Generation of different nucleosome spacing periodicities in vitro

J. Mol. Biol. (1988) 203, 1029-1043

Generation

of Different Nucleosome Spacing Periodicities in V&o

Possible Origin of Cell Type Specificity Arnold Stein-f and Mears Mitchell Department of Biological Sciences Purdue University West Lafayette, IN 47907, U.S.A. (Received 27 October 1987, and in revised form

18 April

1988)

We have bden able to generate. ordered nucleosome arrays that span the physiological range of spacing periodicities, using an in vitro system. Our system (a refinement procedure previously developed) uses the synthetic polynucleotide . the ;bly,d(A-T), . poly[d(A-T)], core histones, purified Hl, and polyglutamic acid, a factor that increases nucleohistone solubility and greatly promotes the formation of ordered nucleosome arrays. This system has three useful features, not found in other chromatin assembly systems. First, it allowed us to examine histones from three different cell types/ species (sea urchin sperm, chicken erythrocyte, and HeLa) as homologous or heterologous combinations of core and Hl histones. Second, it allowed us to control the average packing density (core histone to polynucleotide weight ratio) of nucleosomes on the polynucleotide; histone Hl is added in a second distinct step in the procedure to induce nucleosome alignment. Third, it permitted us to study nucleosome array formation in the absence of DNA base sequence effects. We show that the value of the spacing periodicity is controlled by the value of the initial average nucleosome packing density. The full range of physiological periodicities appears to be accessible to arrays generated using chicken erythrocyte (or HeLa) core histones in combination with chicken H5. However, chromatin-like structures cannot be assembled for some nucleosome packing densities in reactions involving some histone types, thus limiting the range of periodicities that can be achieved. For example, Hl histone types differ significantly in their ability to recruit disordered nucleosomes into ordered arrays at low packing densities. Sea urchin sperm Hl is more efficient than chicken H5, which is more efficient than Hl from HeLa or chicken erythrocyte. Sea urchin sperm core histones are more efficient in this respect than the other core histone types used. These findings suggest how different repeat lengths arise in different cell types and species, and provide new insights into the problems of nucleosome linker heterogeneity and how different types of chromatin structures could be generated in the same cell.

1. Introduction The existence of ordered nucleosome arrays of substantial length is the hallmark of chromatin structure. Order is most often inferred from the existence and quality of the “micrococcal nuclease ladder,” the multiples of a unit DNA length (repeat length) excised from chromatin by the highly preferential cutting of linker DNA over the DNA tightly wound around the histone core of the nucleosome. The numerical value of the nucleosome spacing periodicity in the array is often a

distinguishable feature of the chromatin from a particilar cell type and species. Repeat lengths ranging from about 160 to 250 base-pairs have been observed (Kornberg, 1977). Intracellular repeat length variability was also detected when specific genes were probed (Gottesfeld, 1980; Smith et al., 1983; Gottschling et al., 1983; Benezra et aZ., 1986) suggesting that changes in the repeat length might be of functional significance. The factors and molecular forces that generate nucleosome arrays with particular spacing periodicities are not known. A number of different ideas have been proposed

t Author to whom all correspondence should be sent. CM,22-~8:~6i8Hi”OlOZQ-l.~ $03.00/O

over the last 12 years (for

reviews, see McGhee & Felsenfeld, 1980; Eissenberg 1029 0 1988 Academic> F’rrss Limited

1030

A. Stein and M. Mitchell

et al., 1985). Early on, it was suggested that histone HI, in essence, measures out the distance between adjacent nucleosomes by binding to linker DNA (Noll, 1976; Morris, 1976; Compton et al., 1976). Thus, longer repeat lengths should be associated with longer or more basic histone HI molecules. Consistent with this idea, histone Hl molecules exhibit appreciable variability among the different tissues of an animal (Kinkade & Cole, 1966; Lennox & Cohen, 1983) and among specialized cells of different species (Allan et al., 1980). Inconsistent with this idea, chicken erythrocytes have an unusually long repeat, yet the erythrocyte-specific Hl analog, H5, is substantially shorter than a typical Hl and has about the same number of basic residues (Yaguchi et al., 1979; Hriand et al., 1980; McGhee & Felsenfeld, 1980). Rather than the type of Hl, it is plausible that Hl availability or the state of phosphorylation (Ajiro et al., 1981) during chromatin assembly could influence the nucleosome spacing. Consistent with these ideas, neural cell chromatin, which has a very short repeat length (165 base-pairs) and can condense into an apparently typical higher-order structure (Pearson et al., 1983), contains only 0.45 molecule of Hl per nucleosome (Pearson et aE., 1984). Differences in core histone modifications that occur during replication (Ruiz-Carrillo et al., 1975; Laskey & Earnshaw, 1980), or in the levels and types of nonhistone proteins associated with the nucleohistone of different cell types, could also be the underlying cause of the differences observed. There has been no way to directly test these ideas. Another idea is that periodic signals in eukaryotic DNA, encoded in the base sequence, influence nucleosome spacing (Keene & Elgin, 1984; Mengeritsky & Trifonov, 1984). Even though certain DNA base sequences can precisely position histone octamers (for a review, see Simpson, 1986), this effect alone cannot explain repeat length differences in different tissues of the same animal. it has been suggested that particular Finally, repeat lengths may reflect the average number of nucleosomes formed per unit DNA length, with essentially random spacings (Kornberg, 1981). Why the average nucleosome packing density should vary so much from one cell type to another is not clear. In order to be able to control all of the potential variables that could influence the repeat length, an in vitro chromat,in assembly system is necessary. Recently, we developed a system that generates long physiologically spaced nucleosome arrays, as well as chromatin-like structures, on the synthetic polynucleotide poly[d(A-T)] . poly[d(A-T)] (Stein & Rina, 1984). In this system, purified core histones are first’ reconstituted with the polynucleotide, forming a mixture of disordered and close-packed at any desired average packing nucleosomes, density. Next, histone Hl (or H5) is added in the presence of polyglutamic acid, a component that prevents aggregation and promotes nucleosome alignment. Incubation of this mixture at 37°C

results in the formation of ordered nucleosome arrays. The use of the synthetic polynucleotide eliminates DNA base sequence effects from the analysis, which greatly simplifies the problem; it also results in much better nucleosome alignment than with DNA under the same conditions. Previously, we examined the assembly of chromatin-like structures using chicken erythrocyte core histones plus chicken H5. We have now examined nucleosome array formation in a refined version of this system using core histones and purified histone Hl from several types of cells, in all possible combinations, and over a wide range of conditions. We have been able to generate arrays with spacing periodicities that span the whole physiological range. The results suggest how the different repeat lengths observed in nature may arise, and also provide new insights into understanding the molecular forces that determine the structure of chromatin.

2. Materials and Methods HeLa cells maintained in logarithmic growth at 2 x 10’ to 6 x IO5 cells/ml in suspension using supplemented Minimum Essential Medium (Gibco) and 10% (v/v) calf serum, were kindly provided by Marcia Kremer. These cells were gently spun down, washed, then resuspended in a minimal volume of buffer A (10 mhr-Tris . HCI (pH 8.0). 3 mM-CaCl,, 0.25 M-SUCrOSe) and stored frozen. Chicken blood was purchased from Pel-Freez; freezing was avoided. Sea urchins (Lytechinus pi&s) were purchased Micrococcal Pacific Bio-Marine. nuclease from (Worthington) was dissolved in water at 10 units/PI and stored frozen in small samples. A thawed portion could be stored at 4°C for more than I week without loss of activity. DBase I (Miles) was dissolved in 0.01 M-HCl at 2.0 units/$ and stored frozen in 50-~1 portions; a portion was thawed, added to 450 ~1 of 10 mw-Tris . HCl (pH 7.5), 5 mM-MgCl,, 1 mg bovine serum albumin (Miles Pentex)/ ml, and incubated for I h at 0°C just before use (Rigby et al., 1977); the remainder of this sample was discarded. Proteinase K (Boehringer-Mannheim) was dissolved in water just before use. The large fragment of Escherichia coli DNA polymerase I was purchased from Bethesda Research Laboratories. A 10 mg/ml stock solution (stored at 4°C) of polyglutamic acid of average molecular weight 64,000 (Miles) in 20 mivr-Tris. HCl (pH 7.2) was prepared sodium hydroxide; lower neutralization with by molecular weight polyglutamic acid is not satisfactory for these experiments. Hydroxylapatite (DNA grade) was purchased from Bio-Rad. 2’-Deoxyadenosine-5’-triphosphate and thymidine-5’-triphosphate were purchased from Boehringer-Mannheim. (a) Preparation

