Action of heparin on mammalian nuclei. II. Cell-cycle-specific changes in chromatin organization correlate temporally with histone H1 phosphorylation

486

Biochimica et Biophysica Acta, 517 (1978) 486--499

© Elsevier/North-Holland Biomedical Press

BBA 99093 ACTION OF H E P A R I N ON MAMMALIAN NUCLEI II. CELL-CYCLE-SPECIFIC CHANGES IN C H R O M A T I N O R G A N I Z A T I O N C O R R E L A T E T E M P O R A L L Y WITH HISTONE H1 P H O S P H O R Y L A T I O N

(With an appendix on the Hill analysis of cooperativity of interaction o f heparin with chromatin-bound histones) C.E. HILDEBRAND, R.A. TOBEY, L.R. GURLEY and R.A. WALTERS Cellular and Molecular Biology Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, N.M. 87545 (U.S.A.)

(Received July 7th, 1977

Summary The interaction of the polyanion heparin with the inner histones of chromatin has been used to detect changes in chromatin organization associated with cell-cycle traverse. Synchronized populations of Chinese hamster cells were obtained either in early G1 or near the GI/S boundary. The rate o f interaction of heparin with chromatin-associated inner histones was measured using nuclei isolated from synchronized cell populations in different phases of the cell cycle. A G~-specific decrease in rate of interaction of heparin with inner histones was observed and found to be independent of the presence of hydroxyurea during traverse of G~. A further decrease in heparin-inner histone interaction occurred in late S and G2. These changes correlate temporally with the interphase phosphorylation(s) of histone H1. This correlation is discussed within the framework of current models of higher order chromatin structure (i.e. organization above the nucleosome level). Analysis of the cooperativity of interaction of heparin with inner histones was performed using the kinetic analog of the Hill equation. This analysis suggests that the organization of inner histones on chromatin does n o t undergo large variations during the cell cycle.

Introduction Since the concept of a chromosome condensation-decondensation cycle associated with the cell cycle was proposed by Mazia [1], over a decade ago, growth-phase-specific changes in the organization of chromatin in the eukaryo-

487 tic nucleus have been observed both in quiescent cells which have been stimulated to proliferate and in continuously cycling cells (see refs. 2 and 3 for reviews). Several types of measurements have been used to detect proliferationrelated changes in interphase chromatin organization [2 -4]. This communication provides evidence that cell-cycle-specific changes in chromatin are revealed by the interaction of a polyanion, heparin, with the histone component of chromatin. Studies in this laboratory, as well as results from other laboratories, show that the polyanion heparin is an agent which interacts predominantly with chromatin-associated histones [5]. We have reported earlier that heparin can be used to detect changes in chromatin organization during the G1 phase of the cell cycle [6,7]. We recently described a method [5] for measuring the interaction of heparin with the "inner" or "core" histones (H2A, H2B, H3, and H4) which associate with each other and with DNA to form the basic subunit structure of chromatin. In the present study, this analysis has been applied to the entire cell cycle using highly synchronized populations of Chinese hamster cells. Our results suggest that events occurring in G1 and in late S and G2 decrease the accessibility of inner histones to interaction with heparin. These changes are shown to correspond temporally to G~-specific and late interphasespecific phosphorylations of histone H1. In addition, an analysis of the cooperativity of interaction of heparin with inner histones using a relationship analogous to the Hill equation [8--11] has been performed for different phases of the cell cycle. The results of this analysis indicate that the interactions among the inner histones show only small variations during the cell cycle in spite of the large changes in accessibility of heparin to inner histones. These findings are considered in view of current models for chromatin structure. Materials and Methods

