109

Biochimica et Biophysica Acta, 442 (1976) 109--115 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98656

ION BINDING AND CHROMATIN CONDENSATION

G.A. JACOBS, J.A. SMITH, R.A. W A T t and J.M. BARRY

Department of Agricultural Science, University of Oxford, Oxford, (U.K.) (Received February 2nd, 1976)

Summary 1. The binding o f 4SCa2+ to hen erythrocyte chromatin has been studied to help elucidate how cations induce a reversible condensation of this chromatin. 2. As the u n b o u n d Ca 2÷ of the medium rises from 0.5 to 4 mM, Ca 2÷ is bound to the chromatin with a stability constant of approx. 3.1 and a saturation value of 0.25 Ca 2÷ per DNA phosphate, or one-half the value for pure DNA. Condensation of the chromatin is half complete when this binding of calcium is roughly half complete. Hence the transition from the uncondensed to the condensed state occurs as repulsion between the free DNA phosphates of erythrocyte chromatin is neutralised by bound cations. Genetically active chromatin may be maintained in an uncondensed state in living cells by the presence of different negative groups that remain unneutralised at the u n b o u n d cation concentrations of the cell. 3. That only one-half of the calcium binding sites of DNA are masked in erythrocyte chromatin supports recent models of chromatin structure in which the DNA double helix is wound round a core of histones. 4. Competition for calcium binding sites in the chromatin by other cations was also studied.

Introduction The nucleus in the hen erythrocyte is small and its chromatin is condensed. When isolated hen erythrocyte nuclei and chromatin are suspended in media of low u n b o u n d cation concentration they swell, this change being immediately reversed if the u n b o u n d cation concentration of the medium is raised. An understanding of the mechanism of this change may help to explain how changes in chromatin condensation are induced within living cells. We have previously shown t h a t the change is half complete at concentrations of different cations that should approximately half neutralise those phosphates of DNA that remain uncombined with histones [1]. We concluded that the transition

110 from the uncondensed to the condensed state occurs when repulsion between the DNA phosphates of the erythrocyte chromatin is largely neutralised by bound cations, as it would be in the intact erythrocyte. We now provide further support for this conclusion from a study of the binding of radioactive calcium to hen erythrocyte chromatin. Experience shows that it is first necessary to describe clearly what we mean by th~ binding of cations to chromatin. This is an instance of the general p h e n o m e n o n of specific ion binding, the most familiar example of which is the binding of hydrogen ions to anions to give undissociated acids. In the product, the ions m a y be bound by undefined forces, but essential characteristics of this binding are loss of mobility in an electric field of equal numbers of positive and negative charges; loss of the independent colligative properties of the bound ions; and rapid interchange between the bound and u n b o u n d forms. The law of mass action may be applied to specific ion binding, and each equilibrium is normally characterised by a stability constant, which is lOgl0 of the association constant. Comprehensive tables of stability constants have been published [2]. Binding of cations to DNA has been extensively studied. The binding is primarily to the negatively charged phosphates, although interactions with bases can also occur [3,4]. At physiological pH DNA binds at saturation one divalent cation per two phosphorus atoms [5--10]. The binding of a divalent cation, such as calcium, to a pair of adjacent phosphodiester residues in DNA may be represented by the equation: K =

I X . Ca] [X 2- ] [Ca2*]

where log~0K = the stability constant for the binding. In chromatin, the number of cations bound by the DNA is reduced to about one-half, as discussed'later. Materials and Methods 4SCaCl: was bought from the Radiochemical Centre, Amersham, U.K. Other chemicals were bought from Sigma Chemical Co. Hen erythrocyte nuclei and chromatin were prepared as before [1]. The DNA content of chromatin suspensions was calculated from the concentration of erythrocyte nuclei used (measured in a hemocytometer), and the value of 2.6 • 10 -12 g DNA per nucleus (ref. 11 and Billett, M.A., personal communication). The chromatin was suspended in 4 mM 4SCaC12/20 mM NaC1/10 mM TES (N-tris(hydroxymethyl)methyl-2-aminoethane • HC1) buffer pH 7.4 at approx. 0.5 mg DNA/ml. When determining the calcium bound at a range of calcium concentrations, 0.25 ml portions of this suspension were diluted with 20 mM NaC1/10 mM TES buffer pH 7.4 to the desired concentration. The suspensions were then filtered with suction through weighed membrane filters (25 mM diameter, 0.45 pm pore diameter; Sartorius Co., G.F.R.). The moist filter plus precipitate was reweighed, placed in a vial with 0.5 ml H20 and 5 ml scintillant (butyl-PBD (7 g/l) in a mixture of 2 vol. toluene and 1 vol. Triton X-100), and counted in a Beckman scintillation counter. Portions (0.5 ml) of the 4 mM 4SCaC12 solution were also counted to determine the specific activity, and to enable the counts of chromatin to be corrected for adhering solution. The calcium bound by chromatin in these and other solutions was also deter-