of nuclei

HeLa and chicken erythrocyte nuclei were prepared from HeLa cells or chicken red blood cells, respectively, suspended in buffer A. All manipulations were performed without delay, with samples kept at 0°C. Triton X-100 (from a 20% (v/v) stock) was first added dropwise with gentle stirring to a final concentration of 1% (Hymer & centrifugation for 20 min at Kuff, 1964). After 8000 revs/min using 200 ml bottles in a Sorvall GSA rotor, pellets were homogenized with several strokes of the loose-fitting pestle in a Dounce homogenizer.

Origin of Repeat Length Variability Generally, 2 to 3 more washes using buffer A containing 1o/e Triton X-100 were performed, and the nuclei were then suspended in buffer A without detergent, homogenized and either used directly or frozen. To prepare sea urchin sperm nuclei, first ripe males were identified by intracoelomic injection of all animals with about 0.5 ml of 055 M-KC]. Within about 2 min small amounts of either sperm or eggs could be identified on the bodies of ripe animals. In ripe males, the alimentary viscera were removed and the coelomic cavity was thoroughly rinsed with 055 M-KC] to collect the sperm. This suspension was filtered through 2 layers of cheesecloth, sperm was recovered by low-speed centrifugation and suspended in buffer A. Nuclei were prepared directly. as described above. (b) Preparation

of core histones

To prepare HeLa core histones, nuclei isolated from about 3 1 of HeLa cells in suspension were apportioned into 4 tubes and resuspended, by low-speed centrifuga10 mlcl-Tris HCI tion, in 40 ml of 0.35 M-NaCl, (pH 8.0), 1 mM-Na,EDTA. Samples were then stirred gently for 15 min at 0°C and the washed nuclei recovered by low-speed centrifugat,ion. Next, 10 ml of 060 M-NaCl, 50 m&r-sodium phosphate (pH 68) was added to each causing the nuclei to lyse after gentle stirring. To each tube. 0.4 g of hydroxylapatite was then added, making a paste. Volumes were increased to 40 ml with the same buffer, phenylmethylsulfonyl fluoride was added to a concentration of 0.2 mM. and the suspension was stirred for 10 min at 0°C. The hydroxylapatite-immobilized chromatin was collected by low-speed centrifugation, and subsequently washed 5 t,imes by the above procedure to remove Hl histones. Each pellet was then suspended in 40 ml of 2.5 M-NaCl, 50 miv-sodium phosphate (pH 6.8) by gentle stirring for 15 min at 0°C to elute the core histones from the immobilized DNA. After a low speed spin. the pellets w-ere resuspended in 40 ml of 2.5 M-NaCl-containing buffer and the supernatants were rombined with those of the first extraction. Core histones were concentrated to 1 to 5 mg/ml by ultrafiltration in an Amicon cell using a YMlO membrane. Chicken erythrocyte core histones were prepared the same way except that nuclei, containing about 6 mg DNA/tube, were suspended in 10 ml of 0.70 iwNa(:l. 50 miw-sodium phosphate (pH 6.8) to lyse, and 5 washes were performed as described above using this buffer. The higher ionic strength is necessary to remove histone H5 as well as the Hl histones. Gentle stirring is essential at this salt concent,ration in order to prevent removal of some histones H2A and H2B. Wit,h sea urchin sperm nuclei, we found that it was not possible to remove HI by the above procedure with gentle stirring, and that more vigorous stirring resulted in the removal of a substantial proportion of H2A and H2B. Thus, another procedure was used to selectively remove this unusually tightly associated Hl histone. Nuclei rontaining about 50 mg of DNA were first suspended in 10ml of 0.1 M-NaCl. lOmw-Tris.HCl (pH80), 1 mMNa,EDTA. After equilibration at 37”C, CaCl, was added to 2 mM, 150 units of micrococcal nuclease were added, and the sample was incubated for 30 min. These digested nuclei were then adjusted to 10 mM-Na,EDTA, recovered by low-speed centrifugation, washed with 0.50 M-NaCI, 10 mM-Tris HCI 1 mM-Na,EDTA, and (PH 8.0) suspended in 10 ml of 0.70 M-NaC1, 50 mw-sodium phosphate (pH 6.8). After 30 min at O”C, the nuclei were vortexrd and centrifuged at 8000g for 15 min. The clear

1031

supernatant generally contained about 10 AZ6,, units/ml of predominantly mono- and dinucleosomes, along with some histone Hl. To revove the Hl. the sample was loaded on a 2.5 cm x 90 cm Bio-gel A-5m (Rio-Rad) column equilibrated with 0.65 M-NaCl, 10 mm-Tris . HCI (pH 7.2), 1 mM-Na,EDTA at 4°C. Most of the &4,,c eluted just after the void volume, and the center of this main peak contained mono- and dinucleosomes free of Hl. This material was adjusted to 2.5 M-NaCl. 50 miw-sodium phosphate (pH 68), 0.5 g hydroxylapatite/mg DNA was added, and the suspension was shaken gently for 20 min at 4°C. The hydroxylapatite, which had absorbed most of the DNA, was removed by low-speed centrifugation. Another treatment with a fresh portion of hydroxylapatite reduced the A,,,/A,s, ratio to nearly 0.50, indicating that essentially all of the DNA had been removed. The core histones were then concentrated by ultrafiltration as described above. Analysis of core histone preparations on sodium dodecylsulfate-containing polyacrylamide gels indicated that those from sea urchin sperm and chicken erythrocyte were free of non-histone proteins. HeLa core histone preparations typically contained small amounts of a variety of unknown non-histone proteins. All of the core histone preparations used suffered negligible extents of proteolysis. For sea urchin sperm and chicken erythrocyte core histones the concentration was measured from the ultraviolet light absorbance at 280 nm using an extinction coefficient (A%,) of 4.4 (Stein & Page, 1980). A higher (about 50% to 70%) effective extinction coefficient for each HeLa core histone preparation was estimated approximately, based upon the ultraviolet light absorbance of the protein soluble in 0.1 M-HCI (D’Anna & Isenberg, 1974) after reconstitution of a known volume of sample with DNA. All core histone samples were stored frozen in the 2.5 m-NaCl-containing buffer. (c) Preparation

of HI (and H5) histones

Nuclei were washed twice with 40 ml of 0.35 M-NaCl, 10 miw-Tris . HCI (pH 8.0). 1 mM-Na,EDTA to remove most non-histone proteins. The washed nuclei were then resuspended in 10 ml of 0.7 M-NaCl, 50 mM-sodium phosphate (pH 6.8) and equilibrated 30 min at 0°C with occasional stirring to lyse; then 4.0 g of hydroxylapatite was added, as described above. The volume was then increased to 40 ml with the same buffer. containing in addition 0.2 mM-phenylmethylsulfonyl fluoride. and samples were stirred well for 15 min at 0°C to elute Hl and H5 (contaminated with non-histone proteins and H2A plus H2B to varying degrees in preparations from different cell types) from the immobilized chromatin. After a low-speed spin, the pellet was extracted with an additional 40 ml of buffer and the protein in the combined supernatants was concentrated to 1 to 2 mg/ml by ultrafiltration in an Amicon cell using a YMlO membrane. Histones Hl and H5 were purified by gel filtration on a 1.5 cm x 75 cm column in 0.6 M-NaCl, 50 m&r-sodium phosphate (pH 6.8) using Gephacryl H-200. Superfine (Pharmacia) at 4°C. Histone Hl was fairly well resolved, whereas histones H2A plus H2B generally appeared as a shoulder on the late-eluting side of H5 (in the chicken erythrocyte prep). Appropriate fractions were combined and concentrated using Centricon microconcentrators (Amicon Corp.). All of the Hl samples were pure and intact as judged by sodium dodecylsulfat,e-containing polpacrylamide gel analysis. Two Hl subtypes were

