Nucleosome core particle stability and conformational change

.I. Nol. Rid. (1984) 176, 77-104

Nucleosome Core Particle Stability and Conformational

Change

Effect of Temperature, Particle and NaCl Concentrations, and Crosslinking of Histone H3 Sulfhydryl Groups ?JVAN AUSIO,

DALIA

SEGER ANI) HENRYK

EISENRERG

I’olymer Research Department The Wrizmann Institute of Science Kehovot 76100, Israel (K~wiwd

21 July

198.3, and in revised,form

29 Frbrunry

1984)

1t’r have studied the reversible dissociation of core size DKA from chicken erythrocyte nucleosome core particles in solutions containing 0.1 M to 0.6 M-NaCI. Dissociation increases with increasing Pr’aCl concentration, increasing temperature and dec&reasing particle concentration. ,4t high particle concentrations, no free DNA is observed below 0.3 M-x&I, whereas above 0.3 M-NaCI a lower limit of dissociat,ion is reached. A theoretical analysis based on the migrating-octamer mechanism of Stein is in disagreement’ with his conclusions concerning dependence of core particle dissociation on particle concentration, but provides a good explanation for our observations, and those of others, using salt cboncent’rations up to 1 M-NaCl. It appears that the core particle is not stabilized primarily by electrostatic interactions. DNA length is not critical for core particle stabilization. The conformation of remaining intact nucleosome core particles c-hanges only moderately within the range of NaCl concentrations studied. (Irosslinking by copper phenanthroline of the CysllO histone H3 single sulf~~drgl groups in the intact nucleosome core particle leads to a decrease in stablllty, yet essentially unchanged hydrodynamic properties are maintained at 0.6 M-Ria(Jl, confirming conclusions derived from the behavior of the native core particles. Values for density increments of nucleosome core particles over a range of R’aCl

c*oncrnt,rations are also given. A method is described for studying histones to nucleosome and 260 nm.

core particles

in the ultracentrifuge

binding

by scanning

of

at 230

1. Introduction Histone-histone and histone-DNA interactions have been extensively studied and reviewed (McGhee & Felsenfeld, 1980a; Lilley, 1981: Bradbury et al., 1981; Sperling & Wachtel, 1981). Here we will restrict ourselves to a discussion of those histone-histone and histone-DNA interactions that appear to play a major role in relation to nucleosome particle structure and dynamics. The dramatis personae in nucleosome core particle structure are not individual histones, but rather two well-defined histone complexes that have been studied rather carefully, an H2A,H2R d’lmer and an H3,H4 tetramer. At 2 M-NaCI the 77 ow22 -%836/84/170077-28 oo:i.oo/o

03 1984 A4cadrmic* Press Inc. (London)

Lttl.

78

.I. Al!SIO,

D. SEGEK

ANI)

H. EISfi:SKER(:

core histones (in the absence of DNA) form a weakly stable octamer (Thomas ti Kornberg, 1975; Thomas & Butler, 1977: Philip et al., 1979) apparently identical in structure to the octamer core in the stable nucleosome core particle. W’ith decreasing ionic strength the octamer dissociates by losing first one. then anot,her H2A,H2B dimer. The existence under these circumstances of two heterotypic tetramers has been abandoned (Eickbush & Moudrianakis. 1978; Godfrey rt al.. 1980). At’ salt concentrations propitious for nucleosomr core partic*kb reconstitution the histone core is therefore not in t,he octamer conformation \‘et reconstitution proceeds spontaneously and the separate ent.ities (DNA. H2A.H% B dimer; H3,H4 tetramer) combine to form the nucleosome core. It is likely that 145 base-pairs of DNA complex onto the H3,H4 tetramer “scaffold” into which H2A,H2B dimers can then be neatly fitted to stabilize the complete structure (Klug et ctl., 1980; Finch et d.. 1981; Bentley et al.. 1981). It. is a,lso possible to form a somewhat less-stable complex of one H3,H4 tet.ramer and 145 base-pairs ot DNA, and a more nucleosome core-like particle with two H3,H4 tetramers (Situorl et al., 1978). The salt-dependent histone-DNA interaction involving a strongly negativeI!charged polynucleotide chain and positively charged histones appears superficially, t,o be characteristic of a typical electrostatic interaction. Yet, studies of t,hr melting behavior of nucleosome core particles at, low ionic strength (McGher K: Felsenfeld, 198030b)disclosed that rather few electrostatic bonds are holding the structure in place. Furthermore, it has been shown tha,t core histones. from which the charged, unstructured tails have been removed by the action of trypsin (Whitlock & Simpson, 1977), are capable of reconstituting nucleosome core particles of almost undiminished strength (Whitlook &, Stein, 1978). Thus neither the exact nature of the forces holding the nucleosome core particle together nor the role of the unfolded histone tails is precisely known. It’ has been conjectured that the histone tails may serve to establish connections between nucleosomr units along a folding chromatin chain, thereby serving to stabilize chromat,in higher-order structure. Bemoval of the N-terminal tails, or reduction of t,heir charge by specific acetylation, would therefore tend to weaken the folding of chromatin, at the same time increasing its availability for t,ranxcription. Ichimura et al. (1982) have suggested that a few arginine, rather than lysine, residues play an essential role in the folding of DNA into nucleosome cores. Nucleosome core particles are soluble over a wide range of ?iaC’l concentrations. Their properties are almost, unchanged bet.ween 0.1 M and 0.4 MM-Na.Cl.Yet. they undergo conformational changes both at very low ((Gordon it al., 1978) am1 a,t higher concentrations of monovalent salt. (McGhec c-t nl.. 1980; M’ilhelm 8 Wilhelm, 1980) manifested by moderate increase in frictional resistance to flow at constant, particle composition. Conformational changes at very low ionic strength are difficult to interpret in view of the high residual charge of the complex particles and related charge effects. The conformational transition at 04 M-NaC1, where szO,w decreases to about 10.3 S compared t,o about 11.2 S at 0.1 M-NaCI, has been ascribed to an unfolding of t,he DNA and separation of the histone octamer into two heterotypic tetramers (Dieterich et al.. 1979). Hydrodynamic measurements (Eisenberg & Felsenfeld, 1981) support the view

NUCLEOSOME

CORE

PARTICLE

STRUCTURE

79

that conformational changes in the nucleosome core particle upon increasing the ionic strength to O-6 M-NaCl are not due to an unfolding of the particle, but rather to a moderate increase in molecular size and anisotropy. This conclusion is confirmed by a recent determination of the radius of gyration, R,, of native nucleosome core particles by small angle X-ray scattering between 0.1 M and 0.7 MM-NaCl(Reich, 1982). Over this whole range of salt concentrations R,, which in these experiments is essentially determined by the DNA distribution, was found to be unchanged. The two single sulfhydryl groups in the vicinal H3 histones of nucleosome core particles and of chromatin may be crosslinked to some extent by oxidation in freshly prepared samples. Gould et aE. (1980) have described a method for crosslinking these two groups in histone Hl and H5-depleted chicken erythrocyte chromatin by oxidation with copper phenanthroline. This crosslinking reaction with nucleosomes will be discussed below. Examination of sedimentation patterns of nucleosomes in the analytical ultracentrifuge disclosed the appearance of an additional, slowly moving boundary at concentrations above 0.5 iv-NaCl (Stein, 1979; Eisenberg & Felsenfeld, 1981). This boundary can be identified as free DNA that is derived from nucleosome core particles, particularly with increase in ionic strength. Stein (1979) demonstrated an equilibrium at 0.6 M-NaCl between free DNA, nucleosome core particles and particles containing two rather than one octamer histone core. That nucleosome core particles at moderate ionic strength can absorb more than one additional core histone oetamer had already been demonstrated by Voordouw & Eisenberg (1978). Recent investigations have reported varying amounts of free DNA at different nucleosome core concentrations and ionic strengths (Stacks & Schumaker, 1979; Cotton & Hamkalo, 1981; Vassilev et al., 1981; Yager & van Holde, 1984). In this study we investigate the stability of the nucleosome core particle with respect to temperature, particle and NaCl concentration. Weintraub (1983) has recently suggested that, as a result of nucleosome dissociation from covalently closed chromatin, superhelical turns are released, leading to the generation of potential DNase I- and S,-nuclease hypersensitive sites. No nuclease sensitivity is observed in linear chromatin. The effect, in covalently closed chromatin increases with increasing dilution, or in the presence of added “naked” DKA, presumably because of increased dissociation of nucleosomes from chromatin. These observations provide an interesting functional implication for the results of our present study.