Cell culture synchronization and radioisotope labeling. Chinese hamster cells (line CHO) [ 12] were maintained in suspension culture free from Mycoplasma contamination in F-10 medium (Gibco) supplemented with 10% calf and 5% fetal calf sera (both supplied by Flow Laboratories). Cellular DNA was uniformly labeled during growth in medium containing 0.006 ~Ci/ml [~4C]thymidine (40--60 Ci/mole, New England Nuclear Corp.) for a period equivalent to two culture doubling times prior to application of cell synchronization techniques. Following mitotic selection or growth in isoleucine-deficient medium, cells were cultured in isotope-free medium. The methods employed to obtain highly synchronized cell populations in mitosis [13], early GI [14], or near the G1/S boundary [15 -17] have been described in detail previously. Synchronized populations of cells in early G~ were prepared either by mitotic selection [13] or by growth of cells in isoleu. cine-deficient medium [14]. These early G~ cell populations could be resynchronized near the G1/S boundary by allowing them to traverse G~ in the presence of hydroxyurea for 10 h [15--17]. As an index of synchrony, the fraction of cells synthesizing DNA was determined by standard autoradiographic procedures on parallel cultures which were not prelabeled with [ ~4C]thymidine. Cell concentration was measured with an electronic particle counter.

488

Isolation o f nuclei, treatment o f nuclei with heparin, and analysis of interaction o f heparin with nuclei. All procedures used have been described in detail elsewhere [5,18]. Briefly, nuclei were prepared by treatment of cells at 4°C with 1% NP-40 (a non-ionic detergent from Shell Oil) in ice-cold buffer A (0.01 M Tris • C1, pH 7.4 at 24°C, 0.01 M NaC1, and 0.0015 M MgC12) [18]. Nuclei were collected by centrifugation and subjected to another cycle of 1% NP 40 treatment. Nuclei were washed in buffer A without NP-40, recovered by centrifugation, and resuspended in buffer B (0.25 M sucrose, 0.02 M Tris • C1, pH 7.4 at 24°C). Nuclei were centrifuged again and finally resuspended at 3 • 107 nuclei/ml in buffer B. Samples containing nuclei at 1 . 5 . 1 0 7 nuclei/ml and heparin at indicated concentrations (final concentrations of Tris. C1 and sucrose were 0.01 and 0.125 M, respectively) were incubated at 4°C for 5 min. This treatment causes rapid nuclear lysis and removal of histones (and other chromatin proteins) from DNA in relation to incubation time and heparin concentration as demonstrated previously [18]. The action of heparin was terminated effectively by a 50-fold dilution of the sample. Each sample was mixed vigorously for 15 s with a vortex mixer. The fraction of DNA from which histones were removed during heparin treatment was determined by differential centrifugation as detailed earlier [5,18]. Briefly, samples were subjected to centrifugation at 6 0 0 0 0 × g (average) for 25 min in a fixed-angle rotor. During centrifugation, the dehistonized (deproteinized) DNA remains largely in the supernatant, while the deoxynucleohistone ("chromatin") sediments into the pellet [5]. We have previously shown that the percent of DNA in the supernatant fraction is proportional to, b u t n o t equal to, the percent of total inner histones (H2A, H2B, H3, and H4) removed from the nuclear DNA [5]. For example, we observed that, when 100% of the inner histones were removed from total chromatin, only 75--80% of the dehistonized DNA remained in the supernatant fraction. Also, the 2 0 - 2 5 % of the DNA sedimenting into the pellet was found to be historic free. The sedimentation of a constant fraction of the dehistonized chromatin into the pellet was attributed to the nature of differential centrifugation [5]. Hence, the data presented here have been normalized so that the maximum percent of DNA in the supernatant fraction (75 -80% of the total nuclear DNA at large heparin concentrations where all histone is removed) is set equal to 100%. This normalization is permitted on the basis of control experiments reported previously [5]. Protein determinations. The protein content of isolated nuclei was measured by the m e t h o d of L o w r y et al. [19] using bovine serum albumin (Technicon, certified standard grade) as the standard. Measurements of historic H1 phosphorylation rates. CHO cells were grown and synchronized in the presence of [3H]lysine (50 gCi/1 of culture) for four generations to label their histones totally. Following synchronization, cells were exposed to 20 mCi of H33 2PO4 (carrier-free) per 1 for 1 h at various times in the cell cycle to incorporate 32PO4 into their histones. The histones were then isolated, purified, and fractionated by either preparative gel electrophoresis or Bio-Rex 70 column chromatography as previously described [20,21]. The rate of phosphorylation relative to a m o u n t of histone recovered in the H1 fraction was expressed as the 32P/3H ratio (i.e. relative specific activity).