1032

A. Stein

and

resolved by electrophoresis in the chicken erythrocyte and HeLa preparations, whereas the sea urchin sperm Hl migrated as a single band. Histone H5 preparations contained only trace amounts of Hl and H2A plus H2B, and typically about 95% of the protein was fully intact. (See Stein (1989) for further details and gel analysis of the chicken erythrocyte histones.) The extinction coefficient (A$$) used for Hl or H5 was 2.0 (Johns, 1971). Samples were stored frozen in the same solution used for fractionation. These procedures avoid denaturation or even unfolding of these histones. There is evidence that irreversible changes in purified histone Hl molecules in solution may occur by merely decreasing the pH below 6.0 or by decreasing the ionic strength below about 0.50 M (Brand et al., 1981). We found that acid-extracted HeLa Hl or chicken erythrocyte H5 worked less well in chromatin assembly, and the acid-extracted sea urchin Hl did not work at all (unpublished observations). (d) Preparation

of suitable

PeM-fJ-~~l~ PM~~~-~~l The polymerization reaction was a modification of the procedure described by Stelow et aE. (1972). The reaction mixture contained 66 mnn-potassium phosphate (pH 7.4), 1.5 m&r-dATP, 1.5 mM-dTTP, 1 mM-26.6 mm-MgCl,, mercaptoethanol, 15 pg primer/ml, and 125 units of E. coEi DNA polymerase I Klenow fragment/ml. This mixture was incubated at 37°C for 20 to 30 h. NaCl was then added to a concentration of 1 M, Na,EDTA (from a 0.5 M (pH 8.0) stock solution) was added to a concentration of 10 mM, and the clear solution was dialyzed for at least 20 h against 1.0 M-Nacl, 10 m&r-Tris. HCl (pH 80), 1 mM-Na,EDTA at room temperature. This procedure avoids extraction with phenol, precipitation with ethanol or low ionic strength, conditions that could lead to chain branching by destabilization of base-pairing. After the sample concentration was extensive dialysis, determined in a 1.0 M-NaCl-containing buffer from the absorbance at 260 nm, using a value of 20 AZ6,, units (10 mm path-length cell)/mg of polynucleotide (Inman &, Baldwin, 1962). The same concentration value, typically around 0.65 mg/ml, was obtained when based upon ethanol-precipitable material, indicating that unreacted deoxynucleotides were effectively removed by dialysis under the above conditions. The molecular weight of the poly[d(A-T)] product was strongly dependent upon the size of the primer, and the yield of the reaction was dependent upon the concentration of primer Zhydroxyl ends. To prepare the primer, poly[d(A-T)] at 1 mg/ml was digested with 2 units of DNase I/ml in 10 m&r-Tris . HCl (pH 8.0), 10 m&r-MgCl, for 8 min at room temperature. The reaction was stopped by adjusting the solution to 20 mM-Na,EDTA, 1 y. (w/v) SDS, and the poly[d(A-T)] was purified by extraction with phenol and dissolved in 0.2 mM-Na,EDTA. Freshly renatured (after heating to 106°C) primer and product migrate on a 1% (w/v) agarose gel with average mobilities corresponding to DNA fragments that are about 300 and 12,000 base-pairs long, respectively (Stein, 1989). Samples that are not heated just before loading streak upwards on the gel due to aggregation induced by precipitation with ethanol of the samples in preparation for electrophoresis. This gel serves merely as a convenient method for sample characterization, rather than a measure of actual duplex length. Actual duplex lengths are probably greater than apparent lengths due to secondary structure formation during electrophoresis

M. Mitchell

(and sample preparation for electrophoresis). Finally, the poly[d(A-T)] prepared as described here was much more homogeneous in size than the material used in initial studies (Stein & Bina, 1984) which was prepared by brief sonication of very high molecular weight polynucleotide. (e) Nucleosome

reconstitutionlchromatin

assembly

Poly[d(A-T)] . poly[d(A-T)] was first reconstituted with core histones using conventional salt-gradient dialysis. The polynucleotide in 1.0 M-NaCl, 10 mivr-Tris . HCl (pH 8.0), 1 mM-NazEDTA was mixed with the required amount of core histones (in the 2.5 M-NaCl stock solution buffer) for a final nucleic acid concentration of about 5A 260 units/ml. This mixture (generally, 0.4 to 1.0 ml) was dialyzed (in Spectrapore tubing, 1.0 cm dry width, 12,000 to 14,000 M, cutoff) for 1.5 h against 1.0 1 of 0.80 M-Nacl, 20 rnM-Tris . HCl 0.2 rnM(pH 7.2). NazEDTA, 1 mM-2-mercaptoethanol. The sample was next dialyzed for 1.5 h at room temperature (or overnight at 4°C) against 1 1 of 0.15 M-NaCl, 20 mw-Tris. HCl (pH 7.2), 0.2 mm-Na,EDTA, and finally against 1 1 of 20 mw-Tris . HCl (pH 7.2), 0.2 mM-Na,EDTA for 1.5 h at room temperature (or overnight at 4°C). Usually, greater than 90% of the A,,, was recovered. The product of this reaction was nucleosomes randomly arranged along the polynucleotide (Stein & Bina, 1984). The core histone/polynucleotide weight ratio of many reconstituted samples was measured (in triplicate) using a bicinchoninic protein assay (Pierce) at 60°C for samples adjusted to 0.5% sodium dodecylsulfate; chicken erythrocyte core histones were used as a standard. We found that this ratio was almost always the same, within experimental precision of about f0.05, as the ratio calculated from the concentrations of the components added. Thus, no preferential loss of either core histones or polynucleotide occurred during the dialysis procedure. This material could be kept at 4°C for several days. In the second step of the procedure nucleosomes were aligned and regularly spaced by histone Hl or H5 in the presence of polyglutamic acid. The final reaction mixture contained 20 m&r-Tris. HCl (pH 7.2), 0.2 mM-NazEDTA. 0.15 M or 0.04 M-NaCl (added from 2.0 M or 0.5 M stock solutions, respectively), 2.0 mg polyglutamic acid/ml. core histone-reconstituted polynucleotide at 2.0 A,,, units/ml, and Hl or H5 histones, at the molar ratio (with respect to nucleosomes) stated. A small volume of Hl or H5 histone was added last from a concentrated solution containing 0.6 M-NaCl, which contributed to the final NaCl roncentration. Also, care was taken in adding the other components so that the reconstituted polynucleotide was not exposed to NaCl concentrations greater than about 0.5 M, which could dissociate nucleosomes. After addition of all components, the sample, generally 200 ~1, was mixed well and incubated for 12 h at 37°C in a 400 ~1 capped polypropylene tube without stirring. Reaction mixtures were clear solutions, except for those reactions containing high molar ratios of Hl (greater than 2.5/nucleosome), in the 0.15 iw-NaCl-containing buffer and high weight ratios (greater than 090) of core histones to polynucleotide. These samples were cleared by centrifugation before digestion with micrococcal nuclease. (f) N&ease

digestion/gel

electrophoresis

Generally, a portion containing 5 pg of nucleic acid from the micrococcal nuclease digestion was removed at a time point and added to an equal volume of 0.206 SDS,

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Origin of Repeat Length Variability

were heated for 2 min at 100°C and loaded on a 4% (w/v) polyacrylamide gel containing 7 M-urea, using the Trisborate buffer system as described by Maniatis & Efstratiadis (1980). To obtain even and undistorted gel slots, a polymerization time of 20 min was used and samples were loaded immediately after removing the comb from the electrode buffer-containing apparatus and gently rinsing out the slots. Samples were run into the 15 cm long, 3 mm thick gel at 100 V for 15 min, and then electrophoresis was for 4.5 h at 150 V. Gels were stained with 3 pg ethidium bromide/ml for 15 min. soaked in water overnight, and photographed under ultraviolet illumination using a Kodak no. 9 Wratten filter. We have not been as successful with other denaturing gel systems

in obtaining reliable data.

