Sea urchin sperm chromatin structure as probed by pancreatic DNase I: Evidence for a novel cutting periodicity

DEVELOPMENTAL

BIOLOGY

80,210-224

(1980)

Sea Urchin Sperm Chromatin Structure as Probed by Pancreatic DNase I: Evidence for a Novel Cutting Periodicity ROBERT J. ARCECI AND PAUL R. GROSS~ Department of Biology, University of Rochester, Rochester, New York 14627, and Marine Biological Laboratory Woods Hole, Massachusetts 02543 Received February 1, 1980; accepted April 21, 1980 Chromatin from mature sea urchin spermatozoa is highly compacted and composed almost entirely of DNA and the five histones, although sperm Hl, H2A, and H2B histones differ from those found in embryo or somatic cell nuclei. Release of acid-soluble DNA during pancreatic DNase I digestion is 20-fold slower from sperm nuclei than from embryonic nuclei. Following DNase I digestion, most sperm nuclear DNA remains at high molecular weight, although there appears to be some release of 10 base oligomer fragments. Size analysis of the higher molecular weight DNA reveals a series of fragments that indicate a cutting periodicity of approximately 500 base pairs. This pattern remains when electrophoretic separation is carried out under denaturing conditions. The 500 base pair cleavage pattern was not detected in digestions of embryonic nuclei. Nucleosomes reconstituted with fractionated core histones from sperm gave, upon digestion, a characteristic 10 base “ladder,” with no resistant high molecular weight DNA. Micrococcal nuclease and DNase II digested sperm nuclei to produce DNA fragments with a calculated repeat length of 248 + 3 and 246 + 6 base pairs, respectively. The structural basis for the 500 base pair cutting periodicity in sperm nuclei may reside in the unique sperm Hl histone. INTRODUCTION

A great deal of evidence now supports the view, first proposed by Kornberg (1974), that most chromatins are organized as chains of repeating units, “nucleosomes,” which consist of approximately 200 base pairs of DNA wound externally on an octamer of the histones H3, H4, H2A, and H2B (reviews: Kornberg, 1977; Felsenfeld, 1978). Histone Hl appears to interact primarily with the DNA connecting nucleosomes, “linker” DNA (Van Holde et al., 1974; Varshavsky et al., 1976; Whitlock and Simpson, 1976; Nell and Kornberg, 1977). The mature spermatozoan has an extremely condensed, transcriptionally inert nucleus, which contains all five histone fractions. Sperm Hl, H2A, and H2B are clearly different from those of the embryo or somatic tissue (Ozaki, 1971; Ea.&on and Chalkley, 1972; Ruiz-Carrillo and Palau, 1973; Strickland et al., 1974, 1977a,b, 1978; Brandt et al., 1979; Geraci et al., 1979). It 210 001%1606/80/130210-15$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

is not known whether the sperm has unique H3 and H4 histones as well. As probed by micrococcal nuclease, the sperm nucleus has a basic nucleosomal repeat unit of 240 to 260 base pairs. (Spadafora et al., 1976; Keichline and Wasserman, 1977). Extended digestion with micrococcal nuclease proceeds with a pause at 160 base pairs, then at about 145 base.pairs, and to a limit digest similar to that from sea urchin embryos and rat liver nuclei (Spadafora et al., 1976; Keichline and Wasserman, 1979). Electron microscopic studies confirm that sperm chromatin, like chromatin from embryonic and somatic nuclei, is composed of chains of nucleosomes (Spadafora et al., 1976). Taken together, the condensed nature of this histone-containing nucleus, the frequently proposed relationship between ’ To whom correspondence and requests for reprints should be addressed at Marine Biological Laboratory, Woods Hole, Mass. 02543.

ARCECI AND GROSS

Sea Urchin Sperm Chromatin Structure

211

chromatin condensation and decreased ml and the enzyme at 100 units/ml. The transcriptional activity, and the demon- reaction was quenched in timed aliquots by strated ability of DNase I to digest active the addition of 0.1 vol 0.5 M Tris, pH 8.5, or once active chromatin preferentially 0.17 M EDTA, and the sample immediately (Weintraub and Groudine, 1976; Garel and quick-frozen as described above. Nucleosome reconstitution and electron Axel, 1976), justified a study of sea urchin sperm nuclei in response to DNase I. We microscopy. Fractionated histones were rereport here that the reaction does have constituted with calf thymus DNA (Sigma; novel features, including cleavage of the reextracted with phenol and chloroformDNA with a long periodicity of some 500 isoamyl alcohol) by the step-wise salt dilubase pairs. tion method of Germond et al. (1976). Total core histone (H3, H4, H2a, and H2b) and MATERIALS AND METHODS DNA were combined initially at equal conCollection of gametes from Lytechinus centrations (about 2 mg/ml.). pictus, isolation of nuclei, DNase I and Following incubation, the mixture was micrococcal nuclease digestions, quantitadialyzed for 9 hr (three changes) against tive analysis of acid-soluble DNA, DNA DNase I digestion buffer (10 mM Tris, pH isolation and electrophoretic separation, 7.4, 10 mM NaCl, 0.25 M sucrose, 3 mM DNA fragment size and nuclease cutting MgCL2,0.1 mM PMSF). The dialyzed mixperiodicity measurements, and histone iso- ture was then digested at 37°C with DNase lations were carried out as described in the I at 1 pg/ml. Aliquots were removed, the previous report (Arceci and Gross, 1980). reaction was quenched, the DNA was exHistone fractionation and electrophore- tracted with phenol and chloroform-isosis. Sperm histone fractions were separated amyl alcohol, and finally precipitated with by their differential solubilities essentially ethanol before dissolving in sample buffer according to the procedure of Oliver et al. for electrophoresis. (1972), the only modification being a 30- to Formvar-coated copper grids (300 mesh) 60-min period allowed for the precipitations were carbon coated at 2 x 10e5 Torr by which gave increased yields. We started the heating at 0.125-in. diameter presharpened fractionation routinely with 50 mg of total carbon rod (Fullam Inc.) with just enough histone. current to produce sparking. Following the Sperm histone and total sperm nuclear carbonizing, grids were given a temporary proteins were separated by electrophoresis hydrophilic charge by alternating current on 12.5%polyacrylamide slab gels (11 or 15 glow discharge (at 100 V for 3 min in a lOOcm long) containing SDS at 30-mA con- mTorr atmosphere). stant current, according to Laemmli (1970). An aliquot (20 to 40 ~1) of the reconstiIn the case of total nuclear proteins, pel- tuted nucleohistone (about 5 pg/ml) was leted nuclei were dissolved in the final sam- applied directly to freshly charged grids. ple buffer, heated at 95°C for 4 min, and After 2 min, excess liquid was drained onto then applied to the gel. The gels were filter paper and the grid floated face down stained with Coomassie brilliant blue (Fair- on two successive drops of 0.25 M NaCl(15 banks et al., 1971) and photographed set each). The preparations were then through a yellow filter. stained for 30 set in 0.2% uranyl acetate in DNase II digestion of nuclei. DNase II water. After removal of excess fluid, the (Worthington, HDAC: EC 3.1.22.1) diges- grids were air-dried. They were then rotarytions were performed at 37°C with contin- shadowed at a 7’ angle with platinum/paluous stirring in 10 mM Tris, pH 7.0, 0.1 ladium (7-cm-long wire: 0.008-in. diameter; mM PMSF (Altenburger et al., 1976). DNA Pt/Pd 80/20; Fullam) at 2 x lop5 Torr. was present at a concentration of 0.5 mg/ Grids were examined at 50 kV with an