489 Results

In a previous study using mitotically synchronized CHO cells, we observed a Gl-specific change in interaction of heparin with intact nuclei [6]. In that study, as well as in earlier investigations [18], the interaction of heparin with isolated nuclei was shown to cause both rapid nuclear disruption and release of DNA from chromatin into a slowly sedimenting form which is readily separated from the rapidly sedimenting chromatin by differential centrffugation. We subsequently have reported a direct correlation between the heparin-mediated release of slowly sedimenting DNA from chromatin (lysed nuclei) and the coordinate removal of inner histones (H2A, H2B, H3, and H4) from chromatin [5]. Therefore, the percence of DNA rendered slowly sedimenting during heparin treatment of isolated nuclei indicates how much inner histone is removed from the chromatin [5]. We have applied the differential sedimentation analysis to study further cell-cycle-specific changes in the interaction of heparin with the inner histones. Experiments were performed initially to determine whether G, cells obtained by mitotic selection differed from G~ cells produced by isoleucine deprivation with respect to timing of the G~ change in chromatin organization reported by us previously [6]. To answer this question, synchron!zed populations of early G~ cells obtained by either mitotic selection or isoleucine deprivation were sampled at various times during traverse of the pre-DNA synthetic period. Nuclei isolated from these samples were subjected to heparin treatment, and the percent of inner histone-depleted DNA appearing at several heparin concentrations was measured. Examples of these results appear in Fig. 1 for cells synchronized by the isoleucine deprivation method. The samples were harvested at various times after the G~-arrested cells were placed in complete medium. Data for early GI cells (0.25 h) and mid-S phase cells (8 h) are presented. The cooperative nature of the interaction of heparin with chromatin is suggested by the sigmoidal shape of the heparin concentration dependence curve [5]. I 100 !

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EHe~rin] #g/rnl Fig. I . Heparin c o n c e n t r a t i o n d e p e n d e n c e o f the removal of inner histones from chromatin in intact nuclei isolated from a cell p o p u l a t i o n s y n c h r o n i z e d b y t h e isoleucine d e p r i v a t i o n m e t h o d . T h e curves s h o w n represent samples t a k e n at 0.25 h (e e) or S h ( I i ) after release o f t h e cell population from G I arrest. T h e p o i n t at 100% represents t h e level reached at l o n g r e a c t i o n times (15 rain) or large heparin c o n c e n t r a t i o n (2 mg/rnl) for a 5-min r e a c t i o n [5].

490 We have arbitrarily characterized the cell-cycle dependence of the change in interaction of heparin with chromatin by measuring on a given curve the heparin concentration at the point where 50% of the chromatin is depleted of inner histones (i.e. when 50% of the DNA, relative to the plateau level or 100% point in Fig. 3, appears in the supernatant fraction). We have designated this value as [heparin] 0.s,t where the subscript 0.5 indicates that the heparin concentration was measured at 50% of the effect and t is the time during the cell cycle. Hence, in Fig. I the values of [heparin]0. s,t are approx. 130 and 275 gg/ml for the 0,25- and 8-h samples, respectively. It should be noted here that an increase in [heparin]0. s,t indicates a decrease in rate of interaction between heparin and inner histones [5]. Fig. 2 compares these measurements for cells synchronized by mitotic selection or by isoleucine deprivation. The [heparin]0. s,t values have been determined from curves such as shown in Fig. 1 and have been normalized to the values obtained immediately after all mitotically synchronized cells have divided (1 h) or after isoleucine-deprived cells have been released from arrest into fresh medium (0.25 h). For e c o n o m y of expression, [heparin] 0. 5,t/[heparin]0.s,0 is defined as hi~ 2. These results suggest that the m e t h o d of synchronization alters only slightly the Gl-specific change in chromatin organization detected by heparin. To extend this analysis to the entire cell cycle, the isoleucine deprivation m e t h o d of synchronizing CHO cells was used together with h y d r o x y u r e a to obtain highly synchronized populations of cells near the G1/S boundary. The quality of synchrony was monitored in parallel cultures by autoradiographic and cell-concentration determinations (Figs. 3A and 3B). In Fig. 3C, a comparison is made of the heparin-detected changes in chromatin organization of cells traversing early interphase in the presence or absence of hydroxyurea. These