3. Results (a) Choice of reaction variables Figure 1. Native chromatin samples and a reconstituted sample spanning the physiological range of nucleosome-spacing periodicities. Lane 1, reconstituted close-packed nucleosomes; lane 2, HeLa; lane 3, chicken erythrocyte; lane 4, sea urchin sperm. The reconstituted sample and chicken erythrocyte chromatin were digested in 0.1 M-NaCl, 10 m&r-Tris . HCI (pH 7.2), 0.2 mMNa,EDTA, 1.0 mM-CaCl,. HeLa and sea urchin sperm nuclei were digested in 0.25 M-sucrose, 10 mM-Tris . HCI (pH 8.0), 1.0 mM-CaCl,. Digests that generated similar distributions of nucleosome oligomers were selected. The range of electrophoretic mobilities corresponding to monomers is indicated (M).

20 mM-Na,EDTA solution to stop the reaction. Freshly dissolved proteinase K was added to a concentration of approximately O-2 mg/ml, and the samples were incubated 2 h or more at 37°C. Samples were next adjusted to 1 y0 SDS, 0.1 M-Tris. HCl (pH %O), 10 mMNa,EDTA, and extracted twice with phenol (distilled, saturated with the same buffer; stored frozen in portions), and once with chloroform/isoamyl alcohol (24 : 1, v/v). Samples were finally supplemented with an additional 0.1 M-NaCl and 20 ng of polyglutamic acid (as a carrier), precipitated with 25 vol. ethanol (for 10 min using solid C02), dried under vacuum and dissolved in sample buffer. Polyglutamic acid, in these quantities, does not interfere with electrophoresis or staining with ethidium bromide. It is necessary to run poly[d(A-T)] fragments under denaturing conditions to prevent substantial hairpin formation, which interferes with the analysis (Simpson & Kiinzler, 1979). DNA fragments from a micrococcal nuclease digestion of chicken erythrocyte chromatin were run on the same gel as a reference. DNA restriction fragments ran highly anomalously on this gel system at the loading concentrations required for detection, and provided no useful information. To estimate repeat lengths by the method of Thomas & Thompson (1977) the midpoints of the chicken erythrocyte bands were taken to be multiples of 204 nucleotides for oligomers greater than dimers. We found that this value described oligomer lengths well when chicken erythrocyte chromatin, digested to a similar extent, was analyzed on 1.5% agarose gels using a 123 base-pair ladder (BRL) for gel calibration. Thus, DNA and poly[d(A-T)] samples

In order to examine how different types of histones might influence the nucleosome spacing periodicity, we isolated core histones (an equimolar mixture) and purified HI (and also erythrocyte H5) from three types of chromatin that differ appreciably in repeat length. These are: HeLa, 188 base-pairs (Compton et al., 1976); chicken erythrocyte, 207 base-pairs (Compton et al., 1976); and sea urchin sperm, 241 to 248 base-pairs (Spadafora et al., 1976; Arceci & Gross, 1980). Figure 1 shows DNA “ladders” produced by micrococcal nuclease digestion for these three native chromatin samples, along with a close-packed ladder produced from core histone-reconstituted polynucleotide. These periodicities span the range observed in nature. The close-packed array shown (about 150 base-pair repeat) was produced using chicken erythrocyte core histones (1.0 pg/pg of polynucleotide). However, the same result is obtained using the core histones from the other two cell types (not shown). Although this urea-containing polyacrylamide gel system is capable of resolving only about seven (single-stranded) multiples of the unit repeat, the repeat length differences here are apparent. For example, the length of DNA contained in the sea urchin sperm 5mer is about the same as that of the chicken erythrocyte 6-mer, as expected since (6 x 207)/5 = 248. Similarly, the length of poly[d(A-T)] extracted from the close-packed 4-mer is about the same as that of DNA from the chicken erythrocyte 3-mer, consistent with (3 x 207)/4 = 155. Moreover, it can be seen at a glance that the HeLa repeat length is substantially longer than that of close-packed nucleosomes, but slightly shorter than that of chicken erythrocyte chromatin. In viewing these gels it should be taken into account that the increase in polynucleotide chain length with decreasing mobility is much greater than logarithmic. For example, a mobility difference of about 2 mm at the position of nucleosome dimers corresponds to about 15 nucleotides, whereas a difference of only 1 mm at the position of nucleosome hexamers corresponds to about 80 nucleotides. Therefore, the casual visual impression that all of the polynucleotide oligomers

A. Stein and M. Mitchell

1034 ce

I

I

ce

D

D D

M

M

M

(b)

D D

M M

(e)

Cd)

Fig. 2.

Origin of Repeat Length Variability in lane 2 seem slightly shorter than the corresponding ones in lane 3 is incorrect. Small mobility differences between the chicken erythrocyte standard and a sample, for higher oligomers, indicate substantial chain length differences that are much greater than what would be produced by differences in trimming by exonuclease activity (No11 & Kornberg, 1977). Apart from the histone type, the other variables t,hat might be expected to influence the repeat length are the Hl/nucleosome ratio, the core histone/polynucleotide ratio, the incubation time for nucleosome alignment, and the NaCl concentration. In all cases we have found that the formation of an ordered array requires that an adequate amount of Hl (or H5) is added to the mixture, but addition of an excess does not change the value of the nucleosome spacing periodicity. Adding more HI than needed results in chromatin complexes t,hat are more resistant to micrococcal nuclease digestion and give higher backgrounds in gel ladders. This behavior is very similar to that of native chromatin to which additional Hl was added (unpublished observations). Thus, in what follows, the appropriate amount of Hl (or H5) that was used will be stated. Additionally, we have found that the nucleosome alignment time for some reactions is slower than for others. For example, reactions involving HeLa Hl and those at very low or very high core histone/DNA ratios proceed completely only when incubated at 37°C for nearly 12 hours, whereas other reactions appear to be complete in two to three hours (Stein & Bina, 1984). Thus, we have used the value of 12 hours for all reactions. The extent of histone proteolysis that occurred after t’he 12 hour incubations was found to be very slight in all cases (not shown). Lastly, the effect.s of the NaCl concentration and the core histone to poly]d(A-T)] ratio are described below.

(b) Assembly of nucleosome arrays using homologous core histone-H 1 combinations Previously (Stein & Bina, 1984). it was shown that chicken erythrocyte histones, core plus H5, can compact, poly[d(A-T)] * poly[d(A-T)] into chromatin-like fibers with nucleosome spacings very similar to those in native chicken erythrocyte chromatin. The nucleosome repeat was found to be insensitive to the reaction parameters examined, suggesting that one or more of the histones (for

