Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possibly involved in determining the length of linker DNA

Volume 9 Number 3 1981 Nucleic Acids Research

Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possiblyinvolved in detennining the length of linker DNA

I.A.Zalenskaya, V.A.Pospelov, A.O.Zalensky* and V.I.Vorob'ev

Institute Qf Cytology, Academy of Sciences of the USSR, Leningrad 190121, and *Institute ofMarine Biology, Far East Scientific Center, Academy of Sciences of the USSR, Vladivostok 690022,USSR

Received 16 December 1980

ABSTR-ACT

Comparison has been made between sea urchin and starfishsperm chromatin. The only protein by which chromatins fromthese sources differ significantly is histone B. Sea urchinsperm H2B is known to contain an elongated N-terainal region en-riched in Argo Analysis of the micrococcal nuclease digests ofsea urchin and starfibh nuclei in one- and two-dinsionalelectrophoresis has show that sperm chromatin of both animalsconsists of repeatel units similar in genera-l features to thoseof rat tbymus or liver. However, DNA repeat length in chromatinof sea urchin sperm (23? bp) is higer than that of starfishsperm (224 bp), while the core DElength does not differ andis the same as in tie chromatin of rat liver or thymus. A sug-gestion has been made that the N-terminal region of histoneH2B is associated with the linker DNA and is responsible forthe increased length of sea urchin linker DNA.

INTRODUCTION

In a periodically repeated chromatin subunit, a nucleosome,two main parts can be distinguished: the core particle and theregion between two neighbour core particles which is termed thelinker (for rev, see 1, 2). The core particle consists of DNA"140 base pairs (bp) long combined with an octamer of "Small"histones (H4+H3+H2A+E2B)x2. The linker DNA is probably asso-ciated with histone HI (3-6). While the length of the core DNAis the same in all the cells studied, the length of the linkerDNA varies from 15 to 100 bp depending on the cell type. It hasbeen suggested that the conservation in length of the core DNAis due to the conservation in primary structure of the corehistones, while the variability of the linker DNA is connectedwith changes in the structure of histone HI (6, 7).

© IRL Press Limited, 1 Falconberg Court, London W1V 5FG, U.K.

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At the same time, some recent investigations have shownthat the linker DNA might also be associated with histones H2Aand H2B (8-10). These results as well as data on some variabili-ty of histones H2k and H2B (11) lead to an assumption that his-tones H2A and H2B could, together with HI, define the length ofthe linker DNA (8, 1, 2).

In this paper we report findings obtained using a compara--tive approach on the role of histone H2B in the organization ofa nucleosome. Comparison has been made between sea urchin andstarfish sperm chromatins which contain substantially differenthistones H2B (12-15). At the same time their histones H4, H3,H2A and, what is especially important for this study, histoneHI are very similar (see Results and Discussion). We believethat the impact of histone H2B on the structure of a nucleosomemay be inferred from comparison of parameters of the chromatinsubunits from these two sources. The spexm cells of these ani-mals are apparently very convenient for such analysis: (i) theyrepresent a highly homogeneous cell population; (ii) the con-tent of non-histone proteins in sperm cells is very low, andtherefore their influence on nucleosomal structure can be ex-cluded.

The results presented here are consistent with the pointof view that histone H2B is associated not only with the coreparticle, but also with the linker DNA and affects the lengthof the latter.

MATERIALS AND METODS

Biological material. Sea urchinSe intermedius and starfish A (Sea of Japan) were

used in this study.Preparation of sperm cells and sPerm nualei. The procedu-

res has been described else-where (14, 15). Briefly, the sperm

was collected by centrifugation, washed in STC buffer (0.25 Msucrose, 10 mM Tris-HCl, pH 7.5, 3 mM CaOl2, 0.1 mM phenylme-tylsulfonylfluorid - PIE), and disrupted in the same buffer.The nuclei were washed once i'n STO buffer, containing 0.5%Triton X-100 and twice in the STO buffer without Triton.

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Rat thymus nuclei were isolated by the same method assperm nuclei. Rat liver nuclei were additionally purified bycentrifugation through 2.2 Jl sucrose, 10 mM Tris-HCl, pH 7.5,3 mM GaC12, 0.1 mM PBEF.

Histone isolation. Total histone and histone fractions HIand H2B were isolated according to Oliver et al. (16). The his-tones HI and H2B from sea urchin and starfish sperm have beenobtained in highly purified form by this method (14, 15). Thepurity of the fractions being analysed in the present paper isno less than 98% (as has been judged from electrophoretic data).