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Fig. 3. S u m m a r y o f c e l l - c y c l e d a t a s h o w i n g a c o r r e l a t i o n b e t w e e n p h o s p h o r y l a t i o n o f h l s t o n e H I a n d i n t e r a c t i o n o f h e p a r i n w i t h i n n e r h i s t o n e s . I n p a r t s A , C, a n d E, p o p u l a t i o n s o f C H O cells w e r e s y n c h r o n i z e d i n e a r l y G 1 b y i s o l e u c i n e d e p r i v a t i o n a n d a l l o w e d t o t r a v e r s e G 1 i n t h e a b s e n c e (solid s y m b o l s ) o r p r e s e n c e ( o p e n s y m b o l s ) o f h y d r o x y u r e a . In p a r t s B, D0 a n d F, cell p o p u l a t i o n s s y n c h z o r d z e d in early G 1 b y i s o l e u c i n e d e p r i v a t i o n a n d r e s y n c h r o n i z e d n e a r t h e G 1/S b o u n d a z T b y h y d r o x y u r e a treatm e n t w e r e r e m o v e d f r o m h y d r o x y u r e a a n d a l l o w e d t o traverse late i n t e r p h a s e in d r u g - f r e e m e d i u m . I n d i c e s o f s y n c h r o n y are i n d i c a t e d in p a r t s A a n d B b y t h e f r a c t i o n o f cells s y n t h e s i z i n g significant a m o u n t s o f D N A ( d e t e r m i n e d b y a u t o r a d i o g r a p h y ) o r b y t h e f r a c t i o n o f cells w h i c h has d i v i d e d ( p a r t B). C e l l - c y c l e - s p e c i f i c c h a n g e s in t h e i n t e r a c t i o n o f heparin w i t h c h r o m a t i n - b o u n d h l s t o n e s a r e s h o w n in p a r t s C a n d D. P h o s p h o r y l a t i o n o f h l s t o n e H 1 d u r i n g t h e cell c y c l e is i n d i c a t e d in p a r t s E a n d F.

data were obtained from curves similar to those shown in Fig. 1. It is evident that the Gl-specific change in chromatin takes place at approximately the same rate, independent of the presence or absence of hydroxyurea. The data obtained after release of hydroxyurea-resynchronized cells into drug-free medium are shown in Fig. 3D (note the scale change). These results indicate that, during early and mid-S phase (the first 3 h after release from hydroxyurea blockade), there is very little change in the property of chromatin as detected by heparin treatment. However, between 3 and 6.5 h (late S and G2), a striking increase is observed in the concentration of heparin required to remove 50% of the inner histones. By 9 h, this value decreases to a level approaching the initial early G1 level. Also at 9 h most of the population which will divide has already divided, and 80% of the population is in early interphase of the next cycle. Hence, the remaining 20% of cells may account partially for the fact that the second G1 level for h,n does not quite return to that observed in the first G,. (Note that