1035

example, H5) specified the characteristic chicken erythrocyte repeat. As a logical next step we examined the behavior of the assembly system using two other sources of histones, selected as described above. We found that there were many combinations of parameters that resulted in partial or poor assembly reactions, and that it was necessary to optimize the conditions in each case. The generation of long arrays of physiologically spaced nucleosomes on a large fraction of the polynucleotide should produce a fairly clear MNase ladder, whereas irregularly spaced nucleosomes produce poly[d(A-T)] fragments of non-specific lengths. Thus, optimization involves finding conditions that produce a physiological ladder with the lowest background possible. With chicken erythrocyte histones (core plus H5), the clearest ladder (Fig. 2(a)) was obtained using of 0.15 iwNacl, 0.90 g of core histories/g poly[d(A-T)], and addition of about 2.5 H5 molecules per nucleosome. The spacing periodicity generated on much of the polynucleotide appears indistinguishable from that of native chicken erythrocyte chromatin. Unfortunately, with sea urchin sperm histones a ladder reflecting ordered nucleosome arrays could not be obtained using 0.15 M-NaCl for any core histone/DNA ratio or Hl to nucleosome ratio tested. The nucleoprotein complex formed was generally resistant to digestion (in the 0.15 M-NaClcontaining buffer), and produced predominantly non-specific poly[d(A-T)] fragments and closepacked nucleosome multiples (not shown). However. in 0.04 M-NaCI, 0.60 g of core histories/g of polyCd(A-T)l and about 1.5 Hl molecule per nucleosome, a fairly clear MNase ladder with the characteristic (very long) see urchin sperm spacing periodicity was generated (Fig. 2(b)). The lower NaCl concentration used here was not responsible for the much longer repeat length, because substituting 0.04 M-NaCl for 0.15 M (other parameters unchanged) in the chicken erythrocyte reaction of Figure 2(a) had little effect,, as shown in Figure 2(c). Lowering the core histone/polynucleotide ratio to 0.60, in addition, led to a poor assembly reaction, as evidenced by the high background and small number of multiples in the ladder (Fig. 2(d)). A sea urchin sperm periodicity was not obtained at any H5/nucleosome ratio examined under these conditions. Thus, the sea urchin sperm histone result further supports the hypothesis that one or more of the hist,onrs specifies the repeat length.

Figure 2. Chromatin assembled using homologous combinations of core histones and HI or H5. (a) Chicken erythrocyte (ce) core histones, 0.90 g/g polynucleotide plus H5; 0.15 M-Ku’a(>b The sample was digested for I min using 0% unit MNase/pg of polynucleotide. (b) Sea urchin sperm core histones (0.60 g/g) plus Hl; 0.040 M-NaCl. The sample was digested for 30 s or 1 min (lanes 1 and 2, respect,ively) with 0.10 unit MNase/pg. (c) Chicken erythrocyte core histones (0.90 g/g) plus H5; 0.040 M-NaCl. The sample was digested for 1 min or 1.5 min (lanes 1 and 2, respectively) using 0.25 unit MNase/pg. (d) Chicken erythrocyte core histones (060, g/g) plus H5; 0.040 M-NaCl. The sample was digested 1. 1.5, 2 or 25 min (lanes 1 to 4, respectively) using 0.10 unit MNase/pg. (e) HeLa core histones (0.9, g/g) plus HI; 0.15 M-NaCl. The sample was digested for 1.5 or 2.0 min (lanes 1 and 2, respectively) using 0.15 unit MNase/pg. The positions of the native chicken erythrocyte monomer (M) and dimer (D) are indicated on each gel.

1036

A. Stein and M. Mitchell

.M

M

Figure 3. Chromatin assembled using heterologous combinations of core histones and Hl or H5. (a) Chicken erythrocyte (ce) core histones, 0.75 g/g polynucleotide plus sea urchin Hl, 1.0 molecule added per nucleosome; 0.040 MNaCl. The sample was digested for 1 min using 0.10 unit MNase/mg of polynucleotide. (b) Sea urchin sperm core histones (0.60, g/g) plus chicken erythrocyte H5, 2.0 molecules added/nucleosome; 0.040 M-NaCl. The sample was digested for 1 min or 1.5 min (lanes 1 and 2, respectively) using 0.10 unit MNase/pg. The positions of the native chicken erythrocyte

monomer

(M) and dimer

(D) are indicated

on each gel.

Despite the consistency of these findings, the results using HeLa histones were not in accord with

this simple hypothesis. The clearest physiological ladder, obtained using 0.15 M-NaCl, 0.90 g of core histories/g of [d(A-T)], and about six molecules of Hl per nucleosome, was similar to that of chicken erythrocyte chromatin (Fig. 2(e)); the repeat length was clearly longer than that of native HeLa chromatin (Fig. 1). Thorough investigation of the effect of the amount of Hl added showed that, as with sea urchin Hl and with chicken er:ythrocyte H5, the value of spacing the periodmlty was insensitive to this parameter (although enough Hl must be added to induce nucleosome alignment). Thus, we were left with the question of how the short physiological nucleosome spacing periodicity in HeLa cells is generated. (c) Chromatin assembly using heterologous histone-HI combinations

core

We next examined the chromatin assembled from heterologous core histone-H 1 combinations, again under optimal conditions. Figure 3(a) shows that chicken erythrocyte core histones in conjunction with sea urchin sperm Hl clearly gave a very long spacing

periodicity

similar

to that

of sea urchin

sperm chromatin. Note that the poly[d(A-T)] length contained in the 6-mer for this assembled chromatin is about the same as the DNA length contained in the 7-mer from lightly digested native chicken erythrocyte chromatin. Figure 3(b) shows the reciprocal experiment, sea urchin sperm core histones in combination with chicken erythrocyte H5. Two things seem significant here. First, this reaction proceeded very strongly in 0.04 M-NaCl at the low core histone/poly[d(A-T)] weight ratio of 0.60; a very poor ladder was obtained upon assembly and digestion in 0.15 M-NaCl (not shown). Recall that the homologous chicken erythrocyte 094 M-Naci proceeded poorly reaction, at (Fig. 2(d)). Second, although some of the chromatin assembled appears to have had the chicken erythrocyte repeat length, analyses of gels (not shown) revealed that some of the chromatin had a longer repeat length, approaching that of sea urchin sperm at low extents of digestion. For a mixture of nucleosome arrays with different repeat lengths, longer

repeats

might

be preferentially

detected

at

early digestion times if longer linkers are more frequently cleaved than shorter ones. These experiments show that chromatin assembly with heterologous histones is possible. We have, in fact, obtained physiological nucleosome arrays

Origin

of Repeat Length

1037

Variability

Table 1 Summary

(‘ore histonr typct

HI histone type

Core histone/ polynucleotide ratios yielding periodic arrays

(‘E. HL (‘Fd_ HI 1 (‘IS. HI 1 SW SLB SF’S

HL, CE Hl CE H5 HUS HL CE H5 sus

04x- 1.o 0.45-1.2 0.45-l .o 0.55-0.65 0.45-0.65 0.4550.65

of results

Observed repeat length range (zk 10 WS 19op 220 170->230 200- 240 2W- 215 20& 230 230- 240

Array formation at low core histone/polvnucleotide ratios

0.15M-NC&l + -

044

M-N&l

+ + + +

t Histonr types CE, HL and SUS denote chicken erythrocyte, HeLa and sea urchin sperm. respectively. $ The quality of the gel pattern and the apparent repeat length, in some cases, was a function of the extent of nuclease digestion, in addition to the core histone/poIynucleotide ratio; bp, base-pairs,

using all of the possible combinations of core and Hl (H5) histones from the three cell types. These experiments are summarized in Table 1. (d) Different HI-types differ in their ability recruit nucleosomes into ordered arrays

to

In most of the experiments described thus far we had optimized conditions to obtain the best alignment reaction possible. Here, we examine the relative efficiencies of the Hl-types in generating an ordered physiological array at the low average core histonejpolynucleotide ratio of 0.60 (g/g). At this nucleosomes appear to be initially low ratio, randomly distributed along the polynucleotide, and of naked polynucleotide exist. many regions Because heterologous Hl-core histone combinations permit nucleosome alignment, we have used the same sample of polynucleotide reconstituted with chicken erythrocyte core histones to test the Hl types. In this way effects due to experimental error in the core histone/polynucleotide ratio could be eliminated; the histone Hl-type was the only variable. The results of this experiment are shown in Figure 4. Sea urchin sperm Hl efficiently recruits nucleosomes into ordered arrays (Fig. 4(a)). Ladders generated over a range of digestion times have a sea urchin-like repeat. Chicken H5, under the same conditions (O-04 M-NaCl) appears to recruit nucleosomes into arrays less efficiently (Fig. 2(d)), as evidenced by the higher background in the ladder. However, at 0.15 iw-NaCl, where native chromatin fibers are more highly compacted than in 0.04 MNaCl (Thoma et aZ., 1979; Butler & Thomas, 1980, and references cited therein; Woodcock et al., 1984), and the attractive forces between nucleosomes should be stronger, more bands can be resolved, and the background is lower (Fig. 4(b)). Also, analysis of this gel indicated that the chromatin in this sample had a slightly longer repeat length than native chicken erythrocyte chromatin. Finally, the arrays generated using HeLa Hl, even at 0.15 iv-NaCl shorter than for (Fig. 4(c)), were significantly chicken H5. Long arrays (long ladder with low

