Chromatin condensation Possible dehydrating and stabilizing factors

356

Biochimica et Biophysica Acta, 565 (1979) 356--364

© Elsevier/North-Holland Biomedical Press

BBA 99572 C H R O M A T I N CONDENSATION POSSIBLE D E H Y D R A T I N G AND STABILIZING F A C T O R S

ANDRZEJ JERZMANOWSKI, KRZYSZTOF STAROiQ, BARBARA TYNIEC-KROENKE and KAZIMIERZ TOCZKO Department of Biochemistry, Warsaw University, Zwirki i Wigury 93, 02-089 Warszawa (Poland)

(Received May 15th, 1979) Key words: Chromatin condensation; Spermine; Spermidine; Chromatin hydration; Ethidium bromide

Summary The effect of Na ÷, Mg~ ÷, spermidine and spermine on the dehydration of chromatin gel and precipitation of soluble chromatin has been compared. Considerable differences have been found in the relative ratios within the studied group (Na ÷, Mg 2÷, spermidine and spermine) between the ability to dehydrate (1 : 32 : 53 : 67) and to precipitate (1 : 53 : 800 : 2000) chromatin. On the basis of the dependence of precipitation on initial chromatin concentration it has been suggested that the observed effect as contributed considerably by interparticle aggregation is a relatively good measure of the ability of cation to stabilize higher order structures of chromatin through direct crosslinking or induction of hydrophobic associations at selected sites. In contrary to that the m e t h o d estimating the direct dehydration measures the overall dehydrating effect of a cation exerted on the whole chromatin. It has been suggested on the basis of the above comparative data that the in vivo regulation of the degree of overall chromatin hydration should occur through changes in concentration of free small inorganic cations. Larger organic polycations like polyamines should be mainly involved in stabilization of the higher order chromatin structures. The stabilizing role of larg e polyanions like R N A has been ruled out. It has also been found that the unwinding of chromatin DNA results in considerable chromatin hydration.

Introduction Chromatin condensation is a process occurring universally among eukaryotes. In interphase nuclei various parts of chromatin occur in condensed,

357 heterochromatic regions [1]. Heterochromatin is usually opposed to more relaxed euchromatin which is a part of chromatin open to transcription. The condensed state of heterochromatin is thus often regarded as the reflection of a large scale block of genetic activity. Another well known state of chromatin condensation occurs during eukaryotic mitosis. It concerns the whole chromatin and appears to be a fully reversible process with maximum at about metaphase. There is a large number of evidence that condensation (at least mitotic) involves formation of regular higher order structures of a basic nucleosomal fiber [2]. Some of these structures like for example the 300/~ solenoid can be formed due to the sole action of cations [3] which act as neutralizers of repulsive forces between negatively charged DNA phosphates. On the other hand the formation of every higher order chromatin structure should be accompanied by the overall dehydration. This is due to new hydrophobic interactions which are formed most possibly between nucleosomal protein surfaces. In fact the excessive dehydration of chromatin can alone lead to higher order structures [4]. One can thus say that: (a) agents able to dehydrate chromatin can be very potent condensing factors, and (b) the degree of chromatin dehydration is a good measure of chromatin condensation. However, as was pointed out by Bak et al. [2], it is hardly possible that the higher order chromatin structures consisting of large diameter helices could m ~ n t a i n their compact form due only to small-distance hydrophobic interactions and stabilization provided by small inorganic cations. Certain stabilizing factors of larger molecular weight like protein or RNA were therefore postulated [2]. Bearing in mind the above facts it could be suggested that regular condensation of chromatin results from both the dehydration of the nucleoprotein complex and the crosslinking action of stabilizing factors. In the present work we compared dehydrating and aggregating properties towards chromatin of some physiolo~cally important ions. We estimated the effect of a given ion on the volume of chromatin gel and on precipitation of soluble, sheared chromatin from the solution in order to distinguish its ability to dehydrate and crosslink chromatin. The first m e t h o d provides direct data on dehydrating power of various ions whereas the other reflects much better their ability to crosslink. We found that dehydrating and aggregating . properties of cations present naturally in the cell differ significantly and some of them can act as stabilizing (crosslinking) factors in the concentrations at which their dehYdrating properties are little pronounced. These findings are of relevance to eventual hypotheses a b o u t triggers of mitotic condensation. We also found that partial unwinding of chromatin DNA by ethidium bromide significantly affect the state of chromatin hydration. Materials and Methods

