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Condensation and Precipitation of Chromatin By Multivalent Cations a
a
a
E. Fredericq , R. Hacha , P. Colson & C. Houssier a a
Laboratoire de Chimie Macromoléculaire et Chimie Physique , Université de Liège , Sart-Tilman (B6), B-4000 , Liège , Belgium Published online: 21 May 2012.
To cite this article: E. Fredericq , R. Hacha , P. Colson & C. Houssier (1991) Condensation and Precipitation of Chromatin By Multivalent Cations, Journal of Biomolecular Structure and Dynamics, 8:4, 847-865, DOI: 10.1080/07391102.1991.10507849 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507849
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Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 8, Issue Number 4 (1991), "'Adenine Press (1991).
Condensation and Precipitation of Chromatin By Multivalent Cations E. Fredericq*, R. Hacha, P. Colson and C. Houssier
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Laboratoire de Chimie Macromoleculaire et Chimie Physique Universite de Liege, Sart-Tilman (B6) B-4000, Liege, Belgium Abstract
The condensation and the precipitation of rat liver chromatin upon addition of spermine4 +, spermidine 3+, hexamminecobalt(II1)3+ and Mg2+ cations have been studied using solubility. fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by M!f+ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by M!f+ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.
Introduction Polyamines, in particular spermine and spermidine, are found in almost all eukaryotic cells. They are involved in mitosis, in various cellular syntheses such as those of proteins, RNA and DNA, as well as in growth and differentiation, and in the regulation oftopoisomerase II (1-5). This has stimulated interest in the study of their interactions with DNA and chromatin within the past few years. The condensation and aggregation of DNA by spermine and spermidine have been intensively investigated since about 20 years. Similar studies with chromatin have been developed more recently (6-22). *Correspondence address: E. Fredericq, Laboratoire de Chimie Physique, Universite de Liege, SartTilman (B6), B-4000, Liege, Belgium.
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The main effects of spermine, spermidine and other multivalent cations is to induce condensation of the 10 nm fibre ofchromatin into the 30 nm fibre as well as aggregation into fibrils, and finally precipitation. These effects are generally attributed to the neutralization of DNA phosphate groups by the positive charges of the cations allowing a closer contact between nucleosomes belonging to one single chain or to neighbouring ones ( 11-18). The mechanism of such interactions is far from being elucidated and the specific effects of polyamines have not received any explanation. It is evident that such a specificity requires finer physico-chemical interactions than a simple electrostatic neutralization. Most studies performed in vitro were done before the onset of precipitation. So it seemed worth while to us to extend them to the range of polyamine concentration where two phases separate, and try to characterize by various methods the soluble and the insoluble complexes so obtained. We were able, in particular, to demonstrate an important redistribution of proteins under those conditions as well as significant differences in physico-chemical parameters that we tried to interpret in terms of structural modifications in the two kinds of complexes.
Material and Methods
Preparation and Characterization of Chromatin Nuclei were isolated from rat liver (23) in the presence of 1 mM PM SF. They were stored at -20°C in a special buffer (24). Partial digestion was carried out at 37°C (DNA concentration: 70-100 absorbance units at 260 nm, as measured in (M NaOH) with staphylococcal nuclease (E.C. 3.1.4.7., Worthington) at 10 units/ml of sample and digestion was allowed to progress until a content of acido-soluble residues equal to 3-4% of all soluble chromatin was reached (25). Digestion was stopped by chilling in ice and adding EDTA to a final concentration of O.ol M. Nuclei were pelleted, resuspended in 0.2 mM EDTA pH 7, 0.5 mM PMSF and allowed to lyse by dialysis against the same buffer. The DNA absorbance was then 100-200.The fractionation of chromatin was performed on a 5-30% sucrose gradient in 1 mMTris-HC1,0.2 mM EDTApH 7 byisokineticcentrifugation at4°C, for 15 H at 16,000 rpm (46 000 g) in a SW-27 rotor; 1.5 ml of the sample was loaded on top of each gradient. Mter centrifugation the fractions corresponding to the summit of the main peak were pooled and dialyzed against 0.5 mM EDTA-0.05 mM PMSF pH 7. Chromatin was stored at - 20°C. Calf thymus chromatin was prepared as previously described (26). It was used only in a few solubility measurements. DNA size in chromatin was estimated by electrophoresis (27) and was found to lie in a range of 2,000 to 30,000 base pairs with a predominance of 10,000 to 20,000 base pairs. Proteins from chromatin were analyzed by electrophoresis on polyacrylamide slabgel prepared according to Laemmli (28) with the following modifications: in the separation gel, concentrations of total monomers and of cross-linking agent were 15.1 g/100 ml and 0.66 g/100 g of total monomers respectively; in the stacking gel,
Chromatin Condensation and Cations
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corresponding concentrations were 3.4 g/100 ml and 5.88 g/100 g of total monomers, respectively. Spermine.4 HCl and spermidine.3 HCl (Serva) were used without further purification. Hexamminecobalt (III) chloride was kindly provided by Dr R. Cahay.
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Solubility
Calculated amounts of the precipitant cation solution (1 to 5 mM spermine, spermidine and hexamminecobalt, 10 mM MgC12) were added from a micropipet under stirring, to the chromatin solution (A260 = 4) at 20°C. Mterone hour more stirring the solution was centrifuged 15 minutes in a LMWTH21 machine at 12,000 rpm. (10 000 g). The supernatant was collected as the "soluble fraction" and the precipitate was redissolved in 2M NaCl and dialyzed against 0.5 mM EDTA- 0.05 mM PMSF pH 7. Mter a new similar centrifugation, the supernatant was collected and referred to as the "precipitate". It was checked that precipitation was completed after the one hour stirring procedure. Both fractions were designated by the 1/P ratio at which they were obtained. Spectroscopic Measurements
Circular dichroism spectra were measured on a Jobin Yvon Mark V dichrograph between 250 and 350 nm. Electric dichroism was measured as already described (29,30). Fluorescence intensity was measured on a Baird atomic Fluorispec SF 1OOE in 10 mm path length semi-micro cuvettes at a spectral bandwidth of32 nm, with an excitation wavelength of 290 nm. All data are given in arbitrary units. Fluorescence lifetimes were measured on an Edinburgh Photocounting Instrument Modell99 equipped with a hydrogen lamp. The lifetimes were determined by fitting the experimental decay curve with a two exponentials program provided by the manufacturer. Melting curves were recorded as previously described (13). All spectroscopic measurements were performed on 0.5 mM EDTA solutions at pH 7 with absorbances at 260 nm ranging from 0.3 to 1, preferably around 0.5. All absorbances are reported for a path length of 1 em. Binding of Spermidine
Binding was measured in teflon cells with double 1 ml compartments mounted in an equilibrium-dialysis set up (Dianorm, Zurich), which insures a constant rotation of the cells for stirring. The dialysis membranes were prepared from Visking tubing. A stock solution containing fixed quantities of eH) spermidine (Dupont, NEN Research Products, 32.6 Ci/mmol) and 1 mM cold spermidine in the standard buffer 0.5 mM EDTA pH 7 was prepared. Various dilutions of this stock solution were added to one compartment of the dialysis cells and 1 ml of the chromatin solution
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(A260 = 2) to the other.Rotation of the cells was performed at 20°C for two hours and it was checked that equilibrium was reached after that time. The concentration of labelled spermidine in both compartments was then measured by adding 200 f.Jl aliquots ofthese solutions to 3 ml of Beckman "liquid scintillation cocktail" and counting in a Beckman LS 3801 apparatus. Linearity of dpm with labelled spermidine concentration was checked.