212

DEVELOPMENTAL BIOLOGY VOLUMESO,1980

RCA EMU-3 electron microscope. Magnification was calculated from photographs of a shadowed replica of a 28,800 lines per inch diffraction grating (Fullam).

a first-order reaction, determined by a least-square fitting program (Pearson et al., 1977). In the case of the blastula nuclei, a two-component function gave the lowest GOF (goodness of fit) value (0.0095), i.e., RESULTS the best fit. The first component could be Kinetics of DNase I Digestion of Embryassigned to a 30.9 + 3.1% fraction (not onic and Sperm Nuclei normalized) with a rate constant of 0.507 f 0.116 min-‘. The second and slower comFigure 1, top, shows the release of acidponent was for a 47.9 + 2.8% fraction, with soluble DNA products by DNase I from a rate constant of 0.049 f 0.006 min-‘. The sperm and blastula nuclei under identical terminal value of the reaction was esticonditions, but in separate reactions. Solid mated at 79.4 f 1.0%. lines are drawn to the best fit functions for For the sperm nuclei data, a single component fit was as good as a two-component fit: the rate constant was 0.026 + 0.003 60 min-‘, with an estimated terminal value of 37.1 +- 2.0% and a G.O.F. of 0.0114. To determine whether the kinetics of digestion were intrinsic to each type of nucleus, the following experiment was performed. Blastula nuclei, labeled with [3H]thymidine, were digested in the same reac20 tion with sperm nuclei, which were present at lOO-fold excess (Fig. 1, bottom). For the 0 0 20 40 60 00 loo blastula nuclei, a fast component fraction TIME(MIN1 of 35.3 + 4.0%, with rate constant of 0.11 + 0.164-l, was derived from the data. The slow component represented a fraction of 48.8 + 3.9%, with a rate constant of 0.034 T-----l f 0.005 min-‘; GOF was 0.0145. In the case of the sperm nuclei, a rate constant of 0.025 f 0.002 min-’ and an extent of reaction of 38.0 + 0.9% were obtained; GOF was 0.0096. Thus whether digested separately or together, the relative differences in the rates of acid-soluble DNA production from sperm and blastula nuclei remain. There is clearly no contaminant of one or the other preparation responsible. We cannot be certain whether the obTIYEWINI served kinetic difference is due to actual FIG. 1. Time course of pancreatic DNase I digesrate differences or to differential substrate tion. Top: Nuclei were digested separately at 37°C with 20 pg DNase I/ml and at 1 mg DNA/ml. A, accessibility for the two kinds of nuclei. The Blastula; 0, sperm. Bottom: Nuclei were digested tofact, however, that there is a 20-fold differgether, as described above, with sperm nuclei present ence in the rate constants and only a 2-fold at lOO-fold excess over blastula nuclei. The latter had difference in the calculated final fractions been labeled fo 90 min with 5 pCi/ml [3H]thymidine digested suggests that accessibility differ(Sp Act 6 Ci/mmole; New England Nuclear).

ARCECI AND GROSS

ences alone can probably entire kinetic difference.

not explain

Size Analysis of N&ease-Released Fragments

Sea Urchin Sperm Chromatin Structure

213

the

DNA

When DNA from the nuclease digestions is isolated and electrophoresed on a denaturing, 8% polyacrylamide slab gel in 98% formamide (Fig. 2), the DNA released from blastula nuclei displays a characteristic, prominent series of bands, separated in size by approximately 10 bases (Noll, 1974). With sperm nuclei, however, most DNA remains in high molecular weight form, very little of the product being in the lobase pattern. This is true even after extensive digestion, i.e., 43% acid-soluble DNA. When the sperm DNA was redissolved, redigested with pronase (1 mg/ml at 37°C for 4 hr), and reextracted with phenol and chloroform-isoamyl alcohol, the initial pattern observed in Fig. 2 did not change. It is unlikely that the small amount of DNA that does separate as a lo-base ladder comes from contaminating somatic nuclei. The nuclear isolations were monitored microscopically, and nonsperm histones were not detected on SDS-polyacrylamide slab gels stained with Coomassie brilliant blue. In order to determine the size of sperm double-stranded DNA fragments following DNase I digestion, we ran a nondenaturing 1.8% agarose slab gel of the products. Figure 3 shows that DNase I cut the sperm nuclear DNA into a series of relatively broad but discrete size classes, unlike those produced by micrococcal nuclease (scans a and b). For blastula nuclei bothDNase I and micrococcal nuclease generate double-stranded DNA fragments of similar size classes (Fig. 3, scans c and d). When DNA fragment sizes are plotted as a function of band number, the slope of the regression line yields the average distance in base pairs between cleavate sites (Thoma and Furber, 1976; Morris, 1976a,b; Noll and Kornberg, 1977). Table 1 shows DNA fragment length values and the calculated repeat lengths for the