Nuclease digestion. Nuclei were suspended at a concentra-tion of 1 mg DNA per ml of buffer containing 0.3 M sucrose,10 mM Tris-HCl, pH 7.5, 1 mM CaCOl, 0.1 mM PMEF. Micrococcalnuclease (Worthington) was added up to a concentration of 30units per mg of DNA. The digestion was carried out at 370 0during 3, 15 and 30 min. The reaction was stopped by chillingthe samples on ice. The samples were centrifuged at 6,000 rpmfor 2 min. The nuclear pellet was lysed in 5 mM Tris-HOCl2 mM EDTA, pH 7.5. After 15-20 min the lysed nuclei were cen-trifuged at 6,000 rpm for 10 min. The supernatant, which con-tained soluble chromatin fragments was used for electrophore-tic analysis and isolation of DNA fragments. Rat liver nucleiwere digested with DNase I as described in (17).

DNA isolation. DNA was isolated from the soluble chroma-tin fragments as described in (10).

Gel electrophoresis* The chromatin fragments were separa-ted on 5% polyacrylamide gel in 10 mM Tris-borate buffer, pH8.3 containing 1 mM ETA (TBE buffer) as described in (18).

To determine the DNA repeat length, the DNA fragments iso-lated from nuclei treated with micrococcal nuclease during3 min were separated on 1,8% agarose gel in 20 mM Tris-acetatebuffer, pH 7.7 containing 2 mM EDTA (buffer TAE). The valuesfor the DNA repeat length were obtained from the slope of theregression line from a plot of fragment size versus band numberas described in (7). The DNA fragments, isolated from micro-coccal nuclease digests of rat thymus or liver nuclei were usedas standards. A value of 195 bp was established for both ratthymus and rat liver DNA repeat using RFI DNA fragments from

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phage %1174 produced by restrictase Bsp. The DNA fragments werea kind gift from Dr. R.B3.Streeck and Dr. H.G.Zachau.

To compare DNA length of the core particles the DNA frag-ments were electrophoresed on 6.5% acrylamide gel in TAB bufferand on 10% acrylamide gel in double TBE buffer, containing6.5 M urea. Before electrophoresis on acrylamide-urea gel theDNA samples were dissolved in 8 Ml urea and denatured by boilingfor 5 min.

The electrophoresis of histones was carried out on 15% po-lyacrylamide gel according to Laemmli (19).

A second dimension of electrophoresis was used to analysethe protein and DNA composition of the chromatin fragments. Thestrips of 5% polyacrylamide gel after low ionic strength elec-trophoresis of chromatin firagents were incubated for 30 min in0.0625 M Tris-HOl, pH6.8 containing 0. 2% SDS and laid horizon-tally on the top of the preformed slab of Laemmli gel. Aftercompletion of the run, the gel was washed in 50% ethanol toremove SDB. The gel was first stained with ethidium bromide(0.1 mg/ml) to visualize DNA, and then with Coomassi brilliantblue to stain the proteins.

Amino acid composition was determined on KTaM (Hitachi)amino acid analyzer following 24 h hydrolysis of the proteinsin 6 N HOl at 1100 C. No corrections bave been made for bydro-lytic losses.

RESEITS

Histones from sperm of the sea urchin Strog oentrotusintermedius and the starfish Aphelasterias japonica separatedby SDB-gel electrophoresis are shown in fig. 1 (rat liver andrat or calf thymus are used in this study as standard objects).Histones H3 and H4 have identical electrophoretic properties inthe sperm cells and in calf thymus. Electrophoretic mobility ofhistone H2A from sperm of the sea urchin and the starfish ispractically the same, and it is higher than that of calf thy-mus. Also similar are the sperm histones HI, both migratingslower than calf thymus HI.

On the other hand, histones H2B from sperm of starfish and

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H -

3

H2B -iH3 -- ... --

H2A- U -UH4.

R ~~b c

Fig. 1. SDS gel electrophoresis of histones: (a) sea urchinsperm, (b) starfish sperm, (c) calf thymus.

sea urchin, unlike the histones H4, H3, H2A and HI, differ mar-

kedly. Mol. w. of H2B subfractions from sperm of the sea urchinS. intermedius as determined by SDB-gel electrophoresis are17,550 and 16,200 dalton, while mol. w. of starfish sperm H2Bis close to 13,000 (14, 15).