backgrounds) were only obtained with HeLa Hl at higher core histone/polynucleotide ratios. Figure 4(d), for example, shows that HeLa Hl with chicken erythrocyte core histones at 0.90 g/g polynucleotide gives a longer ladder and lower background. These results indicate that the ability to recruit nucleosomes into ordered arrays, is much greater for H 1 from sea urchin sperm than for HI from HeLa cells. Chicken H5 has intermediate properties, whereas, chicken erythrocyte Hl has properties very similar to those of HeLa Hl (not shown).

(e) The initial nucleosome packing density affects the value of the nucleosome spacing periodicity

strongly

We next investigated to what extent the repeat length depends upon the number of nucleosomes initially deposited per unit length of polynucleotide. This nucleosome packing density is dependent upon the core histone/DNA ratio used in the reconstitution step of the reaction. Although reconstituted samples are probably very heterogeneous in nucleosome density, making it difficult to determine the effect of a particular value of this parameter, the effect of the average core histone/ polynucleotide ratio on the nucleosome spacing periodicity is dramatic. At the fairly low average core histone/polynucleotide ratio of 0.60 (Fig. 4(b)), the spacing periodicity with chicken H5 was slightly longer than at the ratio of 0.90, which was indistinguishable from that of native chicken erythrocyte chromatin (Fig. 2(a)), suggesting that the repeat increases length with decreasing nucleosome density. Of course, when the nucleosome density is decreased further much of the polynucleotide will be nucleosome-free, and it will be more difficult to detect arrays that form because the background from naked polynucleotide regions will be high. Most of the arrays that do form, however, should be free of boundary constraints such as polynucleotide ends or neighboring arrays that’ nucleated

1038

A. Stein and M. Mitchell

(b)

D-

M-M

Cd)

(cl

Fig. 4.

Origin of Repeat Length Variability

1039

M

(b)

Figure 5. Dependence using

chicken

H5/nucleosome polynucleotide

erythrocyte

of the spacing periodicity on t’he initial nucleosome packing density for chromatin assembled (ce) H5. (a) Very low packing density of 0.45 g HeLa core histories/g polynurleotide. The

molar ratio was 2.0 and the NaCl concentration for 1 min. (b) Very

high packing

density

was 0.15

of 1.2 g chicken

M.

Digestion

erythrocyte

was with 0.10 unit MNase/pg

core histories/g

polynucleotide.

The

H5/nucleosome molar ratio was 1.8, and the NaCl concentration was 0.040 M. Digestion was with 0.30 unit MNase/pg for 1.5 or 2.0 min (lanes 1 and 2. respectively). The native chicken erythrocyte monomer (M) and dimer (I)) are indicated on each gel.

independently. Figure 5(a) shows a ladder from chromatin assembled from polynucleotide and HeLa core histones plus H5 at the lowest average core histone/polynucleotide ratio that we have been &le to achieve (O-45). The spacing periodicity was quite long, and similar to the chromatin that’ we assembled using sea urchin sperm Hl (Figs 3(a) or 4(a)). At lower extents of digestion the spacing periodicity was even slightly longer, but t,he background was very high, as expected. At the ot,her extreme of high core histone/ polynucleot~ide ratios, boundary constraints should be exerted. For example, a uniform 200 base-pair nucleosome spacing is not possible for core histone/ polynucleotide weight ratios greater than 0.80. Figure 5(b) shows ladders obtained at the highest average ratio that would allow detection of a periodic array (1.2), using chicken erythrocyte core

histones plus H5. Although the background is high, due to both heterogeneity in the nucleosome density and nucleosome crowding in general, the existence of arrays with a spacing periodicity similar to native HeLa chromatin is evident (compare Fig. 5(b), lane 1 with Fig. 1. lane 2; each relative to chicken erythrocyte chromatin). More extensive digestion of this sample (lane 2) reveals the presence of some molecules containing closely packed nucleosomes, as expected at’ such a high average ratio. These experiments show that a single type of Hl histone, chicken H5, can induce nucleosomes to form physiological arrays with nearly the full range of spacing periodicities found in nature. The value of the periodicity obtained depends upon the initial nucleosome packing density. To examine whether other Hl histones behave in a similar way, we

Figure 4. Ability of Hl-types to recruit nucleosomes into ordered arrays. Polynucleotide reconstituted wit,h 0.60 g chicken erythrocyte (ce) core histone/g was incubated with (a) sea urchin sperm Hl (1.5 moles added/mol nucleosomes) at 0.04 M-NaCI, and digested with 0.20 unit MNase/pg polynucleotide for 30 s. (b) Chicken erythrocyte H5 (2.5 moles added/mol) at 0.15 M-NaCl, and digested with 0.10 unit MNase/mg for 25 min. (c) HeLa Hl (6 mol/mol) at 0.15 M-N&l, and digested with 0.10 unit MNase/mg for 1, 1.5, 2, 2.5 min in lanes 1 to 4, respectively. (d) Polynucleotide reconstituted with 0.90 g chicken erythrocyte core histories/g was incubated with HeLa Hl (5 mol added/mole nucleosomes) at 0.15 MNaCl. and digested with O,I unit MNase/mg for 2.5 min. The positions of the native chicken erythrocyte monomer (M) and dimer (D) are indicated on each gel.

1040

A. Stein and M. Mitchell

D

:3

proportions of all of these species are clearly a function of the average core histone/polynucleotide ratio (not shown). HeLa Hl (as shown above) and also chicken erythrocyte Hl (not shown) do not recruit nucleosomes into arrays at low initial nucleosome packing densities. Therefore, very long repeat lengths cannot be obtained with these Hl types. At high average core histone/polynucleotide ratios (around 1.0) we have not been able to generate to those of nat,ive HeLa ladders equivalent but a significant shortening of the chromatin, repeat can be observed (not, shown). Due to the heterogeneity in the sample, and the low efficiency of array formation with these Hl types, the difficulties in generating, exclusively, a HeLa-like repeat in our system are understandable. Table 1 provides a summary of all our results.