A

1

b MIGRATION -

FIG. 2. DNA fragments produced by DNase I digestion of blastula and sperm nuclei. Nuclei were digested at 37°C with 20 pg enzyme/ml at a DNA concentration of 1.0 mg/ml. Extracted DNA was electrophoresed in an 8% polyacrylamide slab gel in 98% formamide. (A) Photograph of the ethidium bromidestained slab gel. Lanes a, b, and d, blastula nuclei at 9.3, 38.5, and 50% acid-soluble DNA, lanes e and d, sperm nuclei at 9.3, 38.5, and 50% acid-soluble DNA; lanes e and d, sperm nuclei at 8.1 and 43% acid-soluble DNA. (B) Densitometric scans of the corresponding lanes from the negative of the photograph. Restriction fragments from HaeIIIand HincII-cut +X174 RF DNA were used to determine the lengths of the DNase I-produced fragments. Arrows with the suprascripts 70, 80, and 110 refer to the number of bases.

data displayed in Fig. 3. The data yield a cutting periodicity, for DNase I on sperm chromatin, of 498 & 15 base pairs. Averaging five independent experiments gave a value of 513 + 19 base pairs. Figure 4 shows densitometric scans of DNase I-released DNA fragments at two different times during digestion. Fragment sizes and calculated repeat length estimates indicate that although the cutting periodicity remains essentially unchanged, there is some decrease in the size of the oligomers as the digestion proceeds (Table 2). We do not, however, observe any conversion of the

214

DEVELOPMENTAL BIOLOGY VOLUME80.1980

MIGRATION -

FIG. 3. Sizing of DNase I and micrococcal nuclease-produced DNA fragments from sperm and blastula nuclei. Nuclei at a concentration of 1 mg DNA/ml were digested with 20 pg DNase I/ml. Micrococcal nuclease, at 100 units/ml, was used to digest nuclei at 0.5 mg DNA/ml. Extracted DNA was separated in a 1.8% nondenaturing agarose slab gel. (A) Photograph of the ethidium bromide-stained tracks: (a) DNase I-digested sperm nuclei, 33% acid-soluble DNA; (b) micrococcal nuclease-digested sperm nuclei, 2.8% acid-soluble DNA; (c) micrococcal nuclease-digested blastula nuclei, 11.9% acid-soluble DNA; (d) DNase I-digested blastula nuclei, 35% acid-soluble DNA, (e) HueIII-cut +X174 RF DNA, (f) HpaI-cut T7 wild-type DNA. (B) Densitometric scans of the corresponding lanes from the negative of the photograph. Fragment lengths (in base pairs) of the restriction enzyme-cut $X174 RF and T7 DNA are indicated on the abscissa. (C) Calibration curve for the sizing of the DNA fragments; A, HpaI-cut T7 wild-type DNA; n , HueIII-cut +X174 RF DNA. higher molecular weight oligomers into smaller ones. To test the possibility that the 500 base pair DNA fragment pattern might not persist under denaturation, we ran a methyl

mercury agarose gel of those products shown in Fig. 3. The results (Fig. 5) show that even under denaturing conditions, the 500 base pair pattern persists. Little singlestrand nicking has occurred. DNase I-

ARCECI AND GROSS TABLE

215

Sea Urchin Sperm Chromatin Structure 6

1

SIZES (IN BASE PAIRS) OF DNA FRAGMENTS PRODUCED BY MICROCOCCAL NUCLEASE OR DNase I DIGESTION OF EMBRYONIC AND SPERM NUCLEI Multiple

1

2 3 4 5 6 Repeat length (bp)

Sperm micrococcal

Sperm DNase I

239 491

309 834 1313 1796 2316

740 984 1236 1478

Blastula micrococcal

Blastula DNase I

194

162

405 614 828 1041 1257

359 564 776

A abed

248 -t 3 498 + 15 212 + 2 205 + 5

treated blastula nuclei (lane a) undergo, on the other hand, a great deal of single-strand nicking, since most of the DNA shifts to a broad, low molecular weight peak with a mean size of about 146 bases (Fig. 5, curve d). Patterns for sperm as well as blastula nuclei treated with micrococcal nuclease do not show any major changes, which is consistent with little single-strand nicking (Fig. 5, curves b and c, respectively). Table 3 shows DNA fragment sizes and repeat length calculations. As was shown in Fig. 1, bottom, even when embryo nuclei were digested in the same reaction with sperm nuclei, they were digested at a faster rate. In order to determine whether the unusual DNase I cutting periodicity is truly a property of sperm chromatin, rather than of some uncontrolled property of the reaction mixture, we examined the products from the mixed digestion of blastula and sperm nuclei on two different electrophoretic systems. Figure 6, curve a, shows a densitometric scan of the ethidium stained pattern of sperm DNA (in loo-fold excess to the blastula DNA) when electrophoresed on an 8% acrylamide-98% formamide gel. As expected, most of the DNA is in high molecular weight material. In contrast, fluorogruphy of this track shows (curve b) that the [3H]thymidine-labeled blastula DNA has been digested to produce a prominent 10

FIG. 4. Electrophoretic size analysis (1.8% nondenaturing agarose slab gel) of the DNA fragments produced at different times during DNase I digestion of sperm nuclei. Digestion was at 37°C with 20 pg DNase I/ml and 1 mg DNA/ml. (A) Photograph of the ethidium bromide-stained tracks. (a) Sperm nuclei, 12.4% acid-soluble DNA, (b) sperm nuclei, 28.8% acid-soluble DNA; (c) HaeIII-cut +X174 RF DNA, (d) HpaI-cut wild-type T7 DNA. (B) Densitometric scans of the corresponding lanes from the negative of the photograph. The numbers on the abscissa represent the fragment lengths (in base pairs) of the restriction enzyme-cut +X174 RF DNA and wild-type T7 DNA markers.

base pattern. Curve c shows the products of a DNase I digest of prism stage nuclei run in an adjacent slot, to indicate the band positions for a typical 10 base pair pattern. TABLE