The electrophoretic data on similarity and difference ofthe histone fractions was confirmed by the comparison of theiramino acid compositions (Table 1, 2). A characteristic featu-re of histone HI from sperm of both organisms is a high argininecontent - 16.4 and 14.5 mole% as well as a high ratio of basicto acidic amino acids - 9.3 and 8.8 in sea urchin and starfish,respectively. In calf thymus HI arginine content is 2 mole%and basic to acidic amino acids ratio is 5.9 (20). Amino acidcomposition of histone H2A is similar in sea urchin sperm,starfish gonade and calf tbymus (Table 2) (21-23).

Significant difference in amino acid composition is foundbetween histones H2B from sea urchin and starfish sperm. Themost striking variation is in arginine content. The values are:

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Table I. Amino acid composition of some histones from the seaurchin Strongylocentrotus intermedius and the starfish AAelas-t (values are moles %).

Hi HI (15) H2B1+H2b2 H2B (15)sea urchin starfish sea urchin starfish

Lys 27.8 24*.4 12.6 11.7His 1.2 0.8 1.4 1.4Arg 16.4 14.5 18.4 7.7Asp 2.7 1.5 3.9 5.8Thr 1.7 2.4 5.9 7*4Ser 7.7 9*5 11.2 9.4Glu 2.2? 3.0 6,6 9.1Pro 8.1 4.6 4.2 2.0Gly 2.3 4.6 9.4 10.1Ala 21.4 31.4 8.1 11.5Cys - - - -

Val 4.8 1.7 5.8 7.7met - traces traces tracesIle 1.3 0.8 3.6 5.3Leu 2.2 0.7 4.7 5.2Tyr traces traces 2.5 3.3Ph. traces traces 1.4 2.5Lys/Arg 1.7 1.7 0.7 1.5basic/acidic 9.3 8.8 3.1 1.4

Table 2. Iys to Arg and basic to acidic amino acids ratios ofsome histone fractions

Lys/Arg basic/acidicHI Sperm of the sea urchin

Strongylocentrotus intermedius 1.7 9.3Sperm of the starfishAphelasterias japonica 1.7 8.8Calf thymus (20) 15.2 5.9

H2ASperm of the sea urchinPsammechinus miliaris (21) 1.2- 1.4Gonades of the starfishAsterias rubens (22) 1.0 1.4Calf thymus (23) 1.2 1.5

H2BSperm of the sea urchinStrongylocentrotus intermedius 0.7 3.1Sperm of the strafishAphelasterias japonica 1*5 1.4Calf thymus (24) 2.5 1.9

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18.4 mole% in sea urchin sperm in comparison with 7.7 mole% instarfish and 6.2 mole% in calf thymus HR (24).

Tbhe ntlei fromn sea urchin and starfish sperm, rat tbymusand rat liver were digested with micrococcal nuclease. The chro-matin fragments from the digests were separated by low ionicstrength polyacrylamide gel electrophoresis (Fig. 2). To analy-se the DNA and the proteins which are contained in the chroma-tin particles, second dimension electrophoresis was carried out(Fig. 3).

The general electrophoretic pattern of chromatin fragmentsfrom sperm of sea urchin and starfish and from rat thymus issimilar. Zones of mononucleosoeis lacking HI (MN-HI), mononuc-leosomes containing HI (lN+HI), dinucleosomes (DN) and so onare displayed. At the same time, some specific features may benoted. In sperm of both organism (Fig. 2a, c) the zone ofmN-H1 migrates faster than that of rat thymus (Fig. 2b). Fur-thermore, in sea urchin sperm the complete nucleosome particle

DN

MNi+H1 -

MN-Hl

a b c

Fig. 2. Chromatin fragments obtained from 15 mmn micrococcalnuclease digests: (a) sea urchin sperm, (b) rat thymus,(c) starfish sperm.

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_.'I-In~Fi rs

DN+NMN MNE HI HI

t d iren s i o nDN +MN MN

'H _Hi

I11 -I ,

C Co o

4-o

1n1:

t r Z 1

an Z

a b

Fig. 3. Two-dimensional electrophoresis. First dimension - se-paration of chromatin fragments on 5% acrylamide gel. Seconddimension - separation of proteins and DNE on 15% acrylamidegel. (a) sea urchin sperm, (b) starfish sperm, (c) rat thymus.

(MN+HI) separates into two subfractions. The data from two-di-

mensional electrophoresis show that both subfractions contain

the same set of histones but their DNLs differ in length(Fig. 3a). It is not yet clear whether tne fact that the com-

plete nucleosome displays a double zone demonstrates the hetero-

geneity of native chromatin -or this reflects the different ex-

tent of nuclease digestion of the same particle. Since both sub-

fractions have the same protein composition and contain DNA of

different length, the second possibility seems to be more plau-sible.