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A strong binding of the spermidine on the membrane was observed (about 30%). Appropriate corrections were applied to the results but introduced additional errors which reduced somewhat the reproducibility of the results to an estimated accuracy ofca ± 5%. When precipitation occurred in the higher range of the 1/P ratio, the solution from the chromatin compartment was centrifuged for 15 min at 12,000 rpm (10 000 g) in a LMW TH21 machine. The precipitate was redissolved in 2M NaCl. The radioactivity was measured on samples from the supernatant and from the redissolved precipitates. The respective chromatin contents of both fractions were determined from their absorbance at 260 nm.
Results Solubility
The effect of various cations on chromatin solubility is given in Figures 1 and 2, as percentage of initial absorbance at 260 nm, i.e., 4 (0.6 mM chromatin phosphorus) remaining in solution, as a function of 1/P ratio. Moreover in Figure 1 are given solubility data obtained with calf thymus chromatin in the presence of spermine only. Let us point out that all other measurements reported in this work were done
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0~--~d-----~~~~~==~~~~~ 0 0.15 0.20 liP 025 Figure 1: Solubility of various cation-chromatin complexes (as percentage of initial absorbance at 260 nm, i.e. 4, remaining in solution) versus molar ratio (1/P) cation over phosphate: rat liver chromatin-spermine (0), calf thymus chromatin-spermine (e). rat liver chromatin-spermidine (~). rat liver chromatinhexamminecobalt(III) (.).
Chromatin Condensation and Cations
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Figure 2: Solubility of rat liver chromatin-Mg 2+ ion complexes (as percentage of initial absorbance at 260 nm, i.e. 4, remaining in solution) versus molar ratio (1/P) Mg2+/phosphate (lower abscissa) or Mg2+ molarity (higher abscissa). ·
on rat liver chromatin only. There is always a narrow range ofl/P where almost total precipitation by spermine takes place. This was fairly constant for two samples of rat liver chromatin (0.06-0.08), slightly higher for calf thymus chromatin (0.08-0.10). This range around 0.08 is close to the value found by Koch eta/. (16) using chicken erythrocyte chromatin with A 260 = 20, but is half the value of Marquet eta/. (17) found on the basis of turbidity increase, for calf thymus and chicken erythrocyte chromatin with A260 = 0.5. There is no doubt that the 1/P range of precipitation is influenced by several factors: the nature of chromatin, its concentration, its average molecular weight, its protein content etc. Sen and Crothers (11) found a maximum compacting effect of spermine, followed by a macroscopic aggregation at 1/P 0.150.20 for chicken erythrocyte chromatin with A260 = 0.2. The precipitating range of spermidine (1/P 0.13-0.15) is a little higher than that of spermine and in good agreement with data of Koch et a/. (16). For hexamminecobalt(III) the precipitation range is still higher (1/P 0.16-0.20). Similar solubility curves are given for Mg 2+ in Figure 2. Some authors believe that the absolute ion concentration is probably more appropriate to express the results obtained in this case and to compare them with literature (17). We give in Figure 2 the abscissa with both scales, 1/P and Mg2+ molarity. We found a precipitation range of 1/P of 0.4-0.7 or 0.3-0.5 mM under our conditions. A higher solubility is reported in literature for chicken erythrocyte chromatin but results vary from 0.5 to 2 mM Mg 2+ (10,11,16,17). In terms ofi/P values, we may point out the determining influence of valence on the precipitating power of cations.
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Table I DNA and Protein Content of Supernatant and Precipitate Obtained After Precipitation of Chromatin By Spermine at 1/P = 0.10 DNA content" from from analysish absorbance Native Supernatant Precipitate
180 9 155
200 9.5 160
Protein content• from analysisc
Protein/DNA weight ratio
420 36 240
2.3 ± 0.2 4.0 ± 0.25 1.5 ± 0.2
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"in ~Jg/ml. bHubbard et al. (46). cLowry et al. (47).
Figure 3: SDS-polyacrylamide gel electrophoresis of proteins from soluble and precipitated complexes chromatin-cations. Lane 1, native chromatin. Lanes 2 and 3, chromatin-spermine complexes, I/P = 0.12. Lanes 4 and 5, chromatin-spermidine complexes, I/P = 022. Lanes 6 and 7, chromatin-hexamminecobalt(III) complexes, liP = 0.30. Lanes 8 and 9, chromatin-Mg2+ complexes, 1/P = 0.08. In each case, S is for the soluble fraction, P for the precipitate.
DNA and Protein Content Determination ofDNAand protein in the supernatant and the precipitate obtained with spermine indicates an important enrichment of proteins (his tones + NHPs relative to DNA) in the former compared to the latter (Table I). More significant are the electrophoreses made in SDS-polyacrylamide run on various samples at different degrees of precipitation (Figure 3). They display a progressive disappearance ofhistones in the supernatants and theirincrease in the precipitates. Simultaneously a strong increase of all NHPs takes place in the supernatants; in the patterns, two bands in particular (below HI) become relatively stronger in relation with an
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853
enhancement offluorescence (see next paragraph). They can be attributed to HMG proteins 1 and 2 since these two proteins are entirely responsible for the tryptophan fluorescence of chromatin and have been identified in other works at similar places in the electrophoretic patterns of various chromatins (31-33). In the supernatant obtained at 90-95% precipitation by spermine as well as by spermidine, there are practically no histones left and a maximum amount ofNHPs, in particular HMG proteins 1 and 2. Precipitates obtained under the same conditions contain in large amounts histones only, except for faint bands of HMG proteins, particularly the fastest one that we attribute from its position to HMG protein 14 or 17 (32).1t is true that the precipitates are analysed after a redissolution in 2M NaCl. Since everything is redissolved, it seems unlikely that this treatment could modify the constancy of histones distribution, more especially as the main structural features are maintained (vide infra). After precipitation by hexamminecobalt(III) (Figure 3), similar differences appear in the supernatant and precipitate patterns, although somewhat attenuated as regards the enrichment in HMG proteins 1 and 2. The situation is quite different in the case of precipitation by Mg2+ ions. Here the proportions ofhistones and HMG proteins are the same in both fractions, whereas disappearing of a few LMG proteins occurs in the precipitate. In conclusion, precipitation ofchromatin by the tetra- and trivalent cations assayed by us leads to a complete redistribution of proteins whereas Mi+ ions do not bring about any important separation.