111

mined in another way as follows. Portions (1 ml) of the chromatin suspension in 4 mM 45CAC12/20 mM NaC1/10 mM TES pH 7.4 were put into weighed 2-ml plastic tubes and washed three times with the desired solution on a centrifuge. The tube was drained, reweighed, and placed in scintillant for counting as before, the count again being corrected for adhering solution. The two methods gave identical results. Results A feature of the ion binding to chromatin which we study here is the rapid attainment o f equilibrium. This is illustrated by our finding that radioactive calcium which is bound to erythrocytd chromatin is completely removed by three washes on the centrifuge with a medium containing unlabelled 4 mM CaC12. Also, binding of radioactive calcium did n o t increase when chromatin was left in contact with a radioactive solution for one hour rather than a few minutes. Fig. 1 shows the number of Ca 2÷ bound per atom of DNA phosphorus by hen erythrocyte chromatin, at various u n b o u n d Ca 2÷ concentrations in a medium also containing 20 mM NaC1 and 10 mM TES buffer at pH 7.4. Values for calcium bound by whole nuclei were very similar. Scatchard [12] derived an algebraic expression from the mass action equation which may be applied to the experimental values in Fig. 1 to determine the calcium bound to erythrocyte chromatin at saturation. His expression may be represented for present purposes as: n -

= K(N

-- n)

C

where n is the number of Ca :÷ bound to chromatin per atom of DNA phosphorus at an u n b o u n d calcium concentration of c gions/1; N is the number of

i

100~

0.20

7".

0.15 0

0.10

0.0,5

& o

I

1

I

2

I

3

4

[Unbound Ca 2+] (raM) Fig. 1. E f f e c t o f u n b o u n d Ca 2+ c o n c e n t r a t i o n in m e d i u m o n (a) n u m b e r o f C a 2 + b o u n d p e r a t o m o f D N A p h o s p h o r u s b y h e n e r y t h r o c y t e c h r o m a t i n (o o) a n d (.b) t h e A 2 6 0 n m ( c o r r e c t e d f o r s c a t t e r i n g ) of t h e s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n o f a s u s p e n s i o n o f h e n e r Y t h r o c y t e c h r o m a t i n at 3 0 0 0 × g f o r 15 m i n (e "-) ( t a k e n f r o m L e a k e e t al. [ 1 ] ) .

]12

i ~oor o' 7]

(J

0

0.05

0,10

0.15

0.20

0.25

0.30

Ca 2+ bound/atom DNA phosphate F i g . 2. S c a t c h a r d p l o t o f v a l u e s in Fig. 1 f o r C a 2+ b o u n d

by hen erythrocyte

chromatin.

Ca 2÷ b o u n d at saturation; and K is the association c o n s t a n t w h o s e log10 is the stability c o n s t a n t Ks. If n/c is p l o t e d against n, a straight line w o u l d result if Ks remained c o n s t a n t from t h e binding o f the first to the last Ca 2÷ to chromatin. Fig. 2 s h o w s our results in Fig. 1 replotted in this way. The values for 0.5 mM calcium and above lie on a straight line. The intercept o f this line o n the n axis gives the value o f N, namely, 0 . 2 5 3 Ca :+ b o u n d t o e r y t h r o c y t e c h r o m a t i n per a t o m o f D N A p h o s p h o r u s at saturation. This value will be u n a f f e c t e d by the presence of Na ÷ and buffer ions in the solution. The intercept o f the same line o n the n/c axis TABLE