2. Materials and Methods (a) Preparation

of nucleosomecore particles

Kucleosome core particles were prepared from chicken erythrocyte nuclei by 2 different, methods following procedures already described (McGhee 6 Felsenfeld, 1980b; Shindo et al., 1980). In the former procedure, removal of histones Hl and H5 was accomplished using an AG50 W-X2 Bio-Rad ion-exchange resin (Ruiz-Carillo et at., 1980). Core-particle digestion patterns were checked by digestion with DNase I in 0.1 M-NaCl, 50 mM-Tris (pH 8) 1 mM-MgCl,, 1 mM-CaCl, at 20 units of DKase I per mg of core particles, during 1 min, at 37°C.

(b) I’hrnr~n,fhrolinr

rcwc.tio~l

\I’r used o-phenanthrolinr (Gould rt rzl.. 1980) t o rrosslink in situ the 2 H3 histones of intact core particles, via the single (‘ysl 10 sulfhgdryl groups. To minimize aggregation ant1 to optimize the reaction for (‘ore particales. the method was slight17 modified, as follow (:ore particles at, A,,, = 2.5 were dialyzed against 25 rnhl-triethanolamine. 2..5 IYIMEQTA, 0.1 mu-phenylmethylsulfonyl fluoride JJH 8. Aft,er dialysis. 0.2 vol. of’ solution c>ontaining 25 mM-1 .JO-o-~~henanthroline, 3.5 rn~~-cqpe~ sulfatr. w-ere added and tl~c, resulting solution was inctubated for 96 h at 4°C’ under c>onstant agitation. The reaction MW stopped by making the solut’ion to 10 mw-EDTA and dialyzing out the r~~agent againsl I m>f-Tris. HCl (pH 8). 0.1 mnl-EDTA (buffer A). c*ont)aining the desired amount of Sa(‘I

Histonrs were analyzed routinely. either in sodium tlotlrc:~-I sulfate; I.‘,(),, (\I )v ) polyacrylamidr gel slabs (Weintraub rt (11.. 1975) or in ureajacetic acitl-c,ontaining vrr.tic*al gels prepared according to Hurley (1977); DNA4 and nucleosomr COW parti,.lrs lvt’rf’ analyzed on acrylamide gels (Peacock & Dingman. 1967) with different concentration of arrylamide (usually 4”); for core particles and 69;, for I)iY;:I). 1)Iih-Aw-as freed of histontls t)J digestion at, 37°C overnight with proteinase K (at an enzyme to substrate rat,io of l : 10) Following that. the solution was made to 0.20,, sodium dodecyl sulfate and incubated fol one more hour at 37°C. For calibration of DNA sizes, HinfT-digested pBR322 1)X,1 fragments were used. Two-dimensional electrophoresis. performed either in the same slab gel. or by cbombining disc and slab gels, was caarried out according to Todd & Garrarcl (1977). Core DPjA fragments digested h,y Dh’ase I were freed from prot,rin as describrti above and run in acrylamide gels under denat.uring rondit,ions (Maniatis rl nl.. 1975).

Sedimentation-velocity analysis was carried out in a Beckman model E anal~-tic,al equipped wit’h electronic control of sl~eed and temprrature and ;I ult,racentrifuge. photoelectric> scanner. Boundaries were recorded routinely at 265 nm except for the studies on concentration dependence when Aze5 was higher than 0.8. In these cases boundaries were recorded at suitable optical densities in the wavelength range from 270 to 290 nm. .\I1 t,he experiments performed with the ultracentrifuge were varried out at \-ariouh temperatures in 1 mM-Tris HCI (pH X.0). 0.1 rnhl-EDTA (buffer A) with different c~onctentmtions of h’aCl. at 40.000 revsjmin. The measured sedimentation c*oefficirnts. .\ were extrapolated to zero concentration and c.onrertrd to standard conditions. s~~,~. I:or this c>orrrction we used the experimental value for the densit,y inc,remrnt (Cp/Gr), = 0.X apphcable below a NaCl concentration of O-5 M (see Fig. I). Kinding of extra histonr octamers to nucaleosome core particles was studied hy scanning sedimentation pattrrns in the ultracentrifuge at 2 wavelengths. 230 and 260 nm (SW the Appendix). (e) Duterminwtion

of conwntratior~s

DNA concaentrations were measured on a Car>, I IX spectroJ)hotometer by using A DNA= 20 cm2 mg-’ at, 260 nm. iVucleosomr core particxlr c*oncrntrations were evaluated by assuming additivity: where M,,,, is the molar mass, 108,800 (De Lange, 1976) and AOct is the absorption coefficient at 260 nm; 0.23 cm2 mg-’ (Stein. 1979), of core histone octamrrs; MD,, is t,he molar .-lNfP of mass, 96.000, of 145 base-pairs of DNA. For t.hr absorption coeficient riucleosome core particles, we calculated a value ofO.5 (zrn2 rng- ‘.

Xl

0

I

I 0.2

0

I NoCL

FIG.

I 04 Cm)

I

' 0.6

0

and apparent volumes (Eisenberg, 1976) 4’ ( ~ 0 - 0 - ) of chicken erythrooyte nucleosomc core particles, as a function of NaCI concentration, mrasurrtl at WY’ in 1 mM-Tris. HCI (pH NJ), 0.1 rnM-F,DTA buffer. I.

Iknsity

increments

(dp/&),

(-m-O-)

(f) /~etrrmination

of (ap/&),

The density increments (8p/&), (E’ lsenberg. 1976), as function of NaCl concentration and in t,hr ultracentrifuge buffer, were determined at, 20°C in an Anton t’aar DMAO:! Ini~rodcnsit’ometer, fitted with a DMA601 density measuring unit. Thorough dialysis was performed at 4°C in the initial stages and finally at the experimental temperature. These values of (~?p/&), (Fig. 1) were determined in terms of the absorbance and can be recalculated should it become necessary to modify the absorbance concentration ralibrat,ion given in the previous section. Apparent volumes. 4’ E (1 - (3p/&),,)/p, were also c~alrulat~ed.