492 measurements were made on the basis of a fixed volume ot " culture; thus, a small change in the h I/2 value from the first G1 to the second G1 will be attributed to a change in stoichiometry (i.e. the chromatin concentration will double) (ref. 18 and unpublished results)). Results for the cell-cycle-specific phosphorylation of histone H1 are shown in Figs. 3E and 3F. The ratio 32PO4 to [3H]lysine measures the rate of phosphorylation during the 1-h period prior to the time indicated in the figure. For cells released from G~ arrest (Fig. 3E), the data show that the rate of H1 phosphorylation increases rapidly during early interphase. This increase is independent of the presence or absence of h y d r o x y u r e a which blocks cells near the G1/S boundary [15--17]. Following release from h y d r o x y u r e a blockade, the H1 phosphorylation rate increases slightly in early S and then rises sharply in late S and G2 several hours before onset of cell division. It is interesting that the general features of the cell-cycle-specific changes in the interaction of heparin with inner histones (Figs. 3C and 3D) are similar to the changes in H I phosphorylation rate (Figs. 3E and 3F). In early interphase, both the value of hlj 2 and the rate of H1 phosphorylation begin to increase several hours in advance of the entry of cells into S phase. Also, the early interphase H1 phosphorylation and the change in h~/2 occur in the presence of hydroxyurea, as well as in the absence of the drug (Fig. 3E) [6,20]. However, it is evident that the H I phosphorylation rate in cells traversing early interphase without h y d r o x y u r e a continues to increase after approx. 4 h (Fig. 3E), while the rate in cells traversing G~ in the presence of h y d r o x y u r e a appears to reach a plateau at 4 h. After release of cultures from h y d r o x y u r e a blockade, a slow increase in the rate of H1 phosphorylation is observed in early and mid-S phase (0--4 h), followed by a rapid increase in late S and G2 (4--7 h, Figs. 3B and 3F). Similarly, h~/2 shows very little increase in early to mid-S phase (0-.-3 h) and a very rapid change between 3 and 7 h (late S and G2). Upon completion of mitosis and cytokinesis, histone H1 is rapidly dephosphorylated [21], and there is a corresponding decrease in h ~/ 2 (Fig" 3D). It should be noted that phosphorylation of one amino acid residue of H1 decreases the net positive charge of the H1 molecule by one. Since H1 is released from chromatin prior to removal of inner histones [5], it is reasonable to suggest that our observations might be ascribed, in part, to a decreased interaction of heparin with phosphorylated H1 molecules compared with unphosphorylated H1. It follows that a decreased interaction of heparin with phosphorylated H1 may be reflected in a decreased rate of removal of histones from regions of chromatin containing phosphorylated H1 molecules. However, the decreased interaction of heparin with phosphorylated H1 solely on the basis of the decreased positive charge in H1 is unlikely, since phosphorylated H1 and unphosphorylated H I display similar affinities for heparin (unpublished results). Some additional information can be extracted from data of the type shown in Fig. 1. The cooperative nature of the interaction between heparin and inner histone (indicated by the sigmoidal shapes of the curves and supported by our earlier studies [ 5]) was recognized to be analogous to the positive cooperativity exhibited by O2 binding to hemoglobin [10] and by certain allosteric enzymes [10,11]. This analogy is developed in detail in the Appendix. Briefly, the data in

493 Fig. 1 indicate that, as the concentration of a ligand (in this case, heparin) is increased, the rate of removal of inner histones from segments of chromatin increases in a cooperative manner [5] (i.e. initial removal of inner histones facilitates removal of neighboring histones). Initial rates for the heparin-mediated removal of inner histones can be calculated and used to construct a "Hill plot" [8,9,11]. This type of analysis was originally developed by Hill [8] to analyze the "autocatalytic" nature of the binding of O2 to hemoglobin. The Hill analysis has been applied to describe altered cooperativity in the action of allosteric enzymes [10,11] and to characterize changes in the sigmoidal-shaped curves for O2 binding in hemoglobin [22]. Results are shown in Fig. 4 for this type of analysis applied to data from synchronized cell populations in early G1, near the G1/S boundary, and in late interphase (late S and G2). The slope of the line obtained from this analysis is an indicator of cooperativity. A slope of 1.0 indicates no cooperativity, while a slope >1.0 indicates positive cooperativity. The data in Fig. 4 indicate that the slopes for all the curves are approx. 2.0 and that the late S and G2 sample shows a small but significant difference from the slopes of the early G~ and G~/S samples. In contrast, the values of h~j 2 for these samples show large changes in traverse of cells from G~ to G2 (Figs. 3C and 3D). The significance of these observations will be presented in the discussion below.