Fluorescence The only tryptophan-containing proteins of chromatin are HMG proteins 1 and 2 which are responsible for the fluorescence band observed with a maximum around 340-350 nm when excited at 290 nm. These proteins contain each two tryptophan residues per molecule (34). Rat liver chromatin is particularly rich in NHPs and consequently well appropriate for such fluorescence studies. The tryptophan fluorescence can be distinguished from that of tyrosine which is brought about by excitation at 280 nm and gives a very faint band with maximum at 310 nm only detectable by high-sensitivity spectrometers. We observed that, with increasing degree of precipitation by spermine, spermidine and to a lesser extent hexamminecobalt(III), the fluorescence of the supernatant increases very much whereas that of the precipitate remains negligible. Values of specific fluorescence, i.e., fluorescence intensities at 356 nm in arbitrary units divided by the absorbance of the supernatant, are given in Figure 4 for different degrees of precipitation. For spermine and spermidine, initial values are multiplied by a factor of4, for hexamminecobalt(III) by a factor of2. With Mg2+ ions, the variation is negligible. This behaviour is to be related to the increase in HMG proteins 1 and 2 brought
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Figure 4: Specific fluorescence (F/A where F is the fluorescence intensity in arbitrary units and A the absorbance at 260 nm) of chromatin-cation complexes (soluble fraction) as a function of the degree of precipitation, i.e., solubility (see Figure I). Precipitating cations: spermine (0), spermidine (8.), hexammine cobalt(III), Mg2+ (D).
about by the tetra- and trivalent cations. It is difficult to make a precise quantitative correlation because we lack a reliable method for determining the content in these fluorescent proteins. Taking into account the data of Table I, we can estimate the NHP content by subtracting the histone/DNA weight ratio, i.e., around 1, from the total protein/DNA ratio. This gives 1.3 for chromatin. In the supernatant described in Table I, since there are no histones left, the protein/DNA ratio is identical to the NHP/DNA ratio, i.e. 4. The multiplication of the NHP content by a factor higher than 3 may reasonably explain the increase in fluorescence. Modifications of fluorescence could be brought about by structural changes occurring in the environment of the chromophores during the precipitation process and the redistribution of proteins. This point was investigated by the determination of the fluorescence lifetimes oftryptophan in the supernatant resulting from the precipitation of chromatin by spermine. For chromatin the decay curve of fluorescence was very satisfactorily fitted by a two components exponential, the major one representing 66% with a lifetime equal to 5.4 ns and the minor one (34%) with a lifetime of 1.6 ns. These lifetimes are in the range ordinarily found in proteins containing two kinds of tryptophan residues (35). In all the supernatants (Table II), despite the important enhancement of fluorescence reported above, the lifetimes remain identical within
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Chromatin Condensation and Cations Table II Fluorescence Lifetimes of Supernatants After Precipitation of Chromatin By Spermine
1/P
F!A"
t Ib
%tl
~c
% t2
0 0.08 0.18 0.24
2.0 2.3 12 16
5.4 4.8 5 5.4
66 68
1.6 1.0 1.5 1.5
34 32 36 38
64
62
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•Fluorescence intensity over absorbance. bFirst lifetime in ns. csecond lifetime in ns.
experimental error. This is in line with the assumption that the modification of fluorescence intensity is entirely due to an enrichment of the sample in the tryptophancontaining proteins HMG 1 and 2, without any structural change in the environment of these residues. It is likely that the tryptophan residues in HMG proteins 1 and 2 are buried inside the folded part of these molecules (34).
Circular Dichroism The CD spectrum of chromatin between 250 and 350 nm (Fi~re 5) displays a maximum near 282 nm with an ellipticity of 1900 ± 200 deg em dmoC 1 in agreement with the data of Marion et al. (36). The CD spectra obtained with the precipitates at various degrees of precipitation by tetra- and trivalent cations are similar to the chromatin spectrum (results given only for spermine). Only a slight enhancement of maximum ellipticity is observed but this may be due to the fact that the structure has been a little altered by the dissolution in 2M NaCl and recombination at low ionic strength. A blank of chromatin treated in the same way gives also a slightly higher ellipticity at 282 nm, i.e., 2100 deg cm2 dmol- 1• More important changes are observed in the supernatants at high degree of precipitation: the shape of the spectrum is shifted towards that of protein-free DNA (Figure 5) with a significant increase in maximum ellipticity. The lowering of ellipticity between 250 and 300 nm observed for chromatin compared to free DNA is attributed to the combination of DNA to histones (37). So it appears that, under the action of the tetra- and trivalent cations tested, the DNA is partly liberated from protein constraints. This is in line with the release of histones demonstrated by electrophoresis. We assume that the NHPs remaining in the supernatant are still, partly at least, bound to DNA (see discussion) explaining the lowering of ellipticity compared to that of free DNA
Melting Curves The melting curve of our chromatin samples presents the classical monotonic aspect (Figure 6A). The differential curve displays a slightly asymmetric peak, i.e., widened on the side of low temperature, the maximum laying at 80-81 oc. The
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8
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320 Figure 5: Ellipticity of various fractions of chromatin-cation complexes versus wavelength: native DNA (-), native chromatin (-----), soluble complex with spermine, 1/P = 0.10 or spermidine 1/P = 0.30 (······ ), precipitate with spermine, 1/P = 0.10 ( ·- ·- · ).
hyperchromism has the regular value close to 40%. At high degree of precipitation by spermine (Figure 6C), the supernatants curves present two parts: the high temperature peak with its maximum at 81 o C and a wide peak at low temperature whose
857
Chromatin Condensation and Cations
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Figure 6: Melting curves ofvarious fractions ofchromatin~ation complexes. Left ordinate: hyperchromism, right ordinate: normalized derivative. A: native chromatin, B: precipitate with spermine (1/P = 0.12), C: soluble complex with spermine (1/P = 0.09), D: soluble complex with Mg2+ (1/P = 0.9).