I

COMPETITION MATIN

BY OTHER

CATIONS

IN BINDING

OF CALCIUM

TO HEN ERYTHROCYTE

CHRO-

H e n e r y t h r o c y t e c h r o m a t i n (in 1 m l o f 4 m M 4 5 C A C 1 2 / 2 0 m M N a C 1 / 1 0 m M T E S p H 7 . 4 a n d c o n t a i n i n g 0.5 mg DNA) was put into a weighed 2 ml plastic tube and washed three times on the centrifuge with one of the media shown. The tube was drained, reweighed, and its radioactivity corrected for adhering medium. Medium

Calcium bound

45CAC12 2 m M / N a C l 2 0 m M / T E S 1 0 m M p H 7 . 4 + 2 0 m M NaC1 -- 10 mM NaCI -- TES + e t h y l a m i n e HC1 1 0 m M + b u t y l a m i n e HC1 1 0 m M + polylysine (tool. wt. 1000--4000) 2 mM* + polylysine (tool. wt. 4000--15000) 2 mM * + polyserine (mol. wt. 5000--10000) 2 mM * + Histone H1, 200 pg/ml + Histone H3, 200 pg/ml + MgC12 1 m M + MnCl2 1 mM

(45CAC12 a t 1 r a M )

* With respect to amino acid.

Ca2+/DNA phosphate

Relative

0.173 0.159 0.182 0.185 0.166 0.168 0.012 0.012 0.187 0.093 0.135

100 92 105 107 96 97 7 7 108 54 78

0.081 0.059

47 34

113

gives K N , and equals 288. Hence K = 1138 and Ks = 3.06. This value of K s will be greater than the true value owing to the slope of the line being reduced by competition from Na ÷ and buffer ions. However, as seen later, and as expected from published values of stability constants, this competition is n o t great, and the effect on K s will be small. The three experimental values in Fig. 2 at 0.25 mM Ca 2÷ and below lie on a curve of steeper slope. This could mean that the initial Ca 2÷ bind to each chromatin particle with a higher stability constant. However, below about 0.5 mM Ca 2÷ hen erythrocyte nuclei and chromatin, in addition to swelling, tend to clump, possibly as a result of a reversal of net charge from positive to negative. We find that intact nuclei which have been equilibrated in 4 mM 4SCa2+ never lose all their radioactivity when washed in unlabelled CaC12 at below 0.3 mM. It appears that 4SCa2÷ is occluded by the clumped nuclei. If, as here, the nuclei are first disintegrated into chromatin by sonication then all radioactive calcium can eventually be removed. Nevertheless, it is probable that the apparently stronger binding of calcium to chromatin at 0.25 mM 4SCa2÷ and below is due to incomplete removal of ocCluded 4SCa2÷ in three washings at these concentrations. Table I shows the effect of other cations in the medium on the a m o u n t of calcium bound to hen erythrocyte chromatin. Discussion We have previously concluded [1] that the transition of hen erythrocyte nuclei and chromatin from the uncondensed to the condensed state as the cation concentration in the surrounding medium is raised, is due to neutralisation of those DNA phosphates that are uncombined with histones, so eliminating their mutual repulsion which is responsible for the uncondensed state. This conclusion was based on the indirect evidence that the transition is half complete at concentrations of different cations that should approximately half neutralise the DNAphosphates. We now provide more direct evidence. In Fig. 1, in addition to the curve for calcium binding to chromatin, is a curve taken from a previous paper showing the effect of increasing calcium concentration on the sedimentation of hen e r y t h r o c y t e chromatin at 3000 × g for 15 min. This curve has been found to be almost identical to that relating hen erythrocyte nuclear volume to calcium concentration, and the change in sedimentation results from condensation of the chromatin fibres [1]. It is seen that the condensation is in fact half complete when the negative charges are roughly half saturated. That these charges are in fact those of DNA phosphates is proved by their ability to bind 0.25 Ca 2÷ (i.e. 0.5 equivalent of Ca 2÷) per DNA phosphate. Other negative groups in chromatin could n o t provide this total charge equal to one-half of t h a t on the DNA phosphates [13]. Also the stability constant of 3.06 at 0.5 mM Ca 2÷ and above is that expected for binding to DNA phosphates, and is m u c h higher than the constant for binding to carboxyl groups [2]. These results therefore add weight to our previous suggestions as to how decondensation of chromatin is induced in living cells. In most cells, although the concentration of u n b o u n d divalent cations appears to be too low, the concentration of u n b o u n d Na ÷ plus K ÷ appears to be sufficient to neutralise the bulk of those DNA phosphates that are uncombined with histones, and so