Sl

4. Discussion M

Figure 6. Chromatin assembled at high nucleosome packing densities using chicken erythrocyte (ce) core histones in combination with sea urchin sperm Hl. The H 1/nucleosome molar ratio was 1.0, and the core histone/ polgnucleotide weight ratio was 1.0; 0.040 M-NaCl. The sample was digested with 0.32 unit MNase/pg of polynucleotide for 1. 2 or 3 min (lanes 1 to 3. respectively),

examined the other types of Hl histones at extreme values of the nucleosome density. Sea urchin Hl is the most effective in recruiting nucleosomes into arrays at low initial packing densities. Repeat lengths as long as native sea urchin sperm chromatin are the longest that we have been able to observe upon lowering the core histone/polynucleotide ratio. For sea urchin Hl in combination with chicken erythrocyte (or HeLa) a dramatic and fairly uniform core histones, decrease in nucleosome spacing with increasing core histone/polynucleotide ratio was average observed. Figure 6 shows that a periodicity very similar to native chicken erythrocyte chromatin occurs in some of the assembled chromatin at a ratio of 1.0, where boundary constraints due to the polynucleotide ends should exist. The effects of heterogeneity in the core histone/polynucleotide ratio on different molecules, and probably, intramolecular crowding as well, are evident. Sea urchin sperm-like bands corresponding to monomer, dimer and trimer (Sl , S2 and 53) arising from low nucleosome densities can be seen, as well as close packed dinucleosome (CD) and core particle (CP), arising from high nucleosome densities. The relative

We have thoroughly examined how different nucleosome spacing periodicities in the physiological range can be generated by assembling poly[d(A-T)] . poly[d(A-T)] into chromatin-like structures in vitro. In the absence of DNA base sequence effects, which complicate such an analysis, we were able to deduce which other factors influence the repeat length. Because spacing periodicities spanning the physiological range could be achieved, particular DNA base sequences or structural signals (Drew & Calladine, 1987; Travers & Klug, 1987) should probably be viewed as a modulating influence, rather than a requirement for a particular nucleosome spacing periodicity. Our data indicate that histone Hl is necessary t’o generate an ordered nucleosome array with nonzero linker lengths, but that particular Hl types do not directly determine the repeat length. In the absence of Hl, nucleosome close-packing was the only periodicity observed (about 150 base-pairs). This result held true for all three types of core histones we used, under all conditions examined. For samples that were incubated (12 h) with Hl (or H5), the nucleosome spacing periodicity obtained was a function of the initial average nucleosome density of the polynucleotide. This property of the assembly system was overlooked in our initial study using chicken erythrocyte core plus H5 histones (Stein & Bina, 1984) for two reasons. First, our samples are very heterogeneous in nucleosome density, making it difficult to observe the effects of varying the core histone/polynucleotide ratio. Second, the shorter incubation times (2 to 3 h) used were not sufficient for nucleosome alignment at extreme values of the nucleosome density, and poor reactions were obtained. Because of the heterogeneity in nucleosome density in our samples, we cannot deduce the precise functional relationship between this parameter and the repeat length. Much of the chromatin in samples assembled at core histone/polynucleotide ratios between O+!O and 1-O (g/g) possessed a typical

Origin

of Repeat Length

approximately 200 base-pair repeat, irrespective of the Hl type used (Figs 2(a), (c), (e), 4(d) and 6). This result is consistent with the generation of a uniform spacing of nucleosomes throughout the polynucleotide. Figure 7 (continuous curve) shows the calculated dependence of the repeat length on the core histone/DNA ratio for a uniform distribution of nucleosomes. Our data are in fairly close agreement with this plot, when the expected effects of sample heterogeneity are kept in mind, except at very low core histone/DNA ratios, where the value of the repeat plateaued at around 250 base-pairs (represented by a broken extension of the curve). Since 250 base-pairs is also the longest repeat that has been found in nature, this coincidence suggests that this value corresponds to the longest repeat length that is compatible with the 300 A fiber the physiological curve (1 A = 0.1 nm). Clearly, must plateau at some low value of the core histone/ DNA ratio, because the continuous curve diverges to infinity. Although the spacing periodicity is determined by the nucleosome packing density, different Hl types require different nucleosome densities to nucleate arrays. We found that sea urchin sperm Hl is capable of nucleating fairly long arrays at low nucleosome densities. HeLa and chicken erythrocyte HI require high nucleosome densities. Chicken H5 is an int’ermediate case. Similarly, sea urchin sperm core histones, which contain a longer and more basic variant of H2R (Strickland et al., 1977a,b), appear to be significantly more efficient than typical core histones (chicken erythrocyte and HeLa) in forming arrays at low nucleosome densities. This phenomenon is very apparent when nucleosome alignment reactions using chicken H5 with either sea urchin sperm or chicken erythrocyte core histones are compared under the same conditions (Figs 2(d) and 3(b)). The requirement for a critical nucleosome density in order for a particular Hl-type to nucleate a long stable array suggests why different cell types and repeat lengths in bulk species have different chromatin. The generation of a long ordered array should be accompanied by the formation of the 300 a fiber. If we assume that core histone deposition on replicating DNA continues until all of the DKA is folded into the 300 .& fiber, then the repeat length generated should correlate with the nucleosome density requirement for Hl (or an unusual core histone variant). Thus, HeLa Hl, with a high density requirement, should result in core histone deposition until a density corresponding to about 0.90 g/g of newly replicated DNA is attained. Only then can the 300 A fiber be formed; the repeat length would be short. In contrast, sea urchin sperm Hl allows the 300 a fiber to form at low nucleosome densities, before the spacing of the close-packed and randomly arranged nucleosomes becomes constrained by boundaries. This chromatin would have a long repeat length. Hl types that have int’ermediate density requirements should form 300 ,& fibers at intermediate nucleosome

1041

Variability I 380

I

I

I

I

-

Core histoneslpolynucleotide

(g/g)

Figure 7. Calculated dependence of the repeat length on the core histone/polynucleotide weight ratio for a uniform distribution of nucleosomes. The equation of the curve is y = (110,000/660)( l/2), where y is the ordinate, 5 the abscissa, 110,000 is taken as the molecular weight of the core histone octamer, and 660 is taken as the molecular weight of a base-pair (bp). The repeat length values of sea urchin sperm (SUS), chicken erythrocyte (CE), and HeLa, in addition to that of close-packed nucleosomes (Close PKD) are indicated.

densities with intermediate values of the repeat length, generally around 200 base-pairs. The ability of any Hl-type to form 300 A fibers over a range of nucleosome densities suggests that linker length heterogeneity (Prune11 & Kornberg, 1978; Strauss & Prunell, 1982) might reflect heterogeneity in nucleosome density. For example, a 5000 base-pair length of DNA containing 25 nucleosomes, uniformly distributed, should have a repeat length of 200 base-pairs, whereas for 27 nucleosomes, the repeat length should be 185 basepairs. Recently, evidence has been obtained indicating that simian virus 40 (SV40) chromatin is heterogeneous in nucleosome density, with variations as large as eight nucleosomes/molecule

1042

A. Stein and M. Mitchell

(Ambrose et al., 1987). Because of the heterogeneity in nucleosome packing density in our system, we are not able to determine whether the nucleosome linkers within any particular 300 A fiber are heterogeneous in length. It is interesting, nonetheless, that arrays formed using sea urchin sperm Hl (Fig. 3(a)) or core histones (Fig. 3(b)) give the sharpest bands in ladders. Nucleosome spacing in these arrays should be least constrained by boundaries (because arrays form efficiently at low densities), and therefore, least subject to the (differential crowding) effects of density heterogeneity. This result suggests that linkers, in part,icular 300 A fibers, might be fairly homogeneous. DNA base sequence effects, of course, might contribute to linker heterogeneity. The physical basis for the dependence of the repeat length upon nucleosome density is not yet, known but external forces are clearly implicated. We cannot, say whether the value of the overall average nucleosome spacing increase, induced by HI, is restricted solely by the presence of polynucleotide nucleated ends. Independently arrays of sufficient length and stability could also serve as boundaries. Some of the electron micrographs obtained in our initial study (Stein & Bina, 1974) support the latter hypothesis. Polynucleot,ide tails emanating from some 300 a fibers were observed for chromatin with an average core histone/polynucleotide weight ratio of 0.75. Thus, nuclrosome spacing in those particular arrays was not constrained by the presence of both polynucleotide ends. The failure of sea urchin sperm Hl to induce uniform ordering of nucleosomes containing sea urchin sperm core histones! at, high nucleosome densities (Table l)? suggests that very stable independently nucleated arrays, in this case, form boundaries. In any event, a short repeat’ length may imply a nucleosome spacing constraint by a boundary at the end of an array because crowding appears to be necessary to generate short’ repeat lengths. The physical basis for the critical density effect could be the effectiveness of a particular Hl type in reducing the negative charge on the nucleosome. Charge reduction could facilitate the nucleation of a sufficient number of an array by allowing nucleosomes to approach each other closely enough for a sufficient length of time for a higher-order structure to form. Those Hl types that require higher salt concentrations for elution should be the ones that are more effective at, charge neutralization. For example. sea urchin sperm Hl requires a higher salt concentration for elution than HeLa HI (Materials and Methods). Thus, sea urchin sperm Hl -nucleosome interactions may result’ in enough charge neutralization for spontaneous formation of the 300 A fiber, whereas HeLa Hl requires some degree of external constraint to overcome repulsive forces, at least for nucleation. Also, consistent with this hypothesis is our finding that chicken H5 nucleates arrays much more efficiently at 0.15 MNaCl t’han at 0.04 M-Nacl. At O-04 i%r-NaCl, native