2

SIZES (IN BASE PAIRS) OF DNA FRAGMENTS PRODUCED BY DNase I DIGESTION OF SPERM NUCLEI TO DIFFERENT PERCENTAGE ACID SOLUBILITY Multiple 12.4% 28.8% 1

2 3 4 5 6 Repeat length (bp)

391 937 1457 1948

519 + 19

359 882 1341 1874 2405 2941 515 f 25

216

DEVELOPMENTAL BIOLOGY

VOLUME 80,198O TABLE

3

SIZES (IN BASES) OF DNA FRAGMENTS PRODUCED BY DNase I DIGESTION OF SPERM AND EMBRYONIC NUCLEI AS ASSAYED ON A DENATURING GEL Multiple

A abcde

f

Sperm”

Blastulah

1 2 3 4

356 857 1421 1928

146

Repeat length (bases)’

528 + 19

“Determined as described under Materials Methods from data in Fig. 5. ’ Digested to 25% acid-soluble products. ’ Digested to 9.3% acid-soluble products.

FIG. 5. Size analysis of the DNA fragments produced from DNase I and micrococcal nuclease digestions of sperm and blastula nuclei. DNase I at 20 pg/ ml and micrococcal nuclease at 100 units/ml were used to digest nuclei at 1 mg DNA/ml and 0.5 mg DNA/ ml, respectively. Isolated DNA was then separated electrophoretically on a denaturing, methyl mercury, 1.8% agarose slab gel. (A) Photograph of the ethidium bromide-stained tracks: (a) DNase I digestion of sperm nuclei, 33% acid-soluble DNA; (b) micrococcal nuclease digestion of sperm nuclei, 2.8% acid-soluble DNA; (c) micrococcal nuclease digestion of blastula nuclei, 11.9% acid-soluble DNA; (d) DNase I digestion of blastula nuclei, 35% acid-soluble DNA; (e) iYaeIIIcut +X174 RF DNA, (f) HpaI-cut wild-type T7 DNA. (B) Densitometric scans of the corresponding lanes from the negative of the photograph. The numbers on the abscissa indicate the fragment length (in bases) of several of the restriction fragments from lanes (e) and (f ). The very sharp peak present in the higher molecular weight regions is an artifact which can be eliminated by using freshly diluted methyhnercury (R. Angerer, personal communication).

Curve d shows a densitometric scan of the ethidium bromide-stained DNA products from DNase I digested sperm nuclei, electrophoresed on a nondenaturing 1.8% aga-

and

rose slab gel. Here, again, we see the 500 base pair pattern. Fluorography of this slot reveals that the [3H]thymidine-labeled blastula nuclei products have been digested to low molecular weight materials. This means that the unique DNase I cutting periodicity is intrinsic to sperm nuclei. One characteristic morphologic feature of sperm chromatin is its extreme compaction. It was therefore of interest to examine the possibility that swelling sperm nuclei would prevent the appearance of the 500 base pair cutting periodicity. For this purpose, we resuspended sperm nuclei at a DNA concentration of 1 mg/ml in hypotonic buffer (10 mil4 Tris, pH 7.5, 1 mil4 EDTA, 0.1 mM PMSF) and stirred them at 4°C for 12 hr. At the end of this time, the nuclei were swollen to 1.5-2.5 times their original diameters (as measured by light microscopy), although they had not lysed. These nuclei were pelleted, washed several times with DNase I digestion buffer, and finally digested with DNase I as described under Materials and Methods. (Once in DNase I digestion buffer, the nuclei appeared to return to approximately their original size.) The calculated rate constant for a fnstorder, one-component best fit of the course of digestion for such nuclei was 0.0213 f 0.004 min-‘, with a termination value of 47.5 f 2.9%, and a GOF of 0.0262. As shown earlier, unswollen nuclei values were 0.026

ARCECI AND GROSS

Sea Urchin Sperm Chromatin Structure I

MIGRATION -

FIG. 6. Electrophoretic analysis of the bulk and labeled DNA fragments produced by DNase I digestion of unlabeled sperm and [3H]thymidine-labeled blastula nuclei. Sperm nuclei, present in a KM-fold excess, were digested in the same mixture as [3H]thymidine-labeled blastula nuclei (see legend to Fig. 1). Total DNA was at 1 mg/ml and the DNase I was at 20 pg/ml. Curves (a)-(c): An 8% denaturing polyacrylamide slab gel in 98% formamide. (a) Ethidium bromide-stained DNA products (sperm DNA), 5.4% acid-soluble DNA, (b) a scanned fluorogram of the labeled products in the same track as for scan (a), 40.4%acid-soluble DNA (label); (c) another track from the same slab gel, showing a scan of the ethidium bromide-stained products from a DNase I digestion of prism nuclei, 34.6%acid-soluble DNA. Curves (d) and (e): A 1.8% nondenaturing agarose slab gel of the samples used for (a) and (b). Curve (d) is the tracing of stained bulk DNA (sperm), 5.4% acid-soluble DNA. Curve (e) is a tracing of the fluorogram (blastula, [3H]thymidine labeled DNA), 40.4% acid-soluble DNA. The numbers indicated just above the abscissa represent fragment lengths. Those below curves (a)(c) were determined from their migration relative to HaeIII- and HincII-cut restriction fragments from +X174 RF DNA. Those below the curves (d) and (e) show the fragment length (in base pairs) restriction fragments from HaeIII-cut $1X174 RF DNA and ZfpaI-cut T6 wild-type DNA.