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Discussion In several previous investigations, changes in chromatin organization related to cell proliferation have been detected either by (1)measuring changes in the interaction with chromatin of DNA-specific probes or (2) quantitating changes in the morphological features of chromatin (see refs. 2 and 3 for reviews}. The studies described here have demonstrated that changes in chromatin associated with cell-cycle traverse can also be identified using the polyanion heparin which interacts predominantly with histones [5]. In general, the results of these heparin studies show that, as a synchronized population of cells traverses interphase, specific changes in the interaction of heparin with inner histones are observed, first in G1 and again in late interphase (cf. Figs. 3C and 3D). These changes are monitored by the heparin concentration (or by hl/2) which removes the inner histones from one-half of the total nuclear chromatin during the 5-min interval. An increase in this value of hi~ 2 indicates that the inner histones are removed less readily. Hence, in the results shown here, increased phosphorylation of H I correlates with a decreased rate of interaction of heparin with inner histones. This correlation is observed in 01, independently of the synchrony method employed (Fig. 2) and independently of the presence or absence of the resynchronizing agent, hydroxyurea, during traverse of early interphase (Figs. 3C and 3E). A correspondence between H1 phosphorylation and decreased heparin interaction with inner histones is also observed in late interphase (Figs. 3D and 3F). It should be noted here that phosphorylation of inner histones also occurs during interphase [20, 39,40]. However, modifications of the inner histones do not correlated temporally to the changes in heparin-mediated removal of inner histones observed here (Figs. 3C--3F) [20,39,40]. These observations have led us to examine the

495 possibility that the altered (decreased) interaction of heparin with inner histones during cell-cycle progression arises from conformational changes within or between chromatin fibers, with the consequence that the inner histones are less accessible to heparin attack. With regard to this possibility, it is interesting to consider the potential involvement of histone HI modifications in chromatin organizational changes. Before proceeding to this consideration, it is appropriate to summarize the evidence relating to the role of histone H1 in chromatin structure. According to current models for chromatin structure, histone octamers composed of two each of the inner histones (H2A, H2B, H3, and H4) associate with DNA to form repeating structures (chromatin subunits [23,24], nucleosomes [25], or no bodies [26]) at regular (approx. 200 base pairs) intervals along the DNA [27]. Recent evidence implicates the association of histone HI with the short, exposed segments of DNA presumably between nucleosomes [28,29]. The linear array of nucleosomes, the "nucleofilament", is approx. 100 £ (10 nm) thick and has a DNA packing ratio of approx. 7 : 1 relative to the extended length of protein-free DNA [30]. In the presence of a low concentration of Mg2÷, the nucleofilament can assume higher order structures (e.g. supercoils or solenoids) visible in the electron microscope [31] and detectable by neutron scattering [32]. These higher order structures apparently require the presence of histone HI for stability [31]. Hence, it seems likely that HI is involved in maintenance, or perhaps in the formation, of higher order structures of chromatin (relative to the nucleofilament). Since H1 associates with DNA segments which link adjacent nucleosomes [29,30] and is required for stability of higher order "coiling" of nucleosomes [31], modification of H1 might be expected to modulate the transition to or the stability of higher order chromatin structures (e.g. "coils" or "solenoids"). This may occur by (1) altering directly HI-HI interaction on nucleosomes between chromatin strands or within a strand or (2) modifying the interaction of H1 with acidic non-histone chromatin proteins or perhaps with the HMG proteins [33]. A model for the modification of HI-HI interactions by HI phosphorylation has been proposed by Bradbury et al. [34] to describe the condensation of chromatin associated with mitosis. However, it should be emphasized that the interphase HI phosphorylation events described in this report are distinct from the mitotic H1 phosphorylation events [35,36]. Hohmann et al. [35,36] have demonstrated that, in Chinese hamster cells (CHO), the interphase phosphorylation events differ from the mitotic phosphorylation events in (1) the location within the H1 molecule of the phosphorylated amino acid residues and (2) the amino acids which are phosphorylated. Further studies are required to determine if and how changes in chromatin organization correlating with interphase-specific H1 phosphorylations are related to chromatin condensation corresponding to mitotic H1 phosphorylations. A brief digression is in order to consider implications of the Hill analysis presented above (Fig. 4). During the course of these studies, it became evident that the cooperativity of the interaction between heparin and inner histones could be analyzed to provide insight into organization of inner histones on chromatin in different phases of the cell cycle. This analysis was performed using an empirical relationship analogous to the Hill equation [8,10,11], as