One of the main parameters of a nucleosome which supposed-ly could be defined by the proteins it contains is the DNA re-

480

C

... . .. - .__-MWW

WAM

DN +MN MNHl _H1

..P

...Mm.

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peat length.The size of DNA fragments from sea urchin and starfish

sperm micrococcal nuclease digests was determined on 1.8ff aga-rose gel (Pig. 4., Table 3) as described in Material and Methods.The method of nucleosome repeat determination used eliminatesthe effect of UNA degradation from the ends (1, 7). DNA frag-ments from rat liver digest were used as markers (see Materialsand Methods). A value of 237 (±5) bp was found for sea urchinS.intemedius sperm DNA repeat in agreement with data reportedfor other sea urchin species (25, 26). A DNA repeat length of224 (.6) bp was determined for starfish sperm. Thus, DNA con-tained in the complete nucleosome from sperm of the sea urchinSointermedius is about 13 bp longer than that from sperm of thestartish A.ja onica.

In order to find out whether the idea on the core DNA inva-

A B

1353=3:G 1078

872

606

2 341 94

a b c a b c d e

Figl.4. Agarose gel electrophoresis of DNA fragments from micro-coccal nuclease digests. A: (a) rat thymus, 5 min digestion,(b) rat liver, 3 min digestion (c) restriction fragmentsa B:a) and (b) sea urchin sperm, Za) 15 min (b) 3 min digestion,c) rat liver 3 min digestion, (d) and te) starfish sperm,d) 3 min, (eS 15 min digestion.

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Table 3. Sizes of DEA frants isolated from short (3 mi) mic-rococcal nuilease digest of rat liver and sea urchin and star-fish sperm nuclei as detemined from mobilities in agarose gel(see Materials and Methods).

Base pairsBand number Rat liver marker Sea urchin Starfish sperm

sperm12 370 450 4253 570 700 6274 775 930 8505 960 1165 10906 1150 1410 13407 1350 1630 15308 1870

Slope 195.±3 237±5 224±6

riability can be extended to the cells studied here, the DNAframents isolated from 30 min micrococcal nuclease digestswere compared on polyacrylamide gel in two systems. Data fromelectrophoresis of DEA fragments on nondenaturating 6.5% acryl-amide gel show that the position of the sharp edge of the fas-test band which corresponds to the core particle DNA (7) is in-distinguishable from each other in rat liver and sea urchin andstarfish sperm (Fig.5). The same results were obtained when DNAfrom 30 min micrococcal digests denatured by boiling for 5 minin 8 MI urea was electrophoresed on denaturating 10% acrylamidegel containing urea. (Fig.6). The fastest bands of single-stranded DNA from all three sources have a mobility that corres-ponds to 146 bases (27). Thus, there is no doubt that the nuc-leosomes from sea urchin and starfish sperm differ in the Ain-ker DNA length.

DISCUSSION

It is very likely that variation in length of linker DNAis connected with changes in structure of the proteins whichinteract with the DNA. Histone HI might be one of such prote-ins since it is bound to at least a part of the linkr DNA (3-6) and is known to be the most variable of all histones.

At the same time some data exist suggesting that also the

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a b c

Fig. 5. Nondenaturating polyacryl.aide gel electrophoresis ofDNA fragments from micrococcal nuclease digests (30 min di-gestion). (a) sea urchin sperm, (b) starfish sperm, (c) rat li-ver.

108

j6

a b c d

Fig. 6. Comparison of micrococcal and DNase I digests on 10%polyacrylamide-urea gel. Denaturated DNA fragments from (a-c)micrococcal nuclease digests of nuclei; (a) sea urchin sperm,(b) rat liver, (c) starfish sperm; (d) DNase I digest of rat li-ver nuclei. According to corrected data (27) the bands are miul-tiples of 10.4 bases. The band numbers were estimated as in(17 .

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histones of intermediate variability, H2A and H2B, could bytheir N-terminal parts interact with the linker DNA and definetogether with HI, the linker length (8-10). If so, pronouncedchanges in these proteins would have an effect on the linker.