importance increases with 1/P value. Its maximum which in some cases is not clearly defined, lies in the range 55-60oC. The hyperchromism is decreased to 2530%. A similar behaviour is displayed in the case of precipitation by spermidine (results not shown in the figure). With Mg2+ the peak is unique and better defined although still rather wide (Figure 6D). Its maximum lies near60°C for high values of I/P. The curves of the precipitates are all much more regular (Figure 6B). They remain similar to that of chromatin, with a maximum near 81 oc and a hyperchromicity near 40%. The peak is somewhat widened but this is probably due to the dissociation and reconstitution of the precipitates, as may be seen on a curve obtained with chromatin after the same treatment (results not shown). From our experiments it appears that, in the supernatants, there is a slight quantity of the original chromatin giving the peak at 81 oC, progressively disappearing as precipitation is increased. The remaining soluble part contains only DNA and proteins,
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Fredericq et a/.
forming at least in part, heterogeneous complexes, where a small portion of DNA is more or less denatured in view of the reduced hyperchromicity. It is difficult to decide whether the wide low temperature peak is due to complexes DNA-NHP or to complexes polyamine-DNA which present also the type ofheterogeneous curves in the case of spermidine at least (38). In the precipitates there is a chromatin which is deprived of the greater part of its NHPs but has its full content of DNA and histones and keeps its original structural features.
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Electric Dichroism The curve of electric dichroism vs field strength (Figure 7) for rat liver chromatin is similar to that previously found for chicken erythrocyte chromatin (17). Addition of spermine or spermidine brings about a strong decrease of the negative dichroism in the supernatants, already at the beginning of the precipitation (Table III). This is in
0
10 E (kV/cm)
15
Figure 7: Specific electric dichroism of various fractions of chromatin-cation complexes versus field strength: native chromatin (e), reconstituted chromatin (lt.), soluble complex with spermine, 1/P = 0.08 (0), precipitate with spermine, 1/P = 0.07 (V), precipitate with spermine, 1/P = 0.09 (~).soluble complex with spermidine, 1/P = 0.065 (0), precipitate with spermidine, 1/P = 0.12 (()).
859
Chromatin Condensation and Cations Table III Reduced Electric Dichroism at 12.5 kV/em of Supernatants and Precipitates After Precipitation By Spermine and Spermidine 1/P
Supernatants
Precipitates•
0
-0.065h
-0.08c
Spermine
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0.06 0.07 0.08 0.09 0.12
-O.ol
-0.01 +0.02 0 0
-0.11 -0.115 -0.115 -0.115
Spermidine 0.065 0.12 0.16
-0.06 0 0
-0.095 -0.085
"Redissolved in 2M NaCl and dialysed against 0.5 mM EDTA, pH 7. ~ative chromatin. ~ative chromatin treated as in".
line with the effect of various cations on chicken erythrocyte chromatin in the range of solubility of the complexes ( 11, 16,17,21 ). These observations were interpreted on the basis of a transition from the 10 nm to the 30 nm fibre. At a degree of precipitation of 20%, we found that the dichroism becomes slightly positive at intermediate field strength and is no longer measurable at higher I/P ratios. We do not think that the reversal of sign occurring at higher field strength indicates a field-induced stretching, since we observed the same curve after application of several pulses. In fact the pulse signals for that liP value become complex at high field strength, revealing the presence of entities with different dichroism signs and relaxation times. This is in line with the heterogeneity of the samples as evidenced by our other measurements (vide supra). On the contrary, the precipitates obtained by spermine or spermidine action display a more negative dichroism than a blank of reconstituted chromatin (Table III). Although the recombination process may have altered the structure of these precipitates to some extent, it seems likely that here again the two complexes obtained by spermine and spermidine differ considerably in the soluble and insoluble phases: our experimental results show that the former is a much more condensed assembly and that the latter remains closer to the original structure of chromatin. It is interesting to correlate our findings with previous reports: a positive linear
dichroism was found for chromatin at low ionic strength (40) which was attributed to the presence of spermine and spermidine in the preparation (20). On the other hand, Matsuoka eta!. (41) found that under the action of nucleases, two fractions of chromatin may be obtained,one released with a weak positive linear dichroism and one non-released with a negative dichroism.
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860 2
As regards Mg + ions, numerous studies have shown that they bring about a strong
decrease of the negative dichroism and even in some cases a reversal of its sign, i.e., they have an effect similar to that of tetra- and trivalent cations but at higher concentrations (11,17,19,20,39). Binding of Spermidine to Chromatin
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In view of the different properties displayed by the soluble and insoluble complexes of chromatin and tetra- or trivalent cations, it seemed important to determine the binding capacity of the two forms of complexes. This was done for spermidine since we disposed only of eH)spermidine required for binding determination. Bound and free quantities of the cation were measured by the classical equilibriumdialysis method, the titration being made by radioactive counting. A large range of 1/P ratios covering solubility and precipitation domains was examined. In the range of complete solubility, i.e., below 0.018 mM spermidine under our experimental conditions, a fairly regular Langmuir isotherm was obtained (Figure 8) which gave a reasonably straight line for the Scatchard plot, r/c vs r (Figure 9), considering an error estimated to ± 5% in the determination of rand c (r is the number of spermidine bound per chromatin phosphate and cis the free spermidine concen-
r PRECIPITATION RANGE
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6
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•• •
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%~--------~,--------~2--------~3----~ c x 10 5 free spermidine Figure 8: Binding curve of spermidine by chromatin: r (moles of spermidine bound per mole of chromatin phosphate) as a function of c (free spermidine concentration): in the range of total solubility, soluble complex (0); in the range of partial solubility, soluble complex (e), insoluble complex (A).
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Chromatin Condensation and Cations
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r/c x 10-4
0.