+ 0.003 min-’ for the rate constant, 37.1 of: 2.0% for the terminal value, and a GOF of 0.0114. Thus, although nuclei which have been swollen are digested to a somewhat greater extent, the rate of digestion was not significantly different from that for control nuclei. Size analysis of the products from DNase

217

I-digested swollen sperm nuclei is shown (as densitometric scans) in Fig. 7. The left panel, a 1.8% agarose slab gel, shows that DNase I and micrococcal nuclease digestion DNA fragment sizes are similar to those produced from unswollen nuclei (See Fig. 3 for comparison). The right panel is from a separation in an 8%, denaturing polyacrylamide slab gel in 98% formamide. The top curve is for DNA from DNase I-treated swollen sperm nuclei; it shows again little 10 base repeat. The bottom curve, showing the products from DNase I-digested prism nuclei, has a prominent 10 base series. Sperm nuclei that have been swollen are therefore digested by DNase I to give patterns of DNA fragments like those from unswollen sperm nuclei. Unfortunately, it is not possible to carry out DNase I digestions in the absence of bivalent cations. Thus, we cannot say whether the nuclei would, while fully swollen, yield the novel DNase I digestion pattern. We can say, however, that swelling does not destroy irreversibly the structural basis for the 500 base pair cutting periodicity. DNase I Digestion of Reconstituted Nucleohistone. We fractionated the four sperm core histones by the method of Oliver et al. (1972); SDS-polyacrylamide gel electrophoresis of the purified histones is shown in Fig. 8. Nucleosomes were reconstituted with calf thymus DNA by stepwise salt dilution. This procedure has been shown to require the presence of all four core histones (Germond et al., 1976). Figure 9, an electron micrograph of the preparation, shows the beadson-a-string structure characteristic of nucleosomes. DNase I digestion of the reconstituted nucleosomes (Fig. 10) gives rise to a series of bands separated by 10 base increments, with the characteristically prominent 70 and 80 base peaks. Significantly, the core histones did not interact with DNA (at least under these conditions) to yield any nuclease resistant, high molecular weight fragments.

DEVELOPMENTAL BIOLOGY VOLUME80,198O

218

MIGRATIONMIGRATION

-

FIG. 7. Electrophoretic

size analysis of DNA fragments produced from a micrococcal nuclease and DNase I digestion of swollen sperm nuclei. Left panel: A 1.8% agarose slab gel. The curves represent densitometric scans of the ethidium bromide-stained gel tracks. (a) DNase I digestion of swollen sperm nuclei, 29% acid-soluble DNA; (b) micrococcal nuclease digestion of swollen sperm nuclei, 6.3% acid-soluble DNA; (c) HincII-cut $X174 RF DNA; (d) HueII-cut +X174 RF DNA. Fragment lengths of the restriction enzyme-cut $X174 RF DNA are indicated above the peaks. Right panel: An 8% denaturing polyacrylamide slab gel containing 98% formamide. Densitometric scans of the ethidium bromide-stained gel tracks. Top: DNase I digestion of pluteus stage nuclei, 34.6% acid-soluble DNA. The arrows and suprascripts of 80 and 110 represent fragment length (in bases) determined from the relative positions of HueIII- and H&II-cut $X174 RF DNA, run on the same slab gel.

DNase II Digestion of Sperm Nuclei DNase II digests chromatin in a manner similar to the action of staphylococcal nua

“’

H2b

b

c1vImF

‘ii

c

d

e

1

3”

“3

H2a “4

FIG. 8. Electrophoretic separation of fractionated histones from sperm nuclei. A 12.5% polyacrylamide slab gel in SDS stained with Coomassie brilliant blue. Histone fractions were purified by the method of Olvier et al. (1972). (a) Total sperm histone; (b) Hl; (c) H3; (d) H2B (two of the three variants of H2b are resolved here; see Strickland et al., 1978); (e) H2a (top band) and H4 (bottom band). Migration is from top to bottom.

clease and DNase I: it makes both doublestrand and single-strand cuts in DNA. Under certain conditions, DNase II digestion of chromatin leads to a 100 base pair cleavage pattern (Altenburger et al., 1976; Greil et al., 1976). Figure 11 shows that when sperm nuclei were digested with DNase II in 10 mMTris, pH 7.0, 1 mM PMSF at 37°C DNA fragments were cut with a periodicity of 248 + 6 base pairs. The DNA background between the more prominent peaks, however, was very high, and may thus have obscured an underlying 100 base pair cleavage pattern. It has already been shown (Keichline and Wasserman, 1979) that DNase II cuts sperm chromatin into a 10 base series, (as analyzed on 12% polyacrylamide-urea gels). Thus only pancreatic DNase I, among the enzymes used, appears to recognize a special structural feature of sperm chromatin that is related to the 500 base pair cleavage pattern. DISCUSSION

Under

identical

conditions,

release by

ARCECI AND GROSS

Sea Urchin Sperm Chromatin Structure

219

FIG. 9. Electron micrographs of nucleosomes reconstituted from fractionated sperm histones and calf thymus DNA. Histones and DNA were mixed at 1 to 1 weight ratios in high salt and then reconstituted by stepwise dilution. The reconstituted nucleosomes were prepared for electron microscopy as described under Materials and Methods. The solid bar in the photomicrographs represents 0.1 pm.

DNase I of acid-soluble DNA fragments from sperm chromatin is 20 times slower than from embryo nuclei. This is observed whether the two types of nuclei are digested separately or together. The data confirm and extend earlier observations of Spadafora et al., (1976). The size of the fast component in the reaction of blastula nuclei is consistent with estimates of the fraction of the genome being transcribed at this stage (Hough et al., 1975; Kleene and Humphreys, 1977). Under the same conditions, no similar fast component is observed with

sperm nuclei, and they are transcriptionally inert. Our data show further that DNase I, under the conditions employed, cuts the DNA of sea urchin sperm nuclei with a 500 base pair periodicity. Released DNA fragments making up each peak are clearly heterogeneous; the lowest molecular weight peak has several shoulders which appear (consistently) at approximately 162, 212, 255-263, 309-391, and 427-495 base pairs, depending upon the extent of digestion. There is also some decrease in the size of

220

DEVELOPMENTAL BIOLOGY VOLUME80,198O

I 2 mQ g m $

MIGRATION -

FIG. 10. Electrophoretic separation of DNA fragments from a DNase I digestion of sperm histone-calf thymus DNA reconstituted nucleosomes. An 8% polyacrylamide-98% formamide slab gel stained with ethidium bromide and photographed. The reconstituted nucleosomes were dialyzed for 9 hr against 10 m&f Tris, pH 7.4,lO mM NaCl, 0.25 M sucrose, 3 mM MgClz, 0.1 mM PMSF. They were then digested with DNase I as described under Mater& and Methods. (a) Densitometric scan of DNase I digestion products from reconstituted nucleosomes; (b) densitometric scan of DNase I digestion products from pluteus nuclei, 34.8% acid-soluble DNA. The arrows mark the positions of 80 and 110 bases determined by their electrophoretic mobilities relative to Hue111 and H&II restriction fragments from @X174 RF DNA.