3. Results (a) Preparation

and characterization

of nucleosome

core particles

In t)he initial stages of our work nucleosome core particles were isolated in a typical “one-step” procedure (Shindo et al., 1980). Isolated chicken eryt hrocyte nuclei were digested with micrococcal nuclease to 127; to 15% acid-soluble chromatosome particles containing histones DNA. oligonucleosome and Hl to H5 were precipitated by dialysis against 0.1 M-KU (Olins et al.. 1976). Histone Hl to Hti-free nucleosomes were subsequently purified on a sucrose gradient in 0.1 M-NaCl, 50 m;n-Tris .HCl (pH SO), 0.1 mM-EDTA, at 4°C. Core particles isolated in good yield in this fashion may show a somewhat broader DNA length distribution than particles obtained in a “two-step” procedure and csarefully trimmed to homogeneous size (Lutter, 1978); they may also be cont’aminated by “165” base-pair particles in addition to the canonical “145” base-pair size. To investigate whether t,he results presented in this work with respect t,o nucleosome core particle stability are dependent on DNA length in a sensitive way, we therefore prepared core particles that were much more homogeneous in size. In the procedure used (McGhee & Felsenfeld, 19806). isolated nuclei were mildly digested (to 1 to 1.5% acid-soluble DNA) and Iysed. Histones Hl to H5 were then removed from the released chromatin

(a)

H3

-

H2B

-

H2A

-

(b)

FIG. 2. Electrophoretic analysis of (a) the histone and (b) DNA contents of the nucleosomr core particles isolated from sucrose gradients. (a) Lane 1, gel electrophoresis performed according to Hurley (1977); and lane 2, in conventional sodium dodecyl sulfate/l5% polyacrylamide gels (U’eintraub et a/.. 1975). (b) Lane 1, DNA from nucleosome core particles isolated in the one-step procedure; lane 2 DSA from nucleosome core particles isolated in the two-step procedure; lane 3, pBR322, HinfI-digested fragments. fragments at 0.35 M-x&l by a procedure (Ruiz-Carillo et al.. 1980) that avoids core particle sliding and the resulting stripped chromatin was redigested and then fractionated on a sucrose gradient as above. Different fractions along t,he sucrose gradients (coming from both preparations) were subjected to electrophoretic and sedimentation-velocity analysis. This analysis revealed that the first peak observed in both sucrose gradients was due to free DXA. In the one-step method this peak consisted mainly of “5 S” DNA (corresponding t’o core-size “naked” DNA) whereas in the two-step method this peak contained a mixture of a small amount of 5 S DNA plus larger amounts of smaller DNA fragments released during the digestion following removal of histones Hl to H5. Figure 2 shows the electrophoretic analysis of histones and of DNA extracted from nucleosome core particles. The DNA from the two-step procedure is more homogeneous, with one size centered around 145 base-pairs. Core particles prepared by the one-step procedure exhibit two populations with average sizes of 145 and 162 base-pairs. These results are in agreement with similar results previously reported for both methods (van Holde & Weischet. 1978a). Roth preparations have very similar protein compositions, with very low or almost no content of chemically modified core histones and non-histone proteins.

SUCLEOSOME

CORE

PARTICLE

STRUCTIJRE

83

(h) Stability of nucleosome core particles: dependence on ionic strength, temperature and concentration When nucleosome core particles were subjected to analytical velocity sedimentation, both preparations yielded homogeneous populations of particles with the a&,, value decreasing moderately from 11.2 S to 10.3 S when the NaCl with component of the buffer was raised from 0.1 M to 0.6 M-NaCl. Additionally, increase in salt concentration, a minor component in the sedimentation pattern. with s&w = 5.2 S, corresponding to core-size free DNA, gradually increased in relative amount. Since free DNA had been removed during the fractionation in the linear sucrose gradients, the phenomenon observed above must be ascribed to an equilibrium dissociation of core particles, with increasing loss of stability when the salt concentration is raised. Following recent observations by Yager & van Holde (1984) on the dependence of dissociation and reassociation of nucleosome core particles (DNA mean length, 190 + 15 base-pairs) we have re-examined the time dependence of the process with nucleosome core particles prepared by the one-step procedure described above. by experiments rather similar to those described by those authors. Core particles were incubated overnight at 20°C in: (1) 1 mM-Tris. HCl (pH 8.0), 0.1 mM-EDTA buffer at A,,, = 1.1, (2) 0.75 M-NaCl in buffer at A,,, = 1.1; and (3) 2 M-NaCl in buffer at A,,, = 2.9. The NaCl concentration for all three solutions was then adjusted to 0.5 M at A,,, = 0.7 and the percentage of free DNA was determined by ultracentrifugation after various times of incubation at 20°C (Fig. 3). Times of equilibration are faster than those reported by Yager & van Holde (1984) for particles with long DNA, but they also found shorter times with particles of standard core size (van Holde & Yager, 1984). We both found incomplete reversibility (see also Discussion below), in that core particles (Fig. 3) that had not been exposed to high salt concentrations showed a limiting value, at this core

i

20 ~‘--------------*--

--____

t

8 IL’

l

.

IO i

0 0 0

I

I

I

20

40

60

Time

80

Ih)

FIG. 3. Time dependence of nucleosome core particle dissociation and reassociation at 0.5 M-NaCI. 1 mv-Tris’ HCI (pH 8.0) 0.1 mmEDTA buffer, at 2O”C, as followed in the ultracentrifuge (see the text); - - 0 - - l - - , samples stored at 0.0 M-NaCl; - -A-A - - . sample stored at 0.75 MNaCl; - ~ 0 - - 0 - -, sample stored at 2.0 M-NaCl.

84

2nd

particle coneentrat.ion of IS”/;, fret: DNA. whereas in core partkit?s exposed to hot II 0.75 M and :! M-NaCl about gsy{, DNA remained free in solution. More evidence for the fact that> we are not dealing with two distinct populations of core particles, but rather that an equilibrium process is irnplic*ated. comes f’rorn the t,wo-dimensional elect.rophoresis shown in Figure 4. Free I)SA that has movc~l ahead of DNA in irrt)act, nucleosomr core particles in tjhr first elec,troF)hor,e~i~ moves down diagonally in t,he sec~rnd ele~tro~)hor,t~sis. lilt the samta tirnr additional free DNA dissociates from the intact partivIes and sta>‘s irl)~t~ast of thrl f’rr~ 1)X.\ boundary. At this stage the follo\virrg questions ark,. (1) What is the nat,ure of t,he phenomena ittvc~lvetl itr tire apparent 10s~ of &ability of nucleosome core particles with irrcrrasing c.otr~c,rrtrat,ions of SN( ‘I! (2) Does the observed decrease in the sedimentat ion voeficirtrt valueh involvc~ conformational changes in the (‘ore particle. and what is thvir relationship to t tic* dissociation phenomeml?

To answer the first question we pt~t~forrnetl a series ot’ exlrerirnerrts designed t I) study the ternperat,ure- and c.orlc~cnt~ation-tlr~1)enderrc.c. of thta salt-induvchd dissociation process. Here again. experimental solutions were incubated under the appropriate experimental conditions (temperature. c~orrc,rntratiori) fi)r t imc* intervals of two to four hours, sufC:ient to rcducc t)imtx-dependent rReetjs to ;I negligible level. In Figure 5 we show the dependence of the percent,age of fret> DNA as a function of nucleosome core particle DSA concentration, at 0.6 ~?;a(.“. at. 20°C and ICY’. Iksocsiation is higher at the higher temperat.urr and reaches a

Nl~(‘l,EOSOME

0’

0

(‘ORE

PARTJ(:J,E

S’I’RI:(:‘l’17RE

x5

I

I

I

I

I

02

04

06

08

I

IO

C (mg/ml)

1”1(:. 5. I)q)endenw of the percentage of fire DSA on nuclroxome wre particle concentration (in rng:rnl of J)NA); - 0 - 0 -, 0% wNa(:l, 2OY’; - l - l -. WYj M-Xa(X. IW?. Particles were prepar4 acwnding to Shindo et ul. (1980). ISuffer also contained I mwTris (pH 8.1,) 0.1 mix-EDTA. Inset: lwrwntapr of remaining free DXA (in the limit of high core partide vonc*entration) as a function of N:d’I I~oncentration; (0) our data. O+ .n-Na(‘l. PO”?; (m) Vassilev uf al. (1981): (A) Stacks & Sd~rrrnakrr (1979).