114

maintain the condensed portions of the genome in a condensed state [ 1 4 ] . In a few kinds o f cell a general decondensation of chromatin may be induced by the u n b o u n d cation c onc e nt r at i on of the cytoplasm falling to a level at which cations dissociate from DNA phosphates. This appears to be a mechanism by which the egg and sperm chromatin are decondensed on fertilisation of the Xenopus egg [ 1 5 ] . On the o t h e r hand, local decondensation of chromatin associated with gene activation may be induced by the local i n t r o d u c t i o n of negatively charged groups that, unlike DNA phosphates, are unneutralised, and hence mutually repulsive, at the normal u n b o u n d cation concentrations of the cell. Carboxyl and primary phosphate are such groups [2]. It is seen in Table I that the a m o u n t o f c a l c i u m bound to chromatin was little altered by raising the NaC1 c o n c e n t r a t i o n from 20 to 40 mM, by lowering it to 10 mM, or by removing TES buffer from the medium. This was e x p e c t e d from the fact that 85 mM K + is needed to induce half-condensation of hen e r y t h r o c y t e chromatin as opposed to only 0.75 mM Ca 2÷ [ 1 ] . Hence t he value o f 3.06 d ed u ced f r om Fig. 2 for the stability constant for calcium binding t o c h r o matin at 0.5 mM Ca 2÷ and above can be little affected by c o m p e t i t i o n f r o m sodium or buf f er ions. As expected, this value is similar to t hat for calcium binding to phosphodiesters groups such as those in t ri m et aphosphat e [2]. Table I also shows t ha t ethylamine and butylamine had little effect on the q u a n t i t y of calcium b o u n d to chromatin, whereas polylysine (as opposed to polyserine) almost completely displaced the b o u n d calcium. It is clear that an isolated primary amino group has only a low affinity for chromatin similar to t h a t of sodium. But when a n u m b e r of these groups are adjacent in the same molecule, as in polylysine, the affinity of the molecule is very great. It is seen that histones H1 and H3 were m uch less effective t han polylysine in displacing b o u n d calcium. T hey are evidently unable, even when present in excess as here, to bind to ch r o ma t i n in such a way that almost all DNA phosphates are neutralised. Table I also shows t hat replacement of one-half of the Ca > in the medium by Mg > reduced the b o u n d Ca 2÷ to about one-half, showing that t hey have similar affinities for the chromatin. Replacement by Mn > caused a greater reduction. This agrees with half-condensation of e r y t h r o c y t e chromatin being induced by ab o u t 0.8 mM Ca 2+ or Mg > , but by only 0.4 mM Mn > [ 1 ] . It has been shown t hat DNA will bind 0.5 Ca 2÷ for every one of its phosphate groups [ 5--10]. We deduced from Fig. 2 t hat hen e r y t h r o c y t e chrom at i n binds 0.253 Ca 2÷ per DNA phosphate at saturation, or close to one-half the n u m b e r b o u n d by pure DNA. Many ot her instances have been r e p o r t e d in which th e binding of a ligand t o DNA is reduced to precisely [7,8,16--19] or roughly [ 20--22] one-half in chromatin. Recent evidence [ 23,24] suggests that in c h r o m a t i n the DNA doubl e helix is w oun d r o u n d a core of histones (other than H I , and H5 if present). In such a structure only one side of the double helix is in c o n t a c t with these histones, and hence n o t more than one-half o f the DNA phosphates could be b o u n d t o basic amino acids. The basic groups in H1 (and H5 if present) could bind t o only a small p r o p o r t i o n of the remaining DNA phosphates. Hence, the fact t ha t in chrom at i n close to one-half of the phosphates are free to bind Ca 2÷ and o t h e r ligands is good support for this structure of chromatin.