MIGRATION-

FIG. 11. Size analysis of DNA fragments produced by a DNase II digestion of sperm nuclei. The nuclei at a concentration of 0.5 mg DNA/ml were digested with 100 units DNase II/ml in 10 mM Tris, pH 7.0,O.l mM PMSF. Densitometric scans of the corresponding lanes from the negative of the photograph. The fragment length (in base pairs) of the HoeIII-cut +X174 RF DNA are indicated above the peaks on the densitometric scan.

and DNase II produce sperm DNA fragments with a 247 base pair periodicity. Digestion of early embryo nuclei by DNase I and micrococcal nuclease yields, by confragments released initially as digestion trast, DNA fragments with approximately proceeds, although the basic oligomeric the same unit repeat length of 205 f 5 base pattern persists. Conversion of higher oli- pairs and 212 f 2 base pairs, respectively gomers into the fastest running peak was (Table 1). not observed. A possible explanation for In 1975, Spadafora and Geraci concluded these results is that the oligomers come to that DNase I can cut isolated sperm chrobe inaccessible to nuclease after that re- matin into a 10 base series. Interestingly, lease from intact chromatin (perhaps by both sets of data showed mostly high moprecipitation, such as occurs during the lecular weight material remaining after later stages of micrococcal nuclease diges- DNase I digestion. Our own data now add tion) . the fact that DNase I cuts the DNA into a Consistent with the digestion kinetics are 500 base pair series. DNase I does generate electrophoretic data (denaturing condi- DNA fragments separated by about 10 base tions) in Fig. 5, which show that while the intervals, but this pattern is not nearly so DNA from embryo nuclei has been heavily prominent as when embryo nuclei are dinicked, DNA from sperm remains in (rela- gested. We see prominent peaks at 70 and tively) long fragments. 80 bases from DNase I-digested embryo Unlike DNase I, micrococcal nuclease nuclei as well as from sperm nuclei; as

ARCECI AND GROSS

221

Sea Urchin Sperm Chromatin Structure

digestion proceeds, the 80 base peak becomes less prominent. When the four core histones from sperm were used to reconstitute nucleosomes, the products of a DNase I digestion showed the 10 base patterns, but with no remaining high molecular weight DNA. Thus, the four core histones from sperm can organize DNA into nucleosomes capable of being cut at 10 base intervals by DNase I, as is the case for all other organisms studied thus far. Therefore, the 500 base pair cleavage pattern observed for sperm nuclei does not appear to be a property depending solely on the core histone variants of sperm. In any attempt to identify positively the features of sperm chromatin that might give rise to the 500 base pair repeat, several points must be emphasized. First, sperm chromatin is composed of all five histone fractions, although sperm Hl, H2A, and H2B are distinctly different from embryonic and somatic histones. H2B, for instance, is present as three variants, with a repeating pentapeptide in the N-terminal part of the molecule (Strickland et al., 1978). Histone Hl, being both larger and more basic than the embryonic or somatic Hl fractions (Ozaki, 1971; Brandt et al., 1979), is also of particular interest, in light of the compaction of sperm chromatin. We have observed no other proteins in isolated sperm nuclei in amounts similar to those of the histones (Fig. 12). It has already been demonstrated that sperm chromatin consists of chains of nucleosomes (Spadafora et al., 1976; Keichline and Wasserman, 1977; Arceci and Gross, 1980). The repeating unit has been estimated to be 240 to 260 base pairs long, i.e., half that of the DNase I cutting periodicity described in this report. If we assume that the cutting sites preferred by DNase I , in generating the 500 base pair repeat pattern, are in linker DNA, our results can be understood, at least in part, as follows: From the model proposed by Worcel and Benyajati (1971) and Worcel (1977), one

-

HI-1

1(

H2b = H3 H2a H4 -

FIG. 12. Electrophoretic separation of SDS-solubiked nuclei from sperm. A 12.5% polyacrylamideSDS slab gel. Pelleted nuclei were dissolved in the sample buffer of Laemmli (1970), heated at 95°C for 4 min, and applied to the gel. Following electrophoresis the gel was stained with Coomassie brilliant blue. Migration is from top to bottom. The positions of the five histone fractions are indicated.

may visualize the Hl molecules as stretching in an alternating head-to-tail fashion from one linker region to another, interacting with one linker by the basic C-terminal portion and with the other by the shorter N-terminal basic portion. The shorter Nterminal region would protect the linker region less well than the C-terminal region (Bradbury et al., 1975; Singer and Singer, 1976). As mentioned earlier, the Hl from sea urchin sperm is both larger and more basic than somatic Hl (Ozaki, 1971; Brandt et al., 1979). With such an organization, every other linker region would be more accessible to the nuclease, producing an oligomeric series of DNA fragments separated by the number of base pairs in the comparatively stable dimer, i.e., about 500 (see Fig. 13). Symmetric cuts on such products could also account for the shoulders present under the leading peak. We do not understand why only DNase