Iowt~r limiting value at high particle concent,ration. The inset shows t,hat the percentage of remaining free DNA determined here increases dramatically at higher salt, concentrations, as reported by other workers (Stacks & Schumaker. lQ7Q: Vassilev et nl., 1981). In Figure 6 we show the percentage of free DNA as a function of NaCI concentration. Free T)?;A increases with increasing salt, caoncrntration, more so at’ the higher temperature. Finally, Figure 7 shows thtb temperature dependence of the percentage of free DNA at 0.6 M and 0.1 M-XaCl for the two types of preparations of nucleosome core particles described above. The t,rimmed core particles prepared in the two-step method behave identically to thrt (Sort’ particles with broader DNA distribution prepared in the one-step procedure. As the concentration of core particles increases beyond about 0.6 mg

I 11:; ’ j :,:-:I 0

0

I

I

I

I

I

I

01

02

03

04 NaCl (m)

05

06

PIG. A. l’rrrrntagr of free I)NA as a function of Nn(:I wncentration. Total DNA concentration. WO4 mgjml. - 0 - 0 - 2OY~; - l - l ~-, 4Y!. I~ufbr and partidr preparation as in Fig. 5.

X6

I

(0) 50 -

1 /

40

-

I !

4 /

30-

:

,/

20-

0

--__--IO-

0

0~

/

-----

‘0

-i

O,/‘O

!

0

301

4

I

I

I

I

I

I

IL--!

I

1

FIG. 7. Percentage of free IINA as a function of temperature. Tot.al DSA concentration. 0.0-1 my!rnl (a) 0.6 M-NaCl; (b) O-l M-NaCl. BufTer as in Fig. 5: nucleosomr core particles prepared awarding to Shindo et al. (1980) (0). and to McGhee & Felsenfeld (1980b) (0).

I

I

FIG. 8. ISoundary anal.ysis (van Holde & Weischet, nucleouome core particles m O-6 rt-NaCl> 1 mw-T’ris.HCl “0°C.

I

I

I

19786) of the sedimentation velocity i~t (pH X-O), 0.1 mwEDTA; 40.000 revs/mm.

NUCLEOSOME CORE PARTICLE TABLE

STRUCTURE

87

1

Sedimentation coeficients szO,w of intact core particles at various temperatures, NaCl concentrations and particle concentrations s2o.w %aC1 t %A (“C) (mgiml) (S) (M) 0.1

10 20

0.040 0.040

11.3 11.3

0.6

11

0.040 0.1 0.25 0.50 0.74 0.99

10.3 10.4 10.5 10.3 10.4 10.3

20

0.040 0.10 0.25 0.74 0.99

10.4 10.4 10.4 10.1 10.0

Buffer: 1 miwTris (pH 8), 0.1 mM-EDTA. DNA/ml, the percentage of DNA dissociating at 0.6 M-NaCl is small (around 5%) and becomes almost negligible at temperatures below 10°C. The values of the sedimentation coefficients of the core particle boundaries are summarized in Table 1. It can be seen that though the values of sZO,wchange with XaCl concentration, they remain independent of temperature and particle concentration, even at 0.6 M-Nacl. Furthermore, the analysis of the boundaries at, this high salt concentration, carried out according to van Holde & Weischet, (1978b), shows that the non-dissociated particles are homogeneous, as can be seen in Figure 8. (c) Reversibility

of DNA dissociation from nucZeosomecore particles

A series of experiments were performed to establish the reversibility of the observed release of DNA from nucleosome core particles prepared from Hl,H5stripped chromatin. Samples at about 0.3 mg DNA/ml (A,,, = 6) in 0.6 MNaCl, 1 mlvr-Tris (pH 8), 0.1 mM-EDTA, were heated for two hours at 3O”C, and run in the ultracentrifuge at 40,OOOrevs/min at 31°C; the amount of free DNA found was 15%. Overnight cooling on ice and subsequent sedimentation at 6°C reduced the free DNA to 8%. When the sample was diluted to about 0.035 mg DNA/ml (A,,, = 0.7) and run at 27.5”C, free DNA increased to 25%. Overnight cooling on ice and sedimentation at 7°C barely reduced this to 21%. Reassociation is thus observed at high enough particle concentrations and becomes inefficient when the particle concentration is reduced. The following observations were made with respect to concentration of salt. Nucleosome core particles at about 0.75 mg DNA/ml (AzGO = 15) were dialyzed against 0.1 M-NaCl, 1 mM-Tris (pH 8), 0.1 mM-EDTA at 4”C, overnight.

8X

.J. AI1SlO.

I). SE(:ER

.lSI)

H. KIS~:Nl~tCI;~:

l:pon dilution to A 260 = 0.7 with the same solvent. IP,, f’rre l.)XAA was observed in the ultracentrifuge at 21 ‘Y’. (Here wc’ would like to point out that at above 20°C: we usua~lly olkwvt~ a sloping tract 0.1 ~-Na(‘l and at. temperatures ubovt~ the boundary in t>he ult~racentrifugc~, indicating sonlti aggrt~gatioli. Tht, tray I)r~c*omr:i horizont,al at lower t,emperatures alld at higher salt c,c,nc,~ntratiotl~.) ‘I’lrrs c~onc~rntjrat,rd solut,ion was now brought by dilution wit’h approl)riatt’ buffr~~ t(j 0% br-Xa(ll. I m;vl-Tris (pH 8). 0.1 mM-E1>7’.4. and .AZhO = 2.5, This “sioc~k” in 0% M-N&I was diluted to .dzbO = 0.7 in thta samt’ bufier and run at %I (’ iti the SCRII to give Z)‘),, Frye l)KA and a flat traccl in t ht* ultr~*1:rrltrifu~t*. ‘l’ht~ st.ovk was now diluted to .4260 = 0.7 with 0.1 M-Na(‘l and ~~ieltlctl II”),, f’rc~ l)N:I at 21 3 in the, ultrac~c~ntrifilg~,. in accord with t,hc 0.1 n~Nx(‘l c*ont rc~l. A sloping t,rac~ above the boundary indicated aggregation. Anot,hcr sample of the stock was dilutcad to ..126O = 0.7 with 0% M-NaCl and then dialyzt~tl for 2 tr at ‘LO“(’ (iI1 Spec*t~rapor :1 Lubrs to avoid hist,onr Iosst3) against st’veral (‘lli~tl~f?h (II’ 0. I U-Sa( ‘I. 1 rnbl-Tris (pH 8). 0.1 miwElI)‘I’A. (!entrifugation a,t 21”t’tiis;c~losctl >I flat ~rnc~ ~(1 200,, frtLf& l)NA. Thus. tbithchr dialysis was not c*omplt%t’c, or. ah tlotcacl b~~fi~r,t~ reassociation was not efficient at t,his low conc~r~ntrat ion. Finally. rea,ssoc:iation was &ted by concentrating a dilute sanrl)lr in an .-!rlilc,c)ll concentrator with PM10 mornbranr (to avoid histonth losses) from A26o = 2.5 (t,hta st,ock solution in 0% .~Xa(‘l of the previous paragraph) lo .-I LhO = 9. at 20°C was 95 to 97c/,, of thr starting mat,erial afttAr c.onc:erltratioli). (recover!, IJtracentrifugation at 40,000 revs/min at 21°C’ gave :I”, fret> IISA. Thus. reversibility is confirmed with increase in particle concentration and histones werp not “lost” in t)he more dilute stock solution at <-12h0 =T:2.5.

