sea urchin sperm and lily buds (Figures 7 A,B,C) and earlier studied cores from rat liver and mouse ascite tumor cells (7, 8). Specific interaction with DNA of H2B from sea urchin sperm is not shown in this map. The contact H2A,5 (in brackets) was found only in the cores crosslinked after isolation. The arrows point to the main core DNA sites exposed to DNAase I, the arrow length being proportional to the relative accessibility (12, 49, 50). (B) A symmetrical model for the arrangement of histones on two DNA strands in the core particle. The model is based on the data of Figure BA and assumes the presence of a dyad axis of symmetry in the cores (4). Superscripts 1 and 2 denote two copies of each histone in the core. Distances along DNA are given either in nucleotides from the 5'-end or by numbers from -7 to +7 taking the diad symmetry axis in the core particle as the origin, to mark 14 repeats in the 145 bp long double helical core DNA (4).

l--__

#2Bt

_

Figure 9. A three-dimensional model for the symmetrical arrangement of histones on the folded core DNA. The DNA forms a left-handed superhelix containing about 80 bp per turn (3). Positioning of histones H2A (narrow box, broken line), H2B (wide box, broken line), H3 (wide box, continuous line), and H4 (narrow box, continuous line) on two DNA strands is shown by placing histones above and below the DNA line and is taken from Figure 8. 3454

Nucleic Acids Research on of histone H3 with the central (H3,,s,,s) and terminal (H3135,14.) regions of the core DNA located close to each other on the folded DNA (Figure 9) seems to stabilize the DNA superhelix (4, 7). This is consistent with the finding that just one (H3,H4)2 tetramer suffices to make the core DNA fold (57). The fact that the new contact H2A75 appears only in isolated cores brings forth the question of its genesis. This contact may appear as a result of some rearrangement of histone H2A on DNA induced by the absence of either Hi and spacer DNA or internucleosomal contacts in isolated cores. Our preliminary data have also demonstrated the absence of the H2A75 contact in isolated HI-containing nucleosomes crosslinked after preparation, where H2A was not crosslinked to the spacer DNA. This suggests that H2A can be involved in internucleosomal contacts. Participation of core histones in interactions between nucleosomes in chromatin was proposed earlier (2, 58). It is of interest to compare our high resolution map for the arrangement of histones on DNA in the cores (Figures 8 and 9) with the model of three-dimensional structure of the cores based on recent X-ray and neutron diffraction studies of core crystals by Richmond et al. (6) and Bentley et al. (5). Both approaches have led to a conclusion that histones are aligned along the whole DNA length with histone-DNA interactions occurring on the inside of the DNA superhelix. The ±1 and ±4 sites of DNA sharp bending (6) correspond to the region of simultaneous interaction of H3/H4 and H2A/H2B with DNA at 85/65 and 115/35 nucleotides from the 5' DNA ends. The slightly different arrangement of two (H2A-H2B) dimers on DNA and involvement of H2A in the intercore contacts in the core crystals suggested by Richmond et al. (6) agree with our data on the easy rearrangement of H2A (found by comparing isolated cores and cores within chromatin) and with the variable pattern of H2B crosslinking in the area of 20-50 nucleotides from the 5' DNA ends. On the other hand, the assignement of the C-rod-like structure in the region from ±3.5 to 15 to histone H4 is not supported by our data since we observed H2A and H2B but no H4 crosslinking within these DNA segments. The X-ray, neutron diffraction and crosslinking data have been recently supported by direct investigations of topography of histone-DNA interactions in core particles reconstituted from individual histones, labeled by Pt2 (59). Other details of the core structure will hopefully be revealed when further improvements in resolution are complemented with identification of the regions in histone molecules that are crosslinked to each particular segment of core DNA. The ten-nucleotide periodicity in the arrangement of histone crosslinking sites on core DNA (see Figures 7 and 8 in ref. 10) is much sooner determined

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Nucleic Acids Research by the lateral arrangement of histones on the internal side of DNA in the nucleosome than by any specificity in DNA methylation and digestion with micrococcal nuclease (60). In our mock experiments we have found no regularity in crosslinking lysines instead of histones to core DNA (not shown). Functional implications The similarity of the linear arrangement of histones in nucleosomal cores isolated either from the repressed nuclei (sea urchin sperm or chicken erythrocytes) or from the nuclei that are actively involved in transcription and replication (Drosophila embryos and especially yeast) suggests that the overall inactivation of chromatin cannot be reflected and/or regulated at this primary level of chromatin organization. We do not exclude, however, that there can be other structural features in active nucleosomes that have remained undetected by the methods described above. Such processes as histone modification, HMS-protein binding, partial unfolding, etc., might affect histone-histone interactions but not the location of their contacts on DNA. It has been shown that the unfolding of core particles which occurs at high concentrations of urea and NaCl (61) and the binding of two HMG 14/17 molecules to the cores and HI-containing nucleosomes (62) do not lead to any substantial changes in their primary organization. The low conformational stability of yeast nucleosomes (14) is not reflected in their primary organization either. However, all these factors may facilitate removal of histones from transcribed DNA (23, 24,63). Specific features of both active and repressed chromatin may reveal themselves on higher levels of chromatin structure. Thus, the removal of histone HI from moderately transcribed heat-shock Drosophila genes, observed in our laboratory (24) seems to induce unfolding of the 30-nm chromatin fiber into the 10-nm fiber. The presence of spermal variants of histone H2B in sea urchin sperm chromatin hinder such transitions as well as the temporary removal of histone octamers from the sites of transciption and thereby inhibit transcription. The spermal variants of H2B increase the tendency of nucleosomes to aggregate (26) and participate in the organization of linker DNA (64), thus stabilizing the condensed structure of repressed spermal chromatin. Studying the presence of histones in increasingly activated heat shock genes of Drosophila, hsp 22 and hsp 70, it was shown that all HI and some core histones are removed upon moderate transcription while upon active transcription the gene DNA becomes completely free of histones (24, 63). Considering the mechanism by which RNA polymerases could displace histones from DNA and affect the nucleosome structure, one has to take into account the essentially nonoverlapping arrangement of the (H3,H4)2 tetramer and

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Nucleic Acids Research the (H2A-H2B) dimers on the central and terminal regions of core DNA and simultaneous interaction of histone H3 with the both regions in folded cores (Figures 8 and 9). Entering the terminal region of core DNA, RNA polymerase first displaces histone H3 and then one (H2A-H2B) dimer. These displacements will unravel the terminal DNA region or even the whole nucleosome, for example in such a way as was observed in Physarum ribosomal chromatin by Prior et al. (22). Upon its further move, the polymerase will remove the tetramer and finally the second histone dimer. Consistent with this model is the finding by Baer and Rhodes (20) that the RNA polymerase-nucleosomal core complex is deficient in one (H2A-H28) dimer. The low extent of overlapping between the histone tetramer and two dimers on core DNA agrees well with their independent segregation during replication of chromatin (65-69).

ACKNOWLEDGEMENT The authors are grateful to D.Prouss for help in some of the experiments, to V.Korobko for the gift of polynucleotide kinase, to K.Ebralidze and V.Karpov for stimulating discussions and to T.Richmond who acquainted us with his paper before its publication.

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