• 00
0.05
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'r
Figure 9: Scatchard plot of the binding of spermidine by chromatin: r/c versus r. Same symbols as in Figure 8.
tration). An intrinsic binding constant of(2 ± 0.2) .105 mol- 1 was deduced. Tentative extrapolation of the curve gave a maximum amount of binding sites around 0.12, i.e., one spermidine trivalent cation per eight nucleotides. This represents a neutralization of3/8 of DNA negative charges. Accepting a value of60% ofDNAcharges neutralized by histones in chromatin, this would indicate that the saturation of the binding sites occurs with a complete neutralization of the net negative charge. In the range of partial precipitation of the complexes, results were a little more irregular (Figure 8), due to additional errors introduced by the determination of the amounts of soluble and insoluble fractions and to more variability in the binding process in a heterogeneous medium. Nevertheless the data clearly indicate that the amount of spermidine bound by the insoluble complex was close to that found in the soluble fraction, being in excess of 10 to 20%. Scatchard plots did not allow any significant estimation of the binding parameters in that range. We have also investigated by radioactive counting what happens to spermidine bound to precipitates after their dissolution in 2M NaCl and dialysis against 0.5 mM
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EDTA. We found that radioactivity was completely lost and consequently that spermidine had been dialyzed out of dissociated chromatin during its recombination. These results are in agreement with those of Colson and Houssier ( 19) who found that polyamines are dissociated from chromatin at relatively high ionic strength.
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Discussion
As has been already reported in various works, the solubility of chromatin varies along an S-shape curve with the relative concentration of multivalent cations (1/P ratio) in a range which is well defined for a given type of the nucleoprotein at a specified concentration (Figures 1 and 2). A comparison between the cations used here may be done by considering the point of 50% precipitation. These are respectively, in 1/P values, 0.075 (spermine), 0.135 (spermidine), 0.165 (hexamminecobalt(III)), 0.55 (Mg2+). These values are valid only for an initial concentration of chromatin equal to 0.6 mM phosphate (A260 = 4). There is certainly a concentration dependence for the solubility as well as for other parameters studied in this work. The onset of compaction and aggregation in particular must be submitted to the influence of the total concentration of the particles involved. It appears that the cation charge is the determining factor but that s~ecific effects add up, when considering the higher precipitation power of spermidine3 compared to hexamminecobalt(III)3+ ions. However the difference is small and does not account for the important physiological action specifically displayed by the polyamines.
The main objective of this work was to investigate the composition and structural features of the cation-chromatin complexes in the soluble and precipitated phases. The chemical composition revealed in the case of tetra- and trivalent cations an overall enrichment in proteins with the increase ofi/P in the soluble complexes, due to a strong increase in NHPs, whereas histones progressively disappear, the contrary happening in the precipitates. At high precipitation degree by spermine and spermidine, the NHPs are totally concentrated in the soluble complex (except for a very slight amount of some HMG proteins, 14 or 17), a little less by hexamminecobalt(III); at the same time all histones are excluded. The concentration of HMG proteins 1 and 2 is particularly evidenced by tryptophan fluorescence. The strong enhancement of fluorescence in the supernatants occurs without any change in the two tryptophan lifetimes, which indicates that the presence of two classes of residues differing in their environment is maintained even in complexes almost entirely constituted of DNA and NHPs. In this respect the action ofMg2+ ions is quite different: compared to intact chromatin, the soluble Mg 2+-chromatin complexes do not display any change in composition; there is only a slight depletion of LMG proteins in the precipitates. The circular dichroism in the 250-300 nm positive band of chromatin confirms the histone depletion of the soluble complexes, i.e., it is becoming gradually closer to that offree DNA when 1/P increases whereas that of precipitated complexes redissolved and recombined remains practically unaltered.
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From the electric dichroism and the melting curves, we conclude that spermine and spermidine deeply affect the structure of the soluble phase: strong condensation and replacement of the native particles by a heterogeneous mixture ofDNA-NHP complexes, as a result of histone depletion. The insoluble phase, on the contrary, which consists of"nucleohistone" seems to retain the main features of the original chromatin. However it must be recognized that they have undergone a dissociation and recombination for their characterization and that we have no guarantee for an exact reconstitution. Moreover the polyamines have been eliminated by the dialysis process. Are these differences in protein distribution and structural features in relation with a different binding of the precipitating cations in the soluble and insoluble phases? Binding studies made with spermidine at least, reveal that this trivalent cation is bound in similar amount by the soluble and insoluble complexes. Consequently charge effects could not account for the observed differences. Let us now examine to what extent the NHPs found in the supernatants are still bound to DNA or are liberated during the precipitation. We think that at least a part of them remains bound in a protein-DNA complex for the following reasons: 1. The negative electric dichroism at high 1/P values of spermine and spermidine in the supernatant tends to 0 and can even be positive. A solution of protein-free DNA should always display a high negative dichroism even at high spermine to DNA ratio: Marquetet al. (12) found a dichroism equal to -0.3 at 1/P = 0.28. The zero value indicates that DNA has lost its native elongated structure to become a highly condensed particle.
2. The circular dichroism of the supernatants although closer to that of free DNA than to that of native chromatin reaches half the ellipticity of protein-free DNA and this must be due to a binding of proteins since polyamines themselves do not modify the CD of pure DNA at low ionic strength (42,43). 3. Various studies have demonstrated the binding ofHMG proteins to DNA at low and moderate ionic strength (44). In conclusion, spermine and spermidine bind to chromatin free phosphate-groups in a first step, gradually neutralizing the charge and leading to a condensation of the particles from the 10 nm to the 30 nm fibre without disrupting the general assembly. These complexes remain soluble at low ionic strength. In a second step, the addition of higher amounts of these cations brings about precipitation at I/P values where DNA binding sites for cations are close to saturation and the net charge approaching zero value. For spermidine this would happen at a ratio corresponding to one molecule per eight nucleotides where 40% of phosphates are neutralized by the polyamines and the rest by histones. Under such conditions, a further addition of cation brings about a displacement of his tones which are concentrated in the soluble phase whereas part of the particles consisting finally of an
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assembly of DNA and NHPs remains soluble, in a very condensed state, presenting an important heterogeneity. The insoluble particles, after dissociation in 2M NaCl, dialysis and recombination, recover structural features close to those of native chromatin.
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The precipitation of chromatin by Mg2+ ions differs in many respects from these lines. There is no redistribution of proteins and the soluble and insoluble Mg2+chromatin complexes are similar to each other and to native chromatin. Numerous works have already reported the effectiveness of Mg2+ ions in the formation of higher structure and condensation of chromatin (45,46). This condensation is preliminary to aggregation (17,21). Finally the accumulation ofNHPs and the liberation ofhistones in some regions of chromatin under the influence of high amounts of polyamines could contribute to their physiological action since it is well established that histones maintain DNA in a condensed state and that NHPs and in particular HMG proteins play an important role in transcription and regulation (44,47).