For a quantitative interpretation of the dissociation tyuili brium of iIN=\ from of i hfl tnipratiny-o~t,arrl~l, (~orc particles within thr chont’rxt nucleosomr of tlstra histoncl mechanism, we require information conc:tJrning t htt hindinp oct,amers to thta nu&osome carp particle. St.ein (1979) t,stimatrs t.hat the first octarnrr binds with an associat’ion constant K, (St&t%1)iscussion. eyn (4)) roughly greater than 10’ I mo-’ (in unit’s of octamers). From the quilibrium sedimentation data of JGsenherg B Felsenfeld (19X1) in the presence of extra about, 30 x IO6 1 rnol~’ fiJr this binding histones (their Fig. 7) we estimattl constant, though the uncertainty is considerablr. From the sucrost’ gradient data of Eisenberg $ Felsenfeld (their Fig. 1 I ) we cst~irnatr about 106 I mol- ’ only. for to nuc:leosome c+orc’ K,. L%‘z’~conseyuent,ly reinvestigat,ed core histom, binding particles by the mrt,hod described in the Appendix. Figure O(a) shows that) the, increase in sedimentat’ion coefEcientjs, Sag,,,, with increasing Ii (ratio of added histones) is rather similar t,o results histones to nucleosome core particle prcvious1.v reported b>- Voordouw & Eisenberg (197X) and E:isenbrrg & FelsenftM (1981 ). The inset in Figure: 9(a) indicates disappearance of free l>NA in solution at K > 0.4. Figure 9(b) shows that our present, data agretk wrtl with t.hr su(nrostl gradient data (Fig. 11) of Eisenberg & Fetsenfeld (I 981). F rom a r~lot (Fig. IO) of I WTSUS t,he csoncentration (q,) of free histone octamer in solut,ion we clstimate that

XUCLEOSOME I

I 2

I

CORE I

PARTICLE I

I

1

D

I

I

A

1

(a)

.

I

I

.

. T \

89

STRUCTURE

r___

(b)

I 0

I I.0

I

I 2.0

I

I 30

I

I 4.0

I

R FIG. 9. Binding of extra histone octamers to nucleosome core particles at ultracentrifuge at A,,, = 0.7, at 0.5 M and 0% M-NaCl. (a) s20,W versus R, per histone octamers in core particle. Inset: disappearance of free DNA with histone octamers, r, bound per core particle as a function of R; (a) this Eisenberg (1978); (A) Eisenberg & Felsenfeld (1981).

the data are well represented by 2 to 2,5 binding K, value between 1 and 0.8 x lo6 1 mol - I.

(e) Crosslinking

the sulfhydryl

2O”C, as followed in the added histone octamers increase in R. (b) Excess work; (0) Voordoun &

sites (n) per core particle

and a

groups in intact core particles

The two neighboring H3 histones present in the histone octamer core in the rrucleosome core particle were selectively crosslinked via t’heir single sulfhydryl groups by oxidation with o-phenanthroline. After reacting intact nucleosome core particles, 90 to 95% of all H3 histones were crosslinked tjo dimers, as tested by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Chemically reacted core particles were fractionated again in a sucrose gradient. Figure 1 I (a) shows the result as well as the electrophoretic analysis carried out on different regions of the sucrose gradient. As can be seen there, the reaction leads to a complex mixture of several kinds of particles sedimenting at different s values. Three different peaks can be clearly distinguished. The composition of ultracentrifugat’ion each peak is shown in Figure 1 I(b); ( c,) s h ows the analytical analysis. The a values are given in Table 2. Whereas peak 0 corresponds to 5 Sfree DNA, peak 1 contains mainly the crosslinked core particles sedimenting at 10.3 S in 0.6 M-Nacl as well as some free DNA sedimenting at 5 S. To check t’he histonr composition of this 10.3 8 component, two-dimensional electrophoresis was performed as shown in Figure 12. Tt is seen there that the 10.3 S component

90

*I. AUSIO,

D. SEGER

AND

H.

EISENBEKG

0 75 -

2

0

4 c,x107(mol/

FIG. 10. Excess histone free histone octamers.

octamers,

r, bound

per core particle

6 I) as a function

of the concentration.

co, of

contains about 90% of the crosslinked histone H3. Analysis of peak 2 revealed almost the same composition as that of peak 1, by both electrophoresis and sedimentation-velocity analysis, with some increase in the content of a larger particle that becomes clearly visible in the ultracentrifugation and electrophoretic analysis of peak 3. This component has a 16.2 S sedimentation coefficient corresponding to a nucleosome core particle dimer (Tatchell & van Holde. 1977). The origin of this dimer may be due to intercrosslinking of H3 histones of t’wo different core particles, rather than to non-specific aggregation by Cu*+ in t,he reaction. Higher aggregates have not been detected and it can be seen in Figure 12 that the dimer from the histone H3 is present alongside H3 monomers in the band corresponding to the 16 S component. The system becomes more complex, however, when trying to understand the nature of peak 2 in the sucrose gradient (Fig. 11(a)), which has, as alread) mentioned, the same gel electrophoresis and sedimentation pattern as peak 1, yet a higher histone/DNA ratio, as disclosed by the relative values of the A,,,/A,,, ratios in Figure 1 l(a). Another unusual feature, which we believe can be related to some extent to the last one, is the lower degree of stability of the crosslinked particles as compared to the native core particles. Table 2 shows the percentage of free DNA from different fractions along the sucrose gradients as evaluated from the analysis of the boundaries in sedimentation-velocity experiments. From these data, the quantity of free DNA present in peak 2 was evaluated to be 25% higher FIG. 11. (a) Sucrose gradient sedimentation of phenanthroline-crosslinked nucleosomes. (___-, (b) Polyacrylamide (5%) gel electrophoresis of fractions 0, 1, 2, 3 from the A,,,; (- - - -) A,,,. sucrose gradient shown in (a) and from the DNA of the same particles (O’, 1’, 2’, 3’). D corresponds to 145 base-pairs of marker DNA. The inset shows the corresponding sodium dodecyl sulfate; polyacrylamide gel electrophoretic protein analysis of the crosslinking reaction product (C) and fractions 1 and 2; HT, histones before the reaction; Hl, 5. the marker histones Hl and H5. (c) Sedimentation velocity boundaries of fractions 1, 2 and 3 in 0.1 M-NaCl, 1 mi%-Tris.HCl (pH CO), 0.1 mix-EDTA at 20°C and 40,000 revs/min. m, Meniscus; A. free DNA: B, core particles; C, core particle “dimers”.

0

4

0’

I’

8

I2

2’

3’

16

D

0

I

(b)

2 ‘i 3

0 6.1

63

67

65 r (ml Cc) FIG. II.

6-9

92

FIG 12. &dimensional analysis of nucleosomr cow particles cros4inked with phrnatrthroiurr~ reagent. Reacted nucleosomes were first run in a 4 9, polyacrylamide gel (A) and after that thcsy wrcrun in a sodium dodecyl sulfate/lP/, polyacrylamide gel in the second dire&on (13).

than in native (non-crosslinked) core particles under the same c*onditions. I,ow~r stabilit’y of nucleosome core particles with histone H3 internally crosslinked also may bring about a decrease in melting temperatures of these particles (T,vM.is & Chiu, 1980). To explain all these observations we propose a schematic rnod(bl as

Sedimentation coejkients of native and crosslinked nucleosomes nnd Jiactioncltrd materials at 0.1 M und 0.6 WXuU y. Free DNA

Buffer: I mw’l‘ris (pH 8). 0.1 mM-El)?‘A + Nativr cow particles.

N[:(rLEOSOME

(IORE

PARTICLE

STRU(‘TI!RE

93

FIG. 13. Hypothetical scheme proposed to explain the sedimentation behavior of the mixture of r0mponent.s obtained after crosslinking with copper phenanthroline. * Indicates the CysllO positions on the H3 histones. The spatial location of these groups is not implied by the schematic drawing, neither is the spatial location of the histone octamer cores.

shown in Figure 13. According to this model, some of the internally crosslinked core particles (shown in (a)) behave according to the dissociation pattern described above for native core particles (Fig. 11(c), curves 1 and 2), although with a lower degree of stability, probably following the chemical modification introduced. An analogous scheme Figure 13(b) is proposed for crosslinking apparently occurring between individual core particles (the 16 S boundary in Fig. I l(c)). This model explains the experiment’al observations and accounts for the complex situation realized after the crosslinking reaction (Fig. 1 l(a)). In spite of the increase in complexity in the behavior of core particles containing H3 histones internally dimerized by oxidation with copper phenanthroline, these particles still show hydrodynamic behavior identical to that at both 0.1 M-NaCl and 0.6 M-NaCl. Thus it is of intact nat’ive particles, demonstrated that the histone octamer core and the nucleosome core particle remain essentially folded at 0.6 MmNaCl.