496 outlined in the appendix. Briefly, the initial rates (v) of the interaction between heparin and histone were determined at different heparin concentrations and compared with the maximal rate observed at large heparin concentrations. The values were plotted in terms of ~n [v/(V--v)] vs. ~n [heparin] (cf. Fig. 4). The slopes of these lines, determined by linear regression analysis, were obtained for samples from early G~, near the G~/S boundary, and in late S and G2. In general, the slope (designated the "Hill exponent", n) is an indicator of the cooperativity between {or among) subunits of a protein (or enzyme) involved in the binding of substrate to the active site [9,11,37,38]. (A value of n )- 1.0 indicates positive cooperativity, while n = 1.0 indicates no cooperativity.) Hence, a change in the value of the Hill exponent would indicate an alteration which affects the cooperativity of subunits of a protein in binding substrate and, therefore, an alteration in the relationship between (or among) subunits [ 11,37,38]. In this study, heparin is considered to be the substrate which binds to the inner histones in chromatin subunits. When cells from early G~ are compared with cells near the G~/S boundary, it is clear that the value of h~/2 shows a large increase (Fig. 4), while the "Hill e x p o n e n t " shows no significant change. Also, when cells near the G~/S boundary are compared with cells in late S and G2 (Fig. 4), the value of h~: 2 increases more than 2-fold, while the value of the "Hill e x p o n e n t " shows only a small decrease. The approximate constancy of the value of n indicates that the units which determine the cooperativity of the interaction of heparin with chromatin-bound histones do not undergo large variations during the cell cycle. This result suggests that the subunit (or nucleosome) organization of inner histones on chromatin is persistent throughout the cell cycle. This notion is consistent with recent studies which showed that the subunit organization in metaphase chromatin was indistinguishable from that of interphase chromatin [41--44]. Although the preceding analysis of cooperativity of interaction of heparin with inner histones is at an early stage of development, the results obtained suggest that it will provide another approach to the study of organization of inner histones in chromatin and, specifically, effects of inner histone modification on this organization. In general, the results of this study show that heparin can be employed as an agent for detecting changes in chromatin related to cell-cycle traverse. These changes correlate temporally with the interphase phosphorylation of H1. These findings are consistent with a general model which has been proposed to describe the role of histone phosphorylation in condensation of chromatin and chromosomes [45,46]. Further studies are necessary to develop a relationship of the present findings to results of studies utilizing probes which interact with DNA or to measurements of morphological or ultrastructural changes in chromatin during the cell cycle. Acknowledgements The authors thank Drs. W.B. Goad and A.G. Saponara of this Laboratory for valuable discussions of this work. Also, the authors wish to acknowledge the excellent technical support provided by L.T. Ferzoco, J.L. Hanners, and J.G. Valdez. This work was performed under the auspices of the U.S. Energy Research and Development Administration.

497 Appendix

Hill analysis of the cooperativity of interaction of heparin with chromatinbound histones The cooperative nature of heparin-mediated removal of inner histones from chromatin in intact nuclei is suggested by the sigmoidal shapes of the curves shown in Fig. 1. Further evidence for the positive cooperativity of this phen o m e n o n has been presented in a previous communication [5]. The cooperativity of this interaction is similar to that exhibited in the binding of oxygen to hemoglobin [10] and in the action of certain allosteric enzymes [11]. A method for analyzing phenomena such as these was developed by Hill [8] to describe the sigmoidal curves observed for binding of oxygen to hemoglobin at different partial pressures of oxygen. The Hill analysis was modified and extended to studies of the cooperativity observed in the dependence of allosteric enzyme-catalyzed reaction rates upon substrate concentration [11] and has been used to examine the effects of inhibitors of allosteric enzymes [ 11 ]. Since the interaction of heparin with inner histones is irreversible under our experimental conditions, an equilibrium binding study would n o t be meaningful. Therefore, it is necessary to apply the kinetic analog of the Hill analysis to the rate of heparin interaction with inner histones at different heparin concentrations. The Hill equation, as modified to describe kinetic responses [ 10,11 ], can be written as follows: [ v / ( y - v)] = g x " , (1) where v is the initial reaction velocity at a given substrate concentration, x, V is the maximum reaction velocity at substrate concentration where v is independent of x, K is a constant, and n indicates the power dependence of the sigmoidal curve (see below) upon substrate concentration. The value n is designated the "Hill exponent." To apply this analysis to the heparin-mediated removal of inner histones from chromatin, it is necessary to obtain (1) the values for initial rates of inner histone removal at various heparin concentrations and (2) the value of the maximum initial velocity of inner histone removal. The direct relationship between removal of inner histones from ehromatin and appearanee of histone-free DNA has allowed simplification of the description of heparin with intact nuclei as follows: H-DNA + h -~ h-H + DNA,