chromatin fibers are not fully compacted, presumably due to a lesser degree of charge neutralization than at 0.15 M-Nacl (Thoma et al., 1979; Butler & Thomas, 1980; Woodcock et al., 1984). It is plausible that both the dependence of the repeat length on nucleosome density and the critical density effect are cellular mechanisms for controlling gene expression. One possibility is that the local nucleosome density could be dependent upon base sequence effects. Thus, even if nucleosomes in the 300 A fiber are not uniquely positioned, preferred base sequences could conceivably lead to a programmed variability in the initial deposition of core histones on newly replicated DNA. This initial density variation could control nucleation of the 300 1\ fiber in specific regions of the genome. For example, a local high density region might become preferentially associated with a particular Hl subtype that requires a high critical density. Alternatively, a local low density region might not) be able to fold into a cannonieal 300 il fiber. This mechanism could possibly give rise to local repeat length variations, which have been observed in chromatin regions containing particular genes (Gottesfeld, 1980; Gottschling et al., 1983; Smith et al.. 1903; Benezra et aE., 1986). The presence of control proteins on DNA in particular cell types might also influence the density of nucleosomes deposited in particular regions of the genomr. The inter-relationships among factors such as the repeat length value, the presence of DNA-bound non-histone proteins, Hl subt,ypes, and DNA base could be of sequence effects on histones in determining the fundamental importance chromatin structure of particular genes in different cell t,ypes. 1Ve thank Mr James Lauderdale and Dr Minou Bins for helpful discussion. This work was supported by l%PHS grant GM27719 to AX References Ajiro.

K.. Borun, T. W. & Cohen, L. H. (1981).

Biochemistry,

20, 1445-1454.

Allan, .J.. Hartman, P. C., Crane-Robinson, (1. & Aviles. F. X. ( 1980). Nature (London), 288, 675-679. Ambrose. C.. McLaughlin, R. & Bina, M. (1987). AV,uc1. Acids

Res. 15, 3703-3721.

Arrrci. R. J. & Gross, P. R. (1980). Develop. Biol. 80. 186-209. Benezra. R., (Cantor. C. R. & Axel, R. (1986). Call, 44. 697-704. Brand, S. H., Kumar, N. M. bt Walker. 1. 0. (1981). FlCBS Letters, 133, 63-66.

Briand, G.. Kmiecik. D., Sautiere, P., Wouters. D.. BorieLoy. 0.. Biserte, G., Mazen, A. & Champagne, M. (1980). FEBS Letters, 112, 147-151.

Butler, P. *J.G. & Thomas, J. 0. (1980). J. Mol. Biol. 140, m-529.

Compton. *J. L., Bellard, M. & Chambon, E’. (1976). hoc. *Vat. Acad. Sci., 7.l.S.A. 73, 4382-4386.

D’Anna, J. A., *Jr & Isenberg, I. (1974). Biochemistry, 13. 4992 4997.

Drew. H. R. & Calladine, C. R. (1987). J. Mol. Biol. 195, 143-173.

Origin

of Repeat Length

Eissenberg, ,J. C., Cartwright, I. L., Thomas, G. H. & Elgin, S. C. R. (1985). Annu. Rev. &net. 19, 485k536. Gottesfeld, ,J. M. (1980). Nucl. Acids Res. 8, 9055921. Gottschling, D. E., Palen, T. E. & Cech, T. R. (1983). .Vucl. Acids Res. 11, 209332109. Hymer. C. W. & Kuff, E. L. (1964). J. Histochem. Cytochem. 12, 359-363. Inman. R. B. & Baldwin, R. L. (1962). J. Mol. Biol. 5. 172-184. Johns. E. W. (1971). In Histones and Nucleohistones (Phillips. I). M. P.. ed.), p. 37, Plenum Press, London. Keene. M. A. & Elgin, S. (‘. R. (1984). Cell, 36, 121-129. Kinkade, ,J. M.. Jr & Cole. R. D. (1966). J. Biol. Chem.

241, .5790-5797. Kornberg. R. 1). (1977). Annu. Rev. Biochem. 46. 931-954. Kornbrrg, R. (1981). ,Vature (London), 292, 579-580. Laskey. R. A. & Earnshaw. W. C. (1980). Nature (London), 286. 763-767. Lennox. R. FV. & Cohen, I,. H. (1983). J. Biol. Chem. 258. 2622268. Maniatis, T. & Efstratiadis, A. (1980). Methods Enzywbol. 65, 299-305. M&her, .J. D. &, Felsenfeld, G. (1980). Annu. Rev. Biochrm. 49. 1115-l 156. Mmgeritsky. G. & Trifonov, E. N. (1984). Cell Biophys. 6. I-X. Morris, N. R. (1976). Cell, 8, 3577363. Koll. M. (1976). CelZ, 8. 349-355. 109, h-011, M. & Kornberg, R. (1977). J. Mol. Biol. 393 404. Pearson. E. (‘.. Butler, I’. J. (:. & Thomas, J. 0. (1983). EMBO .I. 2. 1367-1372. Pearson. E. C.: Bates, D. L., Prospero, T. D. & Thomas. .J. 0. (1984). Eur. J. Biochcm. 144, 353-360.

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Prunell, A. & Kornberg, R. D. (1978). Cold Spring Harbor Symp. Quant. Biol. 42, 103-108. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg. P. (1977). J. Mol. Biol. 113, 237-251. Ruiz-Carrillo, A., Wangh, L. J. & Allfrey. V. (:. (1975). Science, 190, 117-128. Setlow, P., Brutlag, D. & Kornberg, A. (1972). .I. Biol. Chem. 247, 224-231. Simpson, R. T. (1986). BioEssays 4, 1722176. Simpson. R. T. &, Kiinzler. P. (1979). NucZ. Acids Res. 6, 1387-1415. Smith, R. I)., Scale. R. L. & Yu, J. (1983). t’roc. ,Vat. Acad. Sci., C.S.A. 80, 5505-5509. Spadafora, C., Bellard, M., Compton, J. 1,. & (‘hambon, P. (1976). FEBS Letters, 69, 281-285. Stein, A. (1989). Methods Enzymol.. 170, in the press. Stein, A. & Bina, M. (1984). J. Mol. Biol. 178. 34 1-363. Stein, A. & Page. D. (1980). J. Hiol. Chews. 255, 3629-3637. Strauss, F. & Prunell, A. (1982). Nucl. Acids Res. 10,

2275-2293. Strickland, M., Strickland, W. N., Brandt, W. F. & van Holt, C. (1977a). Eur. J. Biochem. 77. 263-275. Strickland. W. K.. Strickland, M., Brandt, W. F. & van Holt, C. (19796). Eur. J. Biochem. 77, 277?286. Thoma. F., Keller. T. H. & Klug, A. (1979). J. Cell Biol. 83, 403-427. Thomas, J. 0. & Thompson. R. ,J. (1977). Cell, 10, 633-640. Travrrs. A. A. & Klug, A. (1987). Phil. Trans. Roy. Sot.

ser. H, 317, 537-561. Woodcock, C. L. F., Frado, L.-L. Y. 8: Rattner. ,J. B. (1984). J. Cell Biol. 99, 42-52. Yaguchi, M.. Roy. (1. & Seligy, V. L. (1979). Biochem. Riophys. Res. Commun. 90, 1400-1406.

Edited by A. Klug