(a) Material. Fresh calf t h y m u s was cut into small pieces, immediately frozen and stored at --20°C until used. (b) Preparation of chromatin. Soluble chromatin was prepared according to Marushige and Bonner [5] and intensively sheared in a high speed mixer for 6 min. The final soluble preparation was dialysed against 5 mM Tris-HC1, pH 7,

358 claryfied by centrifugation for 20 min at 10 000 × g and the supernatant was diluted to the concentration of about 3 A260 units. Unsheared insoluble chromatin gel was isolated according to Sollner-Webb and Felsenfeld [6] and allowed to swell in 5 mM Tris-HC1, pH 7. Sheared soluble chromatin did n o t have a nucleosomal structure as was checked with micrococcal nuclease, whereas this structure was preserved in unsheared insoluble preparation. On the other hand, if histones were extracted from both preparations and analysed on gels according to Panyim and Chalkley [7] no degradation was observed. (c) Solubility measurements. 1-ml samples of sheared chromatin were mixed with an equal volume of tRNA (Calbiochem, obtained according to Holley et al. [8]) or appropriate salt solution and allowed to stand overnight in a cold room. Then they were centrifuged for 10 min at 10 000 $ g. The absorbance of supernatant was measured at 260 nm except of experiments with RNA when the a m o u n t of DNA in the pellet was estimated after alkaline hydrolysis. (d) Dehydration measurements. 5-ml samples of swollen unsheared chromatin gel were mixed with an equal volume of appropriate salt or ethidium bromide solution in calibrated tubes and allowed to stand for 2 h in a cold room. Then they were centrifuged for 10 min at 2000 × g and the volume of the pellet was estimated.

Results

(a) Differences between systems measuring dehydration and precipitation of chromatin Addition of increasing amounts of dehydration agent to the chromatin gel results in its gradual contraction (decrease of the total volume) which can be directly followed in a calibrated tube upon low speed centrifugation. The other system in which the general effect of different factors on the physical state of chromatin can be measured is based on the determination of the a m o u n t of chromatin precipitated from the solution after high speed centrifugation upon addition of the studied factor. The two systems use rather different initial samples. Chromatin used for dehydration studies is a gel obtained by gentle procedure which resembles the nuclear chromatin as far as its nucleosomal structure is concerned [9]. The gel has its natural volume determined by ionic conditions. In the case of precipitation studies chromatin is usually a sheared preparation additionally claryfied by centrifugation. The origin of its nucleosomal structure is rather problematic [9]. The last system is very sensitive to the initial concentration of chromatin in the sample. As it can be seen from Fig. 1 the shape of the titration curve for chromatin precipitation with Mg2÷ is strongly dependent on chromatin concentration. Positive dependence of precipitation on concentration points that the interparticle aggregation makes a considerable contribution to the observed precipitation. In the case of dehydration one can speak only about the dependence of the process on the ratio of chromatin to the total a m o u n t of ions. For Mg2+ as shown in Fig. 2 it is based on the equilibrium between chromatin bound and free Mg2÷ which favours less free Mg 2÷ if the a m o u n t of binding sites increases [10]. In all further experiments we made the comparisons always for the same initial

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chromatin concentration (in precipitation studies) or at fixed ratio of chromatin to the total volume of solution (in dehydration studies).