Acknowledgments The financial support of the "Services de Programmation de la Politi que Scientifique, Actions de Recherches Concertees, ARC contract no. 80/85-90" and of the "Fonds National de la Recherche Scientifique, IISN contract no. 4.9004.88" is gratefully acknowledged. Thanks are due to Dr. Claire Duyckaerts for her help in the determination of labelled spermidine and to Dr. Francoise Huriaux for electrophoreses of chromatin proteins. References and Footnotes
Abbreviations: PMSF Phenylmethylsulfonyl fluoride, 1/P number of moles of cation over number of chromatin phosphate, NHP non-histone protein, HMG high mobility group, LMG low mobility group, CD circular dichroism, SDS sodium dodecylsulfate. I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16.
Bachrach, U., Functions of Naturally Occurring Polyamines, Academic Press, New York (1983). Tabor, C.W. and Tabor, T.,Ann. Rev. Biochem. 53,749-790 (1984). Nizamuddin, A, Biochem. Educ. 15, 106-110 (1987). Pommier, Y., Kerrigan, D. and Kohn, K., Biochemistry 28,995-1002 (1989). Heby, 0. and Person, L., TTBS 15, 153-158 (1990). Jerzmanowski, A, Staron, K., Tyniec-Kroenke, B. and Toczko, K., Biochim. Biophys. Acta 565, 356364 (1979). Billett, M.A. and Hall, T.J., Nucleic Acids Res. 6, 2929-2945 (1979). Widom, J. and Klug, A., Cell43, 207-213 (1985). Jin, Y.J. and Cole, R.D.,J. Bioi. Chern. 261, 15805-15812 (1986). Wid om, J., J. Mol. Bioi. 190, 411-424 (1986). Sen, D. and Crothers, D.M., Biochemistry 25, 1495-1503 (1986). Marquet, R., Houssier, C. and Fredericq, E., Biochim. Biophys. Acta 825, 365-374 (1985). Marquet, R., Colson, P. and Houssier, C.,J. Biomol. Struct. Dyn. 4, 205-218 (1986). Makarov, V.L., Smirnov, I. and Dimitrov, S.I.,FEBS Lett. 212,263-266 (1987). Fredericq, E., Hacha, R. and Houssier, C.,Int. J. Bioi. Macromol. 10, 51-54 (1988). Koch, M.H.J., Sayers, Z., Michon, AM., Marquet, R., Houssier, C. and Wilffiihr, J., Eur. Biophys. J. 16, 177-185 (1988).
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17. Marquet, R., Colson, P., Matton, AM., Houssier, C., Thiry, M. and Goessens, G.,J. Biomol. Struct. Dyn. 5, 839-857 (1988). 18. Smimov, I.V., Dimitrov, S.l. and Makarov, V.L.,J. Biomol. Struct. Dyn. 5, 1149-1161 (1988). 19. Colson, P. and Houssier, C., FEBS Lett. 257, 141-144 (1989). 20. Hagmar, P., Marquet, R., Colson, P.,Kubista, M., Nielsen, P.E., Norden, B. and Houssier, C., J Biomol. Struct. Dyn. 7, 19-33 (1989). 21. Marquet, R., Thesis, University of Liege (1989). 22. Koch, M.H.J., Sayers, Z., Michon, AM., Sicre, P., Marquet R. and Houssier, C., Eur. Biophys. J 17, 245-255 (1989). 23. Chauveau, J., Maule, Y. and Rouiller, C., Exptl Cell Res. II, 317-321 (1956). 24. Thibodeau, L. and Verly, W., Eur. J Biochem. 107, 555-563 (1980). 25. Mandel, R. and Fasman, G.D., Nucleic Acids Res. 3, 1839-1855 (1976). 26. Houssier, C., Depauw-Gillet, M.C., Hacha, R. and Fredericq, E., Biochim. Biophys. Acta 739, 317325 (1983). 27. Sharp, P.A, Sugden, B. and Sambroek, J., Biochemistry 12, 3055-3063 (1973). 28. Laemmli, U.K., Nature 227, 680-685 (1970). 29. Fredericq, E. and Houssier, C., Electric Dichroism and Electric Birefringence, Clarendon Press, Oxford (1973). 30. Houssier, C. and O'Konski, C.T., in Molecular Electro-Optics (Krause, S., Ed.) NATO ASI Ser. B64, 309-339 (1981). 31. Seyedin, S.M. and Kistler, W.S.,J. Bioi. Chem. 254, 11264-11271 (1979). 32. Craddock, V.M. and Henderson, AR., Carcinogenesis I, 445-450 (1980). 33. Butler, AP., Mardian, J.K.W. and Olins, D.E.,J Bioi. Chem. 260, 10613-10620 (1985). 34. Baker, C., Isenberg, 1., Goodwin, G.H. and Johns, E.W., Biochemistry 15, 1645-1649 (1976). 35. Beechem, J.M. and Brand, L., Ann. Rev. Biochem. 54, 43-71 (1985). 36. Marion, C., Roux, B. and Coulet, P.R., FEBS Lett. 157, 317-321 (1983). 37. Watanabe, K. and lso, K,J Mol. Bioi. 151, 143-163 (1981). 38. Morgan, J.E., Blankenship, J.W. and Matthews, H.R.,Arch. Biochem. Biophys. 246,225-232 (1986). 39. Me Ghee, J.D., Rau, D.C., Charney, E. and Felsenfeld, G., Cell22, 87-96 (1980). 40. Tjemeld, F., Norden, B. and Wallin, H., Biopolymers 21, 343-358 (1982). 41. Matsuoka, Y., Nielsen, P.E. and Norden, B., FEBS Letters 169, 309-312 (1984). 42. Gosule, L.C. and Schellman, J.A,J Mol. Bioi. 121,311-326 (1978). 43. Wilson, R.W. and Bloomfield, V.A, Biochemistry 18,2192-2196 (1979). 44. Johns, E.W., The HMG Chromosomal Proteins, Academic Press, London (1982). 45. Thoma, F., Koller, Th. and Klug, A, J Cell. Bioi. 83, 403-427 ( 1979). 46. Watanabe, K. and Iso, K., Biochemistry 23, 1376-1383 (1984). 47. lgo-Kemenes, T., Harz, W. and Zachau, H.G.,Ann. Rev. Biochem. 51, 89-121 (1982). 48. Hubbard, R.W., Matthew, W.T. and Dubovik, D.A,Anal. Biochem. 38, 190-201 (1970). 49. Lowry, O.H., Rosebrough, N.J., Farr, AL. and Randall, R.J.,J Bioi. Chem. 193,265-275 (1951). Date Received: August 10, 1990
Communicated by the Editor C. W. Hilbers
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Condensation and Precipitation of Chromatin By Multivalent Cations a
a
a
E. Fredericq , R. Hacha , P. Colson & C. Houssier a a
Laboratoire de Chimie Macromoléculaire et Chimie Physique , Université de Liège , Sart-Tilman (B6), B-4000 , Liège , Belgium Published online: 21 May 2012.