Sea urchin sperm H2B is substantislly different from itscounterparts from other sources (12-15). H2B subfractions ofsea urchin sperm have mol. w. markedly higher than that ofstarfish sperm H2B. The amino acid composition of sea urchinsperm H2B is characterized by a very high arginine content.The primary sequence analysis of six H2B subfractions fromsperm of three sea urchin species has shown that the increasein mol. w. is due to the elongation of the N-terminal regions(12). These extensions contain basic repetitive pentapeptidesPro-Thr-Lys-Arg-Ber or Pro-Arg-Lys-Gly-Ser and bear some resem-blance to protamines (28).

To find out how the alteration in N-terminal part of thehistone H2B from sea urchin sperm could be reflected in chroma-tin structure we compared using micrococcal nuclease some para-meters of sea urchin and starfish sperm chromatin. Starfishsperm cells have been chosen for such analysis since all thehistones they contain, for exeption of histone H2B, seemed tobe very similar as judged from their amino acid compositionsand electrophoretic behavior in two systems. To verify the abovewe considered also the data available on the primary structuresof these proteins. Histones H3 and H4 are evolutionary highlystable. Only limited chanes have been round in variable N-ter-minal part of sea urchin sperm H2A (23).

The very important point is histone HI since it is one ofthe proteins that according to current hypothesis might be res-ponsible for variation of linker DNA length. Electrophoreticdata show that histone HI from sea urchin and starfish spermhave very similar (if not identical) mol. w. and charge (14,15). Amino acid compositions of the both are also alike, thepeculiarities that distinguish them from somatic HI being ofthe same character. Moreover, frou the vast body of the data onprimary structures of histone HI family (including sea urchinsperm HI), Von Holt et al. (12) concluded that HI variants iso-lated from comparable cells show high degree of homology.

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Contrary to the histone HI, the histone H2B from starfishsperm is approximately 20 amino acid residues shorter than thatfrom sea urchin sperm and does not contain the specific N-ter-minal domain that ha been found in sea urchin sperm 12 (13).In primar structure and amino acid composition starfish spermH2B resembles that from somatic cells.

Thus, the only protein in which sea urchin and starfishsperm nucleosomes significantly differ is histone H2B. At thesame time the linker DNA from sea urchin sperm nucleosome isabout 13 bp longer than that from starfish sperm. On the basisof the data presented in thiis paper it can be suggested thatthe increase in linker DNA length of sea urchin sperm nucleo-some might be due to c es in the N-terminal region of histo-ne H2B: its elongation and enrichment by basic amino acid resi-dues with a high affinity to DNA. The structural homologies bet-ween the reiterated pentapeptides present in N-terminal part ofH23 and the protamines suggest that this region can be involvedin condensing the genetic material (29). In this sense the ba-sic N-terminal region of sea urchin sperm H2B may play a rolesimilar to that of histone HI.

The influence of the basic N-terminal region of H2B on thelinker length can be obviously exerted if this region interactswith the linker DNA. A suggestion that the basic regions of H2Band H2A have binding sites outside the core partiole has alsobeen made on the basis of PIE data (9). In addition, the analy-sis of Serratia endonuclease digest of trypsin-treated chroma-tin also demonstrates a possibility of interaction between N-ter-minal regions of core histones and the linker DNA (10).

It should be noted that histones H2A, and H2B with markedlychanged electrophoretic properties were found in plants (30).The repeat DNA length in plant chromatin is however about 200 bpwhich is characteristic of the cells with the "usual" set ofcore histones. (31). This apparent contradiction to our conclu-sion may be explained if we take into consideration data on ami-no acid composition and partial amino acid sequence of planthistones (32). Like sea urchin sperm histone H2B, pea histonesH2B and H2A both contain evolutionary conserved middle and car-boxyterminal region, whereas their N-terminal regions are vari-

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able. However changes in N-terminal regions in pea and sea ur-chin histones are of different character. The basicity and argi-nine content of pea H2A and H2B are virtually the same or evenlower than these values in their calf thjmus counterparts (32).Therefore, these data are not inconsistent with our suggestionthat the specific changes in H2B molecule, namely those whichare connected with the affinity of the N-terminal region to DNA,influence the linker DNA length.

Our findings support the assumption that conservation inlength of core DNA might be due to the invariability of histo-nes H3 and H4 and conserved hydrophobic central and C-terminalregions of nistone H2B (and probably U2A) responsible for histo-ne interactions (33) while the linker DNA changes would be de-fined by evolutionary variable histone HI and N-terminal regi-ons of histone H2B at least.

ACKNOWLEDGEMENT

We thank Dr. R.Ch. Ibragimov for amino acid analysis.

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