(b) Factors affecting the state of chromatin hydration The most important unspecific dehydrating agents in the cell are small cations of different valency (or amount of positive charges) which act through simple neutralization of negative charges of chromatin DNA phosphates. The dehydrating power of four of such naturally occurring cations (Na ÷, Mg2÷, spermidine and spermine) are compared in Fig. 3. The general range of concentration at which the dehydration occurs for a particular cation is in good agreement with the earlier observations of Leake et al. [11] and Bradbury et al. [12], and with the studies of Chang and Carr [13] on the competition among cations in binding to DNA. The concentration of particular cations necessary to half-contract the chromatin gel are: 8 mM Na ÷, 0.25 mM Mg2÷, 0.15 mM spermidine and 0.12 mM spermine. In contrast to large difference in the dehydrating power (30-fold) between mono- and divalent cations the difference between di- and multivalent (i.e. trivalent spermidine and tetravalent spermine) cations is by far less pronounced. As regards factors which act in opposite direction to cations, i.e. will cause the increase in chromatin hydration, one could expect that conformational changes in chromatin DNA caused by its unwinding would result in extra hydration due to unmasking of new DNA phosphates. This could be relevant to the findings that chromatin regions

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Fig. 4. E f f e c t o f e t h i d i u m b r o m i d e on the v o l u m e o f c h r o m a t i n gel in 5 mM Tris buffer (~ ~) and in 0.3 mM MgC12, 5 mM ~ b u f f e r (o o). T h e a m o u n t o f c h r o m a t i n D N A in l O - m l s a m p l e was 12.5 rag. Gel v o l u m e w i t h o u t e t i h i d i u m and MgC12 is 100%.

involved in transcription are more relaxed than the rest of chromatin whether or not the nascent R N A is actually formed [14]. To check this possibility we used an artificial D N A unwinding agent, ethidium bromide, which intercalates between stacked D N A bases and partially unwinds the right-handed double helix [15]. When applied to chromatin it results in structural changes which as far as the features revealed by micrococcal nuclease are concerned resemble the transition from the transcriptionally inactive to the active state [16]. If ethidium is added to chromatin gel the increase in hydration is readily observed both for the fully decondensed chromatin and for chromatin partially dehydrated with Mg 2+ (Fig. 4). The effect is most pronounced at ethidium bromide/nucleotide ratio of about 0.05. It is worth noting that this value is close to the highest dose of ethidium bromide which does not yet affect the basic nucleosomal structure of chromatin as revealed by micrococcal nuclease [ 16]. Further increase in ethidium concentration results in gradual dehydration of chromatin. The last effect is probably due to the cationic nature of ethidium itself which, at higher concentrations is known to bind electrostatically to D N A phosphates [17].

(c ) Precipitation studies; factors stabilizing higher order structure of chromatin As can be seen in Fig. 1 precipitation of diluted chromatin is mostly due to

361 interparticle aggregation. It could result either from direct crosslinking of different particles by the cationic molecule (this however could not be the case for Na ÷) or from neutralization of enough charges on the surface of neighbouring particles for the aggregation of hydrophobic sites to take place. In either case the effect should be the formation of large multiparticle aggregates which would precipitate from the solution. Fig. 5 compares the ability of different cations to precipitate chromatin. The concentration necessary for half-precipitation are: 40 mM Na ÷, 0.75 mM Mg 2÷, 0.05 mM spermidine and 0.02 mM spermine. One can present these values in order to show the increasing ability for precipitation as 1 : 53 : 800 : 2000. The appropriate values showing the dehydrating ability of the same cations are 1 : 32 : 53 : 67 (see preceding section). It can be seen that the ratios of cation concentration necessary for half-precipitation to the concentration necessary for halfdehydration are rather similar for mono- and divalent cations whereas they are completely different for multivalent cations. The latter points to the extraordinarily high ability of multivalents to aggregate chromatin. It is important that at the range of concentrations at which multivalent cations strongly aggregate chromatin they only slightly affect chromatin hydration. It can therefore be concluded that the aggregation here results probably from neutralization of charges at selected, single areas on the surface, the process resembling very much the stabilization of higher order structures through fixing junctions at selected points.