4. Discussion (a) Comparison of nucleosome core particle dissociation with results from other studies Sk:in (19’79) reported erythrocyte nucleosome

a reversible core particles

mechanism for the dissociation (with a DNA length distribut.ion

of chicken of 140 to

94

.J. AUSIO.

D. YEGER ,ASD H. EISENI3EK.C:

160 base-pairs) and in size-trimmed particles derived from HeLa cells. whereby an equilibrium is established, in particle preparations at NaCl’concent,rations around 0.6 M, between intact core particles (in preponderance). free DNA and core particles containing an additional bound “migrating” octamer. St’ein dissociated nucleosome core particles by exposure to 2.5 MM-XaCland t~hen quickly decreased He thus studied dissociation followed by a the salt concentration to 0.6 M-?r’aCl. reconstit’ution system, in which he observed an initial fast and then a slowrr step. the origin of which is not clear. Erard et nl. (1982) reported t ha,t caorc-part,i(sle DNA folds and unfolds in processes taking less than a millisec~ond. In another contribution, Stacks & Schumaker (1979) studied by ana,lyt,ieal ultracentrifugation analysis the dissociation of chicken erythrocytr nucleosomes;. with DNA ranging in size between 130 and 180 base-pairs and containing trace amounts of histones Hl and H5. at 22”C, over a range of NaCl concentrations between 0.25 M and 1 M and as a function of part,icle concentration. Increasing dissociation of free DNA was reversibly observed with increasing SaC”l concentrat)ion; and decreasing dissociation. approaching a residual plateau \vaw observed with increase in particle concentration. They also studied t’he transfer of core histones and nucleosome reconstitution at high NaCl c~oncentrat~ions (0.6 M to 0.8 M) to recipient, bacteriophage T7 DNA. which further emphasizes core histom mobility under these ionic conditions. Piucleosome dissociation has also been observed more recently by Cotton & Hamkalo (1981), at) physiological ionic strengths and at, low particle concentrations, by isokinetic sucrose gradient centrifugation. Nucleosomes prepared from mouse L929 cells contained approximately equimolar amounts of the core histones, slightly lower a,mounts of histone Hl and l>XA ranging in length from 145 to 190 base-pairs. Dilution of monomers at 0.14 M-NaCl resulted in rapid conversion of 10 to 400/A of the [3H]thymidine-labeled material from 11 S to 5 S, corresponding to t,he s value of monomer-length DNA. Recycling 11 6 monomers remaining after dissociation through a second dilution in salt generated an equivalent proportion of 5 S material, as seen after the initial dilut,ion. Thus, in agreement with other workers. the existence of a subset of less-stable nucleosomes does not appear likely. A difficult,y exists with respect to underst’anding the separation of the core particle and free DKA peaks in both the analyt,ical ultracentrifugat,ion and sucrose-gradient centrifugations. If t’he equilibria are “instantaneous” (rapid as compared to the relatively slow migration and separation times in thp ultracentrifuge) then. in keeping with basic ult’racentrifugation t)heory (Cann. 1970; van Holde. 1975). only one. more or less broadened or skewed. boundary should be observed in t’he sedimentation patt,erns. Separation of components into distinct patterns may indicate a slower time scale. or a more complex ligand-induced reaction. Ts stoichiomet,ry indeed preserved? Cot*ton & Hamkalo (1981) found by analysis of nucleosome monomers labeled with [ “C]lysine that the 5 S DXA peak was free of histones. yet in the material sedimenting at I I F the c,ount corresponding to the histones was considerabl,v reduced and part of it \vits recoverable from the walls of the centrifuge tube. This is somew-hat ill disagreement with the migrating-octamer hypothesis of Stein (1979). yet it ma)

NUCLEOSOMECORE

PARTICLESTRUCTURE

95

again be related to the diluted state of the system. In our present work we have also looked, by sedimentation boundary analysis according to van Holde & Weischet (1978b), for particles containing two histone octamers rather than one, within the sedimentation boundary (Fig. 8). Such a component has not been observed though it should be characterized by an s value of 13 to 14 S (Stein, 1979: Eisenberg & Felsenfeld, 1981) as distinct from 11 S for the native nucleosome particle (these values should be normalized in dependence on the NaCl concentration). Va,ssilev et al. (1981) claim heterogeneity of Ehrlich ascites tumor nucleosomes, upon dissociation with 1 M-NaC1 and other salts and analysis on metrizamide gradients. They studied mononucleosomes prepared in a one-step digestion containing equimolar amounts of all the histones and nucleosome cores prepared by stripping histone Hl before digestion with nuclease and crosslinking of the core mononucleosomes with dimethylsuberimidate. The latter procedure is rather drastic and modifies about 70% of the lysine groups. DNA length distribution is not specified. Though the systems are rather different, these authors, in keeping with results previously discussed, report particle-concentration-dependent onestep dissociation. Nucleosome heterogeneity, claimed on the basis of the coexistence of intact and fully dissociated oligonucleosomes, may be discounted (see below) in our interpretation of the migrating-octamer mechanism. It has not been truly established that either modification (acetylation) or the presence of histone variants is responsible for altered nucleosome structure and stability. Very recently, Yager & van Holde (1984) have studied nucleosomes containing long DNA (190+ 15 base-pairs) and inner histones only. They also found increasing dissociation, with free DNA increasing with increasing salt and decreasing particle concentration. One study (Erard et al., 1981) clearly disagrees with the idea that DNA size and size distribution are not critical in determining the stability at high NaCl concentration, by claiming that core particle stability critically depends upon a small number (two base-pairs) of terminal nucleotides. (b) Analysis of core particle dissociation by the migrating-octamer mechanism A crucial observation for the interpretation of core particle dissociation is that, though dissociation strongly increases with decreasing particle concentration, some dissociated DNA (the amount depending on temperature and ionic conditions) remains even at the highest particle concentrations (Stacks & Schumaker, 1979; Vassilev et al,, 1981; this work). Stein (1979) assumed that all histone octamers released are complexed with core particles (thus no free histones are present in solution), which led him to believe that core particle dissociation is independent of core particle concentration. We shall show, below, that the migrating-octamer mechanism proposed by Stein correctly describes the results, when applied without the above simplifying approximation. It is difficult to make a precise comparison between the work of Stein and our own approach. He raised the NaCl concentration to 2.5 M, at which DNA and

96

.I. AliSlO.