115

Acknowledgements We are grateful to Mr. P.H. Nye for helpful advice. This work was supported by the Medical Research Council and the Cancer Research Campaign. References 1 L e a k e , R . E . , T r e n c h , M.E. a n d B a r r y , J.M. ( 1 9 7 2 ) E x p . Cell Res. 7 1 , 1 7 - - 2 6 2 Sillen, L . G . a n d Margell, A . E . ( 1 9 6 4 ) S t a b i l i t y c o n s t a n t s o f m e t a l - i o n c o m p l e x e s , Special p u b l i c a t i o n No. 17, T h e C h e m i c a l S o c i e t y , L o n d o n 3 L u c k , G. a n d Z i m m e r , C. ( 1 9 7 2 ) E u r . J. B i o c h e m . 29, 5 2 8 ~ 5 3 6 4 S h i n , Y . A . ( 1 9 7 3 ) B i o p o l y m e r s 12~ 2 4 5 9 - - 2 4 7 5 5 F e l s e n f e l d , G. a n d H u a n g , S.L. ( 1 9 6 1 ) B i o c h i m . B i o p h y s . A c t a 51, 1 9 - - 3 2 6 S a n d e r , C. a n d T s ' o , P.O.P. ( 1 9 7 1 ) J. Mol. Biol. 55, 1 - - 2 1 7 C h a n g , K . Y . a n d C a r t , C.W. ( 1 9 6 8 ) B i o c h i m . B i o p h y s . A c t a 1 5 7 , 1 2 7 - - 1 3 9 8 S c h m i d t , G., C a s h i o n , P.J., S u z u k i , S., J o s e p h , J.P., D e m a r c o , P. a n d C o h e n , M.B. ( 1 9 7 2 ) A r c h . Biochem. Biophys. 149, 513--527 9 Wiberg, J.S. a n d N e u m a n , W . F . ( 1 9 5 7 ) A r c h . B i o c h e m . B i o p h y s . 7 2 , 6 6 - - 8 3 10 Mathieson, A.R. and Olayemi, J.Y. (1975) Arch. Biochem. Biophys. 169, 237--243 11 D a v i d s o n , J . N . ( 1 9 6 0 ) T h e B i o c h e m i s t r y o f t h e N u c l e i c A c i d s , 4 t h E d n . , M e t h u e n & Co. L t d . , L o n d o n 1 2 S c a t c h a r d , G. ( 1 9 4 9 ) A n n . N . Y . A c a d . Sci. 5 1 , 6 6 0 - - 6 7 2 13 F r c d e r i c q , E. ( 1 9 7 1 ) in H i s t o n e s a n d N u c l e o h i s t o n e s (Phillips, D.M.P., e d . ) , P l e n u m Press, L o n d o n 1 4 B i n e t t , M.A. a n d B a r r y , J . M . ( 1 9 7 4 ) E u r . J. B i o c h e m . 4 9 , 4 7 7 - - 4 8 4 1 5 B a r r y , J . M . a n d M e r r i a m , R.W. ( 1 9 7 2 ) E x p . Cell Res. 7 1 , 9 0 - - 9 6 16 J u r k o w i t z , L. ( 1 9 6 5 ) A r c h . B i o c h e m . B i o p h y s . 1 1 1 , 8 8 - - 9 5 1 7 Y o n u s c h o t ~ G. a n d M u s h r u s h , G.W. ( 1 9 7 5 ) B i o c h e m i s t r y 1 4 , 1 6 7 7 - - 1 6 8 1 1 8 Klein, F. a n d S z i r m a i , J . A . ( 1 9 6 3 ) B i o c h i m . B i o p h y s . A c t a 72, 48---61 1 9 M i u r a , A. a n d O h b a , Y. ( 1 9 6 7 ) B i o c h i m . B i o p h y s . A c t a 1 4 5 , 4 3 6 - - 4 4 5 2 0 K l e i n m a n , L. a n d H u a n g , R.C.C. ( 1 9 7 1 ) J. Mol. Biol. 55, 5 0 3 - - 5 2 1 2 1 Clark, R . J . a n d F e l s e n f e l d , G. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 3 6 2 2 - - 3 6 2 8 2 2 I t z h a k i , R . F . ( 1 9 7 1 ) B i o c h e m . J. 1 2 2 , 5 8 3 - - 5 9 2 23 Kornberg, R.D. (1974) Science, 184,868--871 2 4 Noll, M. ( 1 9 7 4 ) N u c l e i c A c i d Res. 1, 1 5 7 3 - - 1 5 7 8

Ion binding and chromatin condensation.

109 Biochimica et Biophysica Acta, 442 (1976) 109--115 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 98656...
409KB Sizes 0 Downloads 0 Views