To cite this article: E. Fredericq , R. Hacha , P. Colson & C. Houssier (1991) Condensation and Precipitation of Chromatin By Multivalent Cations, Journal of Biomolecular Structure and Dynamics, 8:4, 847-865, DOI: 10.1080/07391102.1991.10507849 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507849
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Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 8, Issue Number 4 (1991), "'Adenine Press (1991).
Condensation and Precipitation of Chromatin By Multivalent Cations E. Fredericq*, R. Hacha, P. Colson and C. Houssier
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Laboratoire de Chimie Macromoleculaire et Chimie Physique Universite de Liege, Sart-Tilman (B6) B-4000, Liege, Belgium Abstract
The condensation and the precipitation of rat liver chromatin upon addition of spermine4 +, spermidine 3+, hexamminecobalt(II1)3+ and Mg2+ cations have been studied using solubility. fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by M!f+ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by M!f+ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.
Introduction Polyamines, in particular spermine and spermidine, are found in almost all eukaryotic cells. They are involved in mitosis, in various cellular syntheses such as those of proteins, RNA and DNA, as well as in growth and differentiation, and in the regulation oftopoisomerase II (1-5). This has stimulated interest in the study of their interactions with DNA and chromatin within the past few years. The condensation and aggregation of DNA by spermine and spermidine have been intensively investigated since about 20 years. Similar studies with chromatin have been developed more recently (6-22). *Correspondence address: E. Fredericq, Laboratoire de Chimie Physique, Universite de Liege, SartTilman (B6), B-4000, Liege, Belgium.
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The main effects of spermine, spermidine and other multivalent cations is to induce condensation of the 10 nm fibre ofchromatin into the 30 nm fibre as well as aggregation into fibrils, and finally precipitation. These effects are generally attributed to the neutralization of DNA phosphate groups by the positive charges of the cations allowing a closer contact between nucleosomes belonging to one single chain or to neighbouring ones ( 11-18). The mechanism of such interactions is far from being elucidated and the specific effects of polyamines have not received any explanation. It is evident that such a specificity requires finer physico-chemical interactions than a simple electrostatic neutralization. Most studies performed in vitro were done before the onset of precipitation. So it seemed worth while to us to extend them to the range of polyamine concentration where two phases separate, and try to characterize by various methods the soluble and the insoluble complexes so obtained. We were able, in particular, to demonstrate an important redistribution of proteins under those conditions as well as significant differences in physico-chemical parameters that we tried to interpret in terms of structural modifications in the two kinds of complexes.
Material and Methods
Preparation and Characterization of Chromatin Nuclei were isolated from rat liver (23) in the presence of 1 mM PM SF. They were stored at -20°C in a special buffer (24). Partial digestion was carried out at 37°C (DNA concentration: 70-100 absorbance units at 260 nm, as measured in (M NaOH) with staphylococcal nuclease (E.C. 3.1.4.7., Worthington) at 10 units/ml of sample and digestion was allowed to progress until a content of acido-soluble residues equal to 3-4% of all soluble chromatin was reached (25). Digestion was stopped by chilling in ice and adding EDTA to a final concentration of O.ol M. Nuclei were pelleted, resuspended in 0.2 mM EDTA pH 7, 0.5 mM PMSF and allowed to lyse by dialysis against the same buffer. The DNA absorbance was then 100-200.The fractionation of chromatin was performed on a 5-30% sucrose gradient in 1 mMTris-HC1,0.2 mM EDTApH 7 byisokineticcentrifugation at4°C, for 15 H at 16,000 rpm (46 000 g) in a SW-27 rotor; 1.5 ml of the sample was loaded on top of each gradient. Mter centrifugation the fractions corresponding to the summit of the main peak were pooled and dialyzed against 0.5 mM EDTA-0.05 mM PMSF pH 7. Chromatin was stored at - 20°C. Calf thymus chromatin was prepared as previously described (26). It was used only in a few solubility measurements. DNA size in chromatin was estimated by electrophoresis (27) and was found to lie in a range of 2,000 to 30,000 base pairs with a predominance of 10,000 to 20,000 base pairs. Proteins from chromatin were analyzed by electrophoresis on polyacrylamide slabgel prepared according to Laemmli (28) with the following modifications: in the separation gel, concentrations of total monomers and of cross-linking agent were 15.1 g/100 ml and 0.66 g/100 g of total monomers respectively; in the stacking gel,
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corresponding concentrations were 3.4 g/100 ml and 5.88 g/100 g of total monomers, respectively. Spermine.4 HCl and spermidine.3 HCl (Serva) were used without further purification. Hexamminecobalt (III) chloride was kindly provided by Dr R. Cahay.
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Solubility
Calculated amounts of the precipitant cation solution (1 to 5 mM spermine, spermidine and hexamminecobalt, 10 mM MgC12) were added from a micropipet under stirring, to the chromatin solution (A260 = 4) at 20°C. Mterone hour more stirring the solution was centrifuged 15 minutes in a LMWTH21 machine at 12,000 rpm. (10 000 g). The supernatant was collected as the "soluble fraction" and the precipitate was redissolved in 2M NaCl and dialyzed against 0.5 mM EDTA- 0.05 mM PMSF pH 7. Mter a new similar centrifugation, the supernatant was collected and referred to as the "precipitate". It was checked that precipitation was completed after the one hour stirring procedure. Both fractions were designated by the 1/P ratio at which they were obtained. Spectroscopic Measurements
Circular dichroism spectra were measured on a Jobin Yvon Mark V dichrograph between 250 and 350 nm. Electric dichroism was measured as already described (29,30). Fluorescence intensity was measured on a Baird atomic Fluorispec SF 1OOE in 10 mm path length semi-micro cuvettes at a spectral bandwidth of32 nm, with an excitation wavelength of 290 nm. All data are given in arbitrary units. Fluorescence lifetimes were measured on an Edinburgh Photocounting Instrument Modell99 equipped with a hydrogen lamp. The lifetimes were determined by fitting the experimental decay curve with a two exponentials program provided by the manufacturer. Melting curves were recorded as previously described (13). All spectroscopic measurements were performed on 0.5 mM EDTA solutions at pH 7 with absorbances at 260 nm ranging from 0.3 to 1, preferably around 0.5. All absorbances are reported for a path length of 1 em. Binding of Spermidine
Binding was measured in teflon cells with double 1 ml compartments mounted in an equilibrium-dialysis set up (Dianorm, Zurich), which insures a constant rotation of the cells for stirring. The dialysis membranes were prepared from Visking tubing. A stock solution containing fixed quantities of eH) spermidine (Dupont, NEN Research Products, 32.6 Ci/mmol) and 1 mM cold spermidine in the standard buffer 0.5 mM EDTA pH 7 was prepared. Various dilutions of this stock solution were added to one compartment of the dialysis cells and 1 ml of the chromatin solution
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(A260 = 2) to the other.Rotation of the cells was performed at 20°C for two hours and it was checked that equilibrium was reached after that time. The concentration of labelled spermidine in both compartments was then measured by adding 200 f.Jl aliquots ofthese solutions to 3 ml of Beckman "liquid scintillation cocktail" and counting in a Beckman LS 3801 apparatus. Linearity of dpm with labelled spermidine concentration was checked.