I). SE(:ER

ANI)

H

EISE:SISER(:

histones are completely dissociated from each other. and then decreased thr Ka(ll concentration quickly back to 0% M. About XOo,i, of the DNA and histones “instantaneously” refolded into proper nuclrosome core part,icles and core particles containing an additional histonr octamer, followrd by a “slow” step at the conclusion of which only 9 t,o IO:,, free DNA remained. independent of part,icle concentration, according to St’ein (1979). The slow step is a consequence of the competition by DNA (D) and core particles ((I) for hist,onr oct.amers (0) to J-ield either C‘. or core particle-histom octamer caomplrxes (( ‘0). Kinetic and equilibrium constants were derived by Stein from the rrlaxation of ;1 reconstituting DSA-histonr mixture to equilibrium. at 0% .M-KaC1. rather than from direct observation of t,he properties of native (‘ore particles under a varier) of conditions, as studied by us and by ot,her authors. The migrating-ocbtamer mechanism (Strin, f 979) assumrs the t,wo equilibria: DSO F! C’ and governed by the equilibrium

(‘+o

2 c:o

iI)

(2)

association constants: K, = IC]/II>][Ol

(3)

K, = [C’O]/lC:j[ O]

ill

and We have further: [ I)]-

IO] = [C’O]

and --[Cl = [D]+lc’o where c is the starting concentrat’ion of undissociated nucleosomes, which can 1~ calculated from the total DNA concentration and known core-particle composition. We also define a constant: K = K,iK2

= 1C]2/1f)]lC‘O],

(7i

which describes t,he eyuilibrium: 1) + co e Lx’

(8)

characteristic of the migration of a hist.one octamer bet.ween t.wo core particles?. Assuming 0 to be approximately zero, which is the equivalent of saying that all free histones are complexed into the CO form, Stein argued that the fra,ction x = D/c of dissociated core particles is independent of c. Experimental evidence though shows that x increases at low c values; yet reaches a constant limiting value at high c values, which is in good agreement wit’h the mechanism proposed t We should recall here that though the histones complex as octamers (Eisenberg & Felsenfeld. 1981) they coexist as H3,H4 tetramers and H2A,H2B dimers in 0.5 M or 0.6 iM-NaCl solution. We cannot provide a complete analysis of all binding equilibria. Following t,he observation (Eisenberg & Pelsenfeld, 1981) that H2A,H2B histones do not complex with nucleosome core particles we could argue that the equilibrium is between core particles and H3,H4 tetramers; the H2A,H2B dimers. available in stoichiometric amounts, fitting into the sites formed by the core particle H3,H4 trtramer complex. Reference to histone octamers in solution is thus to a nominal species. the molar concentration of “oetamers” and H3.H4 tetramers being identical.

NUCLEOSOME

CORE

PARTICLE

STRUCTURE

97

(it is indeed not necessary to assume a subpopulation of non-dissociating core particles, for which no good evidence exists, to understand that, at high c values. x does not go to zero). We solve equations (3) to (7) to yield: K=

(l-2x)2

(9)

which, in the limit of large c values, reduces to: K = (1 - 2S,in)‘/X~in, allowing evaluation of K from the limiting minimum value, xmin. K, can then be derived from the complete quadratic equation (9); K, is given by equation (7). If xmin tends to zero, K goes to infinity and equation (9) reduces to the simple form: K, = (1 -x)/cx2

(11)

applicable to the reaction given by equations (1) and (3) only. A summary of the calculations is given in Table 3. For core particles, at 20°C and at 10°C (Table 3A(l) and (2)) and at 0.6 M-NaCl, we have calculated K from x,~,, using equation (10) and K, using equation (9). For core particles at various temperatures in 0.1 M-NaCl (Table 3A(3)) we assume .rmin = 0, reducing the analysis to the simpler equation (11). For the temperature dependence in 0.6 MNaCl (Table 3Af4)) for which K is- not known (except at 10°C and 20°C; see Table 3A(l) and (2)) we took K2 = 0.19 x lo6 1 mol- ‘, independent of temperature. Indeed K, at 10°C is rather close to its value at 20°C. We then calculated K, by equation (9) remembering that K = K,/K,. The work of Stacks & Schumaker (1979) is summarized in Table 3B, at 0.6 M, 0.8 M and 0.9 M-N&l. using equation (9) and a value of K at 0.8 M and 0.9 M-h’aC1 derived from xminr as reported by these authors. In Table 3C(l) we calculated K, from the data of Cotton &. Hamkalo (1981) in 0.15 M-NaCl using equation (II), assuming xmin = 0 at t.his salt concentration. This is indeed also observed by these authors. In Table 3C(2) we calculated K, from the data of Cotton & Hamkalo for various l;aC’l concentrations. As the ?jaCl concentration does not exceed 0.35 M we still assume xmin = 0. The summary presented in Table 3 is of great interest. In our work we have used chicken erythrocyte core particles and have precisely specified the experimental conditions used. We compared t’hese data (Fig. 14) with results on core particles from various sources, with some uncertainty in the temperat’ure specification, in a plot of In K, against In mNaC,. Wit’hin the relatively large error that we expected from this combination of experiments, Figure 14 discloses consistency beyond expectation. From the slope of the curve we derived AnNac,, being the number of moles of Na released per dissociated mole of core particle (Record et al., 1978). We calculated AnNaC,eq ual to about 2. which is far less than the number of possible interactions commensurate with 145 base-pairs of DXA. 4

98

J. AUSIO,

D. SEGER

AND

TABLE

Dissociation

H. EISENBERG

3

constants of nucleosome core particles NaCl concentration and core particle

as a function concentration

A. This work (chicken erythrocyte cow pwrticles) (1)

0.6 iwNaCI xmin = 0.05

Suffer A K = 324

K, x 1O-6 K, x IO-” -AC; (1 mole ‘) (kcal mol ’ )

(mg D!bA/ml)

.r

0.035 0.2 0.4

0.185 0.100 0.075

69 515 68

Average

64

(2)

0.6 M-NaCl Zmin = 0.035

(mg DiA/ml)

10.5

10-C’

- AG’”1 K, x 1W6K, x 1OF6 (I mol-‘) (kcal mol-‘)

0.135 0.085 0.055 0.045

137 72 122 185

Average

129

0.18

IO.6

0.1 M-NaCl Buffer A emin = 0 K-P02 c = 0.035 mg DNA/ml

(3)

(4)

0~20

Buffer A K = 706 z

0.035 0.2 0.4 0.6

“o”( ’

(4,

x

K, x 1V6 (I mol-‘)

5 10 15 20 25 30

0.035 0.05 0.065 0.095 0.14 0.185

2170 1050 609 276 121 66

-A(;‘; (kcal molF’) I I.9 11.8 Il.7 11.4 11.1 10.9

0% M-NaCl Buffer A K, =0.19x IO6 1 mol-’ c = 0.035 mg DNA/ml K, x lo-’ (I mol-‘)

5 10 15 20 25 30 Buffer A EDTA.

-Ac:; (kcal mol-‘)

0.125 161 10.5 0.135 137 10.6 0.150 109 10.7 0.185 68 10.6 0.275 Pi.1 10.2 0.400 IO.5 9.8 is 1 mrt-Tris.HCI (pH 8), WI rn~

of temperatuw.

NUCLEOSOME

CORE

PARTICLE

STRUCTURE

99

TABLE 3 (continued) B. Stacks & Schumaker (1979) particles)

(chicken erythrocyte core

t = 25Y’, c = 0.025 mg DNA/ml Buffer: 10 mat-Tris HCl (pH 8) N&Cl (M)

K, x 1o-6 (1 mol-‘)

K

o+.l

0.165 0.295 0.405

0.8 0.9

324t 181 181

24.0 12.7 9.6

-AG; (kcal mol-‘) 10 9.7 9.5

t From our work. 1 From z,,,~” = 0.16.