(2)

where h indicates heparin, H-DNA is the inner histone • DNA complex in intact c h r o m a t i n , h - H is the heparin • inner histone complex formed upon treatment of intact nuclei with heparin, and DNA is the inner histone-depleted DNA. Kinetic studies of the interaction of heparin with inner histones have been presented in previous reports [5,18]. In analyzing these data, we found that the removal of inner histones can be described by first-order kinetics within the accuracy of our measurements. It is possible to analyze such data according to first-order kinetics with respect to the appearance of h-H (i.e. the rate of removal of inner histones from chromatin) (see Fig. 5). Initial rates, v, for different heparin concentrations can then be obtained as described below. It should be emphasized that this kinetic analysis is applied only to facilitate

498

determination of initial rates of removal of histone from chromatin. We have found empirically that, at a given heparin concentration, the time dependence of removal of inner histones from chromatin can be represented, to a first approximateion, by l n [ H ( t ) / g ( o ) ] ~- - - k t ,

(3)

as indicated in Fig. 5 where H(0) is the total initial a m o u n t of chromatinassociated inner histories; H ( t ) is the a m o u n t of inner histones remaining chromatin-associated after incubation at 4°C for time t; and k is the rate constant. The validity of this relationship at different heparin concentrations has been verified by measuring the rate of inner histone removal from chromatin at heparin concentrations ranging from 50 to 400 t~g/ml. Eqn. 3 can be rewritten as~ H(t)/H(O) = e-kt.

(4)

The initial rate of removal of inner histones can be calculated by taking the derivative of Eqn. 3 with respect to t and obtaining the value of this derivative at t = 0. It follows from Eqn. 4 that d / d t [ H ( t ) / H ( O) ]t: o = - - k .

(5)

Hence, the initial rate of removal of inner histones is the negative of the rate constant. For much of the data presented here, the inner histone removal from chromatin was measured at a single time (i.e. at 5 min). The rate constant can be calculated from these data by use of the relationship in Eqn. 3 so that k = --ln [ H ( t ) / H ( O ) ] / t = v .

(6)

The initial rate of formation of the heparin • histone complexes (v) is just the negative of the rate of removal of inner histones from chromatin. Since it is customary to use initial velocity of the appearance of product (in this case, heparin • inner histone complex) in the Hill analysis, we have used the value for the rate constant as the initial rate. The initial rates were determined for several concentrations of heparin using nuclei from cells in different phases of the cell cycle. V was estimated from kinetics of heparin-mediated removal of inner histones from chromatin at large heparin concentrations where v did n o t change significantly with increased heparin concentration. After determining values of v at known heparin concentrations, x, and of V, these data can be analyzed according to the modified Hill equation. The analysis is simplified by rewritting Eqn. I as follows: In [ v / ( V - - v)] =ln K -- n In x .

(7)

This form of the Hill equation indieates that the value of n can be determined from the slope of a plot of ~ n [ v / ( V - v ) ] vs. ~n x, as shown in Fig. 4. Over the range of heparin concentrations employed in this study (50 -800 pg/ml), the linear fit to the data was obtained by linear regression analysis. The value of n is an indicator of interactions between (or among) the inner histones (either within a nucleosome or between nucleosomes) so t h a t a change in this value would reflect a change in the organization of ehromatin at the level of the inner histones. This m e t h o d for analysis of the cooperativity displayed in the inter-

499

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