222

DEVELOPMENTAL BIOLOGY VOLUME80,198O

the DNA fragments seen in the 500 base pair repeat are generated as discrete nucleoprotein particles. Altenburger et al. (1976) have shown that, under certain conditions, DNase II can cut DNA in chromatin with a 100 base pair periodicity, but they were unable to isolate nucleoprotein particles with lengths of 100 base pairs. It will also be important to determine how widely varied ionic conditions may affect the observed periodicity reported here. Geraci and Noviello (1979) have shown that varying salt concentration affects the digestion of sperm nuclei by micrococcal nuclease. For locating more precisely the cutting sites of DNase I, digestions prior to or following exposure to micrococcal nuclease may prove informative. The role of Hl and FIG. 13. Possible arrangement of Hl molecules in the other histones can be investigated by chromatin. Shown is the alternation of more nuclease selective removal procedures (Lawson and susceptible linker DNA (from Worcel and Benyajati Cole, 1979; Weischet et al., 1979), as well as (1971) and Suau et al. (1979)).The larger rectangles by reconstitutions employing and substitutrepresent the nucleosomal cores as seen from the side. ing histones (including Hl) from different The coiled line represents DNA. The three portions sources. of histone Hl are depicted as a small rectangle for the C-terminal region (binding to the proposed less accesFinally, although we have not observed sible linker DNA), a circle for the N-terminal region in embryo nuclei any DNase I cutting pe(binding to the proposed more accessible linker DNA), riodicity larger than that of the nucleosome and a straight line for the central hydrophobic region. repeat length, it remains possible that it The diagram is not drawn strictly to scale. might exist, but be missed due to the extremely rapid digestion of such nuclei. It I is able to cut sperm chromatin with such remains to be determined whether the 500 a 500 base pair cleavage pattern, while mi- base pair cutting periodicity is: specific for crococcal nuclease and DNase II do not. It sea urchin sperm nuclei; characteristic of is clear, however, that the actions of differ- other histone-containing sperm nuclei as ent enzymes on chromatin vary consider- well (Adler and Gorovsky, 1975); related to ably (Weintraub and Groudine, 1976; Garel extreme forms of chromatin compaction and Axel, 1976; Gottesfeld et al., 1974; Bel- (e.g., avian erythrocytes); or related instead lard et al., 1977; Wallace et al., 1977;Alten- to some more general feature of chromatin burger et al., 1976; Sollner-Webb et al., organization. 1978). No particular functional role for the We would like to thank Dr. Martin Gorovsky, Dr. core histone variants emerges from the Robert Angerer, and Mr. Stuart Moss for continuing scheme presented above, but they may and helpful discussions of this work. The computer prove to have important specific interac- program used in analyzing the nuclease kinetic data tions with Hl (Hardison et al., 1977;Bonner was kindly made available by Dr. Robert Angerer. We and Steadman, 1979). Clearly, such a are grateful to Dr. Claiborne Glover, who carried out the reconstitutions and prepared the grids used for scheme is only one of many possibilities electron microscopy. Support has been provided by (Thoma et al., 1979; Lobr and Van Holde, grants from the National Institute of Child Health and 1979). Human Development (HD-08652) and the Rockefeller It will be of interest to determine whether Foundation (GA PD 7812).

ARCECI AND GROSS REFERENCES

ALDER, D., and GOROVSKY, M. A. (1975). Electrophoretie analysis of liver and testis histones of the frog

Rana pipiens. J. Cell. Biol. 64, 389-397. ALTENBURGER, W., Hij~z, U., and ZACHAU, H. G. (1976). Nuclease cleavage of chromatin at 100-nucleotide pair intervals. Nature (London) 264, 517-

525. ARCECI, R. J., and GROSS, P. R. (1980). Histone variants and chromatin structure during sea urchin development. Deuelop. Biol., 80, 186-209. BONNER, W. M., and STEADMAN, J. D. (1979). Histone 1 is proximal to histone 2A and Az~. Proc. Nat.

Acad. Sci. USA 76,2190-2194. BRADBURY, E. M., CHAPMAN, G. E., DANBY, S. E., HARTMAN, P. G., and RICHES, P. L. (1975). Studies on the role and mode of operation of the very lysinerich histone Hl (Fl) in eukaryote chromatin. Eur.

J. Biochem. 57,521-528. BRANDT, W. F., STRICKLAND, W. N., STRICKLAND, M., CARLISLE, L., WOODS, D., and VAN HOLT, C. (1979). A histone programme during the life cycle of the sea urchin. Eur. J. Biochem. 94, l-10. EASTON, D., and CHALKLEY, R. (1972). High resolution electrophoretic analyses of the histones from embryos and sperm of Arbacia punctulata. Exp. Cell

Res. 72, 502-508. FAIRBANKS, G., STECK, T. L., and WALLACH, D. F. H. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10,2606-2617. FELSENFELD, G. (1978). Chromatin. Nature (London) 271, 115-122. GAREL, A., and AXEL, R. (1976). Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proc. Nat. Acad. Sci. USA 73, 3966-

3970.

HARDISON, R. C., ZEITLER, D. P., MURPHY, J. M., and CHALKLEY, R. (1977). Histone neighbors in nuclei and extended chromatin. Cell 12,417-427. HOUGH, B. R., SMITH, M. J., BRITTEN, R. J., and DAVIDSON, E. H. (1975). Sequence complexity of heterogeneous nuclear RNA in sea urchin embryos.

Cell 5,291-299. KEICHLINE, L. D., and WASSERMAN, P. M. (1977). Developmental study of the structure of sea urchin embryos and sperm chromatin using micrococcal nuclease. Biochim. Biophys. Acta 475.139-151. KEICHLINE, L. D., and WASSERMAN, P. M. (1979). Structure of chromatin in sea urchin embryos, sperm and adult somatic cells. Biochemistry 18, 214-219. KLEENE, K. C., and HUMPHREYS, T. (1977). Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos. Cell 12,143-155. KORNBERG, R. D. (1974). Chromatin structure: A repeating unit of histones and DNA. Science 184,868-

871. KORNBERG, R. D. (1977). Structure

of chromatin.

Annu. Rev. Biochem. 46,931-954. LAEMMLI, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LAWSON, G. M., and COLE, R. D. (1979). Selective displacement of histone Hl from whole HeLa nuclei: Effect on chromatin structure in situ as probed by micrococcal nuclease. Biochemistry 18, 2160-2166. LOHR, D., and VAN HOLDE, K. E. (1979). Organization of spacer DNA in chromatin. Proc. Nat. Acad. Sci.

USA 76,6326-6330. MORRIS, N. R. (1976a). Nucleosome

structure

in As-

pergillus nidulans. Cell 8,357-363. MORRIS, R. N. (1976b). A comparison of the structure of chicken erythrocyte and chicken liver chromatin.

Cell 9, 627-632.