C. Cotton particles)

& Hamkalo

0.15 M-NaCI Zmin = 0

(1)

(mg DkA/ml) 0.010 0.005 0.002 0.001

(2)

(1981)

(mouse L929

cell core

Buffer: 10 mllr-Tris (pH ‘i’.3), 1 mrvr-EDTA

z

K, x lO-6 (I mol-‘)

0.220 0.234 0.274 0.400

155 270 466 361

Average

313

-AG; (kcal mol-‘)

11.5

c = 0.001 mg DNA/ml Zmin = 0 Buffer: 10 miwTris (pH 7.3), 1 mrt-EDTA NaCl (M)

32

0.05 o-15 0.25 0.35

0.29 0.38 0.56 0.56

K, x 1O-6 -AG; (1 mol-‘) (kcal mol-‘) 813 414 230 135

12.1 11.7 11.4 11.0

One must conclude that the electrostatic aspects of the interaction between DNA and the core histones are overshadowed by interactions of a different nature. From the values of AG” (in Table 3) and their dependence on temperature, we could calculate heats and entropies of interaction, yet it does not appear likely that strong statements can be made at present. The values of K, derived from the analysis of all the results in Table 3 are much smaller than the values reported by Stein (1979, Table 2) from an analysis of his reconstitution experiment. He thus reported an equilibrium constant of K = 3 x 10” 1 mol-’ at 26°C and 0.6 MM-NaCl (pH g), whereas we find (Gable 3A(l)) K, = 6.4 x 10’ 1 mol-’ at 20°C for the same process; K, is subject to t’he same discrepancy and only the values of K are of the magnitude expected

100

.I. AUSIO.

1). SEGER

AND

H. EISENKtSK(:

FIG. 11. Double logarithmic plot of equilibrium constant K, against Sa(‘l molarity work: (A) Starks & Schumaker (1979); (0) Cotton 8~ Hamkalo (1981).

VICAR,.(0~ this

from his work. We find K, equal t’o 0.X to I.0 x IO6 1 tnol- ’ by a direct binding study (Results, section (c)), i.e. approaching the value (about 0.2 x IO6 1 mol-‘) resulting from the present analysis. The reconstitution experiment described h> Stein may not, necessarily be comparable to thr straightforward dissociation studies analyzed here. Although we believe that the analysis of core pat%ic~lc dissociation emtbrges qualitatively correct from the analysis we have presented on the basis of our work and of others, variability in c>ore particle stability may persist’ depending on t hts presence of histone variants and possibly on post-synthetic modificat,ion (RuinCarillo et al.. 1975).

In a recent study, Daban bz Cantor (1982) attached the fluorescent, reagent N-(1-pyrene)maleimide to the sulfhydryl cysteine 110 group of histone H3 and t’hen studied reconstitution of pyrene-labeled core particles by salt-. jumping from 2 M-NaCl to between 0.6 M and 0.2 M-NaCI. The measurements were particle concentration. They performed at very low (3.3 x lop3 mg DNA/ml) report (their Fig. 3) a sharp transition in fluorescence intensity between 0% M and (b4 M-Nacl, which they ascribe to a strong nucleosome core-particle conformational transition in this range of NaCl concentrations. From our analysis. above, of core particle dissociation at low particle concentration, we calculate (at the concentration reported by Daban & Cantor) a rather smooth increase in the value of x from about 0.4 at 0.25 M-NaCl to about 0.7 at 0.8 M-l?;aCl. Though the particles are partially dissociated, no sharp transition of t,he type reported t)>

NIICLEOSOME CORE PARTICLE

STRt~r(:TlrRE

101

Daban & Cantor is expected for native core particles. It seems to us that it is essential, in particular when studying systems as sensitive as nucleosome core particles. whenever attaching a bulky perturbing probe, to establish (by accessory hydrodynamic or scattering experiments) that the attachment of the probe is not affecting the behavior one wishes to establish. Thus Dieterich et al. (1979), using a) similar approach, previously reported strong unfolding of nucleosome core particles at 0.6 x-NaCl, which has not been corroborated by hydrodynamic (McGhee d al., 1980; Eisenberg & Felsenfeld, 1981) or small-angle X-ray scattering (Reich, 1982) measurements. Tt could be that high salt concentration promotes a rearrangement of histones with little change in the overall size of the pa.rGle. We have noted above that in situ crosslinking of the histone H3 Cysl 10 groups leads to some further destabilization of the core particle DXA-histone interaction, though the non-dissociated internally crosslinked core particles behave similarly to native core particles at both 0.1 M and 0.6 M-N&l. Having thus separated the processes of core particle dissociation and conformational change, we believe that DNA conformation is unchanged between 0.1 M and 0.6 M Na~Cl and that moderate changes in frictional parameters may be due to unstructured tails of histone loosening with increased NaCl concentration. Gary d al. (1978) found by high-resolution proton magnetic resonance studies that between 0.3 M and 0.6 M-Nacl there is further release of basic regions of histones HS and H4 from the core particle complex. The polar tails of histones H2A and H212 arc more mobile at lower ionic strength. This may also be in accord with the observations by Eshaghpour et al. (1980) of a constant distance from the 11XA t,crminus to CysllO of hist’one H3 in chicken erythrocytc core particles (in very low concentrations of salt: in 0.6 ivr-NaCl, and in high concentrations of urea). provided that the structural equivalence of labeled and native particles at low and high salt is established.

Appendix Binding of extra histones to nucleosome core particles was studied by scanning ultracentrifuge sedimentation patterns at 230 nm and 260 nm. To determine optical densities at. the two wavelengths we used a collimator (Flossdorf, 1980), manufactured by Dr W. Schrader, Rraunschweig, attached to the Beckman model E ultracentrifuge. Considerably increased light intensity in the ultraviolet, allows scanning down to 220 nm. Histone and DNA concentrations in the plateau and in the particle-depleted supernatant can be calculated by using the known absorption coefficients of the two components at the two wavelengths (Stein. 1979). A typical experimental result. obtained following sedimentation at 10.000 revs/min at 20°C for 60 min, is shown in Figure 15. Tn Figure 15, J: is A,,, in the plateau region and y is excess AZSO (over A,,, in the supernatant), also in the plateau. From the ratio y. measured at various times t and ext,rapolated to t = 0 (to allow for radial dilution), we calculated T, the number of excess histone octamers bound per nucleosome core particle. To determine the concentration of “free” hist,ones in the plateau, identified with

102

J. AUSIO,

D. SEUER

AND

H. EISENBEK(;

-

0230

FIG. 15. Schematic representation of sedimentation pattern of nucleosome core particles, with itdded histones; R z 2; (~ ) A,,,; (- - - -) A,,,; for details. see the text.

histone concentration in the supernatant, we should determine y*. t,he AZ30 value above the sediment,ating boundary. The base-line O,,, can be obtained by scanning the double-sector cell at 230 nm. containing buffer in both compartments. An alternative simple procedure is to determine R. the input ratio of added histone octamers to nucleosome core particle histone ortamers by analysis in a spectrophotometer before the experiment. Free histones in solution can then be obtained by multiplying the difference (R-r) by the nucleosome core particle concentration. Calibration of the optical system was undertaken wit,h nucleosome (lore particles at R = 0, in 0.1 M-NaCl, 1 mM-Tris. HCl (pH 8.0). 0. I mm-EDTA. clarified by centrifugation for 20 min at 12,000 g. The values of AzjO and A,,, were determined in a Cary 118 spectrophotometer and then in the ultracentrifugr at 10,000 revs/min. The base-lines O,,, and OzeO had previously been determined with the same cell at the same velocit,y, with buffer in both compart,ments of the cell. The nominal factor A,,,(ultracentrifuge)/dz,,(spectrophotometer) - 1.2 WLLS determined precisely. We found that, this factor divided by the corresponding fact,or at 260 nm was equal t,o O-974, rather than unity. This may be due to the difference in the spectral profile of the light source (high-pressure mercury--xenon lamp) in the ultracentrifuge to the light source used in t,he spectrophotometer. It is thus demonstrated that binding studies can be undertaken conveniently in the ultracentrifuge by scanning at more than one wavelength. We thank Hanna Gould for introducing us to the topic of specific crosslinking of histone H3 in the nucleosome and for initial experiments. We acknowledge permission from Tom Yager and Ken van Holde to quote results in the course of publication. We also thank Yeheskiel Haik for expert technical assistance with the ult.racentrifuge. This study was supported by grants from the Tsrael-U.S. Binationat Foundation.
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by A. Klruy