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The question arises whether larger molecular weight physiologic anions, which obviously cannot dehydrate chromatin, can stabilize the higher order structures through crosslinking at selected sites. To check this possibility we examined the effect of soluble t R N A on chromatin precipitation (Fig. 6). It can be seen that up to the concentration corresponding to a R N A / D N A ratio of 0.4 R N A has no effect on the solubility of chromatin independently on whether it acts alone or in the presence of Na ÷ or Mg 2÷. Higher concentrations o f R N A were not used because of the danger of unspecific effect on solubility due to the removal of H1 histone from chromatin [18]. Discussion

It is rather accepted that shrinking of nuclear chromatin induced by small cations can to some extent resemble the naturally occurring process of chromatin condensation [11]. The mechanism of such cation-induced condensation is presumably the closer packing of chromatin nucleosomes due to reduction of repulsive forces between DNA phosphates. The observed dehydration results most possibly from new hydrophobic interactions between chromatin proteins. Therefore the cation-induced condensation is in fact equivalent to the dehydration of nucleoprotein. On the other hand, the small cations, except of being excellent dehydrating agents, do n o t provide the long-range stabilization which, as pointed out by Bak et al. [2] is necessary to maintain the large diameter helical structures postulated in condensed mitotic chromosome. In studies dealing with the effects of various agents on chromatin condensation two basic methods have been used the results of which are usually treated interchangeably. One of them is based on the measurements of the effect on the relative chromatin volume [12], the other measures the ability to precipitate chromatin from solution (using turbidity or sedimentation analysis) [11, 19,20]. In fact both methods differ considerably in the effect they measure. Whereas the first measures directly the .degree of dehydration and informs a b o u t the dehydrating ability of a given agent the second measures mostly the ability to produce interparticle aggregates or crosslinks. In the present work we found that for a series of cations, e.g. Na ÷, Mg 2÷, spermidine and spermine, the ratio of dehydrating abilities is considerably different from the ratio of the abilities to produce interparticle aggregation. The multivalent cations (spermidine and spermine) which are extremely effective in producing interparticle aggregation are far less effective in producing dehydration. As compared to Mg 2÷ their dehydrating power is less than 2-fold. On the other hand, the cellular concentration of total spermidine and spermine does n o t exceed 2 mM [21] as compared to a b o u t 10 mM total cellular magnesium [10]. Although the binding constant towards DNA is higher for polyamines than for divalent cations it can be concluded from the above data that the participation of polyamines in overall chromatin dehydration in the cell should be considerably lower than that of magnesium. In contrast they would be rather favoured as stabilizers of higher order structures. Using the precipitation system we found that small size R N A was n o t able to induce interparticle aggregation. Thus at least as far as unspecific effect is concerned R N A should be ruled out as possible stabilizer of condensed chroma-

363 tin, a suggestion which was put forward by Bak et al. [2]. It seems that stabilizer should in general be of cationic rather than anionic nature. In postulating the triggers of the overall (for example mitotic) chromatin condensation one should consider both the need for dehydration without which the formation of more closely packed nucleosomes can be hardly imagined, and the stabilizing factors maintaining the rigidity of higher order structures as postulated by Bak et al. [2]. From the data presented in this work it seems that moderate changes in physiologic concentration of free small cations should provide a better way of regulation of the overall chromatin hydration than the similar changes in the concentration of free larger cations like spermidine and spermine. The last group, however, occurring in small concentration can provide an excellent stabilization for the higher order structures formed as a result of dehydration caused for example by the increase in small free cations. In the present work we also found that chromatin hydration was strongly affected by the degree of unwinding of chromatin DNA. The characteristic feature of transcriptionally active extended chromatin is the protein/DNA ratio different from that of inactive, condensed chromatin [11]. One can imagine that this difference alone could account for different degrees of condensation. On the other hand, the consequence of DNA unwinding is the increased accessibility of chromatin DNA to proteins as checked by nuclease probe [16], an effect which is also observed naturally for transcriptionally active regions of chromatin [14]. It seems therefore possible that the observed overall decondensation of transcriptionally active fragments of chromatin may partly result from partial unwinding of DNA.

Acknowledgement This work was supported by the Polish Academy of Sciences within the project 09.7.1.

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