GERACI, G., MARCHI, P., and NOVIELLO, L. (1979). The sea urchin (Sphaerechinus granularis) codes different H2b histones to assemble sperm and embryo chromatin. Cell Differ. 81, 187-194. GERACI, G., AND NOVIELLO, L. (1979). DNase hydrolysis of chromatin DNA in intact sea urchin sperm cells. Effect of the ionic strength on the digestion parameters. Cell. Differ. 8, 203-210. GERMOND, J. E., BELLARD, M., OUDENT, P., and CHAMBON, P. (1976). Stability of nucleosomes in native and reconstituted chromatins. Nucleic Acids

Res. 3,3173-3192. GOTTESFELD, J. M., GARRARD, SON, R. F., and BONNER, J., cation of the template-active A preliminary report. Proc.

223

Sea Urc*bin Sperm Chromatin Structure

W. T., BAGI, G., WIL(1974). Partial purififraction of chromatin:

Nat. Acad. Sci. USA

71,2193-2197. GREIL, W., IGO-KEMENES, T., and ZACHAU, H. G., (1976). Nuclease digestion in between and within nucleosomes. Nucleic Acids Res. 3, 2633-2644.

NOLL, M. (1974). Internal structure of the chromatin subunit. Nucleic Acids Res. 1, 1573-1578. NOLL, M., and KORNBERG, R. D. (1977). Action of micrococcal nuclease on chromatin and the location of histone Hl. J. Mol. Biol. 109, 393-W. OLIVER, D., SOMMER, K. R., PANYIM, S., SPIKER, S., and CHALKLEY, R. (1972). A modified procedure for fractionating histones. Biochem. J. 129,349-353. OZAKI, H. (1971). Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Stronglylocentrotus purpuratus. Develop. Biol. 26, 209-219. PEARSON, W. R., DAVIDSON, E. H., , and BRITTEN, R. J. (1977). A program for least squares analysis of reassociation and hybridization data. Nucleic Acids Res. 4, 1727-1737. RUIZ-CARRILLO, A., and PALAU, J. (1973). Histones from embryos of the sea urchin Arabacia lixula. Develop. Biol. 35, 115-124. SINGER, D. S., and SINGER, M. F. (1976). Studies on

224

DEVELOPMENTALBIOLOGY

the interaction of Hl histone with superhelical DNA: Characterization of the recognition and binding regions of HI histone. Nucleic Acids Res. 3, 2531-2547. SOLLNER-WEBB,B., MELCHIOR,W., JR., and FELSENFELD, G. (1978). DNase I, DNase II and staphylococcal nuclease cut at different, yet symmetrically located sites in the nucleosome core. Cell 14, 611627. SPADAFORA,C., BELLARD, M., COMPTOM,J. L., and CHAMBON, P. (1976). The DNA repeat lengths in chromatins from sea urchin sperm and gastrula cehs are markedly different. FEBS Lett. 69,281-285. SPADAFORA,C., and GERACI, G. (1975). The subunit structure of sea urchin sperm chromatin: A kinetic approach. FBBS Lett. 57, 79-82. SPADAFORA,C., NOVIELLO,L., and GERACI,G. (1976). Chromatin organization in nuclei of sea urchin embryos. Comparison with chromatin organization of the sperm. Cell Differ. 5, 225-231. STRICKLAND,W. N., STRICKLAND,M., BRANDT, W. F., MORGAN, M., and VON HOLT, C. (1974). Partial amino acid sequence of two new arginine-serine rich histones from male gonads of the sea urchin. (Parechinus angulosus). FEBS Lett. 40,161-166. STRICKLAND,M., STRICKLAND,W. N., BRANDT, W. F., and VON HOLT, C. (1977a). The complete-aminoacid sequence of histone H2B(l) from sperm of the sea urchin Parechinus angulosus. Eur. J. Biochem. 77,263-275. STRICKLAND,W. N., STRICKLAND,M., BRANDT, W. F., and VON HOLT, C. (1977b). The complete aminoacid sequence of histone H2b(2) from sperm of the sea urchin Parechinus angulosus. Eur. J. B&hem. 77,277-286. STRICKLAND,M., STRICKLAND,W. N., BRANDT,W. F., VON HOLT, C., WITTMANN-LIEBOHL, B., and LEHMANN,A. (1978).The complete amino-acid sequence of histone H2b(3) from sperm of the sea urchin

VOLUME 80,198O

Parechinus angulosus. Eur. J. Biochem. 89, 443452. SUAU, P., BRADBURY, E. M., and BALDWIN, J. P. (1979). Higher-order structures of chromatin in solution. Eur. J. Biochem. 77, 593-602. THOMAS, F., KOLLER, TH., and KLUG, A. (1979). Involvement of histone Hl in the organization of the nucleosome and of the salt-dependent superstructures of chromatin. J. Cell. Biol. 83, 403-427. THOMS,J. O., and FURBER,V. (1976).Yeast chromatin structure. FEBS Lett. 66, 274-280. VAN HOLDE, K. E., SAHASRABUDDHE,C. G., and SHAW, B. R. (1974). A model for particulate structure in chromatin. Nucleic Acids Res. 1, 1579-1586. VARSHAVSKY,A. J., BAKAYEV, V. V., and GEORGIEV, G. P. (1976). Heterogeneity of chromatin subunits in uito and location of histone H2. Nucleic Acids Res. 3,477-492. WALLACE,R. B., DUBE, S. K., and BONNER,J. (1977). Localization of the globin gene or the template active fraction of chromatin of Friend leukemia cells. Science 198, 1166-1168. WEINTRAUB, H., and GROUDINE,M. (1976). Chromosomal subunits in active genes have an altered conformation. Science 193, 848-856. WEISCHET,W. O., ALLEN, J. R., RIEDEL, G., and VAN HOLDE, K. E. (1979). The effects of salt concentration and Hl depletion on the digestion of calf thymus chromatin by micrococcal nuclease. Nucleic Acids Res. 6,1843-1862. WHITLOCK, J. P., JR., and SIMPSON, R. T. (1976). Removal of histone Hl exposes a fifty base pair DNA segment between nucleosomes. Biochemistry 15, 3307-3314. WORCEL, A. (1977). Molecular architecture of the chromatin fiber. Cold Spring Harbor Symp. Quant. Biot. 42,313-324. WORCEL,A., and BENYAJATI, C. (1971). Higher order coiling of DNA in chromatin. Cell 12,83-100.