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A strong binding of the spermidine on the membrane was observed (about 30%). Appropriate corrections were applied to the results but introduced additional errors which reduced somewhat the reproducibility of the results to an estimated accuracy ofca ± 5%. When precipitation occurred in the higher range of the 1/P ratio, the solution from the chromatin compartment was centrifuged for 15 min at 12,000 rpm (10 000 g) in a LMW TH21 machine. The precipitate was redissolved in 2M NaCl. The radioactivity was measured on samples from the supernatant and from the redissolved precipitates. The respective chromatin contents of both fractions were determined from their absorbance at 260 nm.
Results Solubility
The effect of various cations on chromatin solubility is given in Figures 1 and 2, as percentage of initial absorbance at 260 nm, i.e., 4 (0.6 mM chromatin phosphorus) remaining in solution, as a function of 1/P ratio. Moreover in Figure 1 are given solubility data obtained with calf thymus chromatin in the presence of spermine only. Let us point out that all other measurements reported in this work were done
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0~--~d-----~~~~~==~~~~~ 0 0.15 0.20 liP 025 Figure 1: Solubility of various cation-chromatin complexes (as percentage of initial absorbance at 260 nm, i.e. 4, remaining in solution) versus molar ratio (1/P) cation over phosphate: rat liver chromatin-spermine (0), calf thymus chromatin-spermine (e). rat liver chromatin-spermidine (~). rat liver chromatinhexamminecobalt(III) (.).
Chromatin Condensation and Cations
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Figure 2: Solubility of rat liver chromatin-Mg 2+ ion complexes (as percentage of initial absorbance at 260 nm, i.e. 4, remaining in solution) versus molar ratio (1/P) Mg2+/phosphate (lower abscissa) or Mg2+ molarity (higher abscissa). ·
on rat liver chromatin only. There is always a narrow range ofl/P where almost total precipitation by spermine takes place. This was fairly constant for two samples of rat liver chromatin (0.06-0.08), slightly higher for calf thymus chromatin (0.08-0.10). This range around 0.08 is close to the value found by Koch eta/. (16) using chicken erythrocyte chromatin with A 260 = 20, but is half the value of Marquet eta/. (17) found on the basis of turbidity increase, for calf thymus and chicken erythrocyte chromatin with A260 = 0.5. There is no doubt that the 1/P range of precipitation is influenced by several factors: the nature of chromatin, its concentration, its average molecular weight, its protein content etc. Sen and Crothers (11) found a maximum compacting effect of spermine, followed by a macroscopic aggregation at 1/P 0.150.20 for chicken erythrocyte chromatin with A260 = 0.2. The precipitating range of spermidine (1/P 0.13-0.15) is a little higher than that of spermine and in good agreement with data of Koch et a/. (16). For hexamminecobalt(III) the precipitation range is still higher (1/P 0.16-0.20). Similar solubility curves are given for Mg 2+ in Figure 2. Some authors believe that the absolute ion concentration is probably more appropriate to express the results obtained in this case and to compare them with literature (17). We give in Figure 2 the abscissa with both scales, 1/P and Mg2+ molarity. We found a precipitation range of 1/P of 0.4-0.7 or 0.3-0.5 mM under our conditions. A higher solubility is reported in literature for chicken erythrocyte chromatin but results vary from 0.5 to 2 mM Mg 2+ (10,11,16,17). In terms ofi/P values, we may point out the determining influence of valence on the precipitating power of cations.
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Table I DNA and Protein Content of Supernatant and Precipitate Obtained After Precipitation of Chromatin By Spermine at 1/P = 0.10 DNA content" from from analysish absorbance Native Supernatant Precipitate
180 9 155
200 9.5 160
Protein content• from analysisc
Protein/DNA weight ratio
420 36 240
2.3 ± 0.2 4.0 ± 0.25 1.5 ± 0.2
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"in ~Jg/ml. bHubbard et al. (46). cLowry et al. (47).
Figure 3: SDS-polyacrylamide gel electrophoresis of proteins from soluble and precipitated complexes chromatin-cations. Lane 1, native chromatin. Lanes 2 and 3, chromatin-spermine complexes, I/P = 0.12. Lanes 4 and 5, chromatin-spermidine complexes, I/P = 022. Lanes 6 and 7, chromatin-hexamminecobalt(III) complexes, liP = 0.30. Lanes 8 and 9, chromatin-Mg2+ complexes, 1/P = 0.08. In each case, S is for the soluble fraction, P for the precipitate.
DNA and Protein Content Determination ofDNAand protein in the supernatant and the precipitate obtained with spermine indicates an important enrichment of proteins (his tones + NHPs relative to DNA) in the former compared to the latter (Table I). More significant are the electrophoreses made in SDS-polyacrylamide run on various samples at different degrees of precipitation (Figure 3). They display a progressive disappearance ofhistones in the supernatants and theirincrease in the precipitates. Simultaneously a strong increase of all NHPs takes place in the supernatants; in the patterns, two bands in particular (below HI) become relatively stronger in relation with an
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enhancement offluorescence (see next paragraph). They can be attributed to HMG proteins 1 and 2 since these two proteins are entirely responsible for the tryptophan fluorescence of chromatin and have been identified in other works at similar places in the electrophoretic patterns of various chromatins (31-33). In the supernatant obtained at 90-95% precipitation by spermine as well as by spermidine, there are practically no histones left and a maximum amount ofNHPs, in particular HMG proteins 1 and 2. Precipitates obtained under the same conditions contain in large amounts histones only, except for faint bands of HMG proteins, particularly the fastest one that we attribute from its position to HMG protein 14 or 17 (32).1t is true that the precipitates are analysed after a redissolution in 2M NaCl. Since everything is redissolved, it seems unlikely that this treatment could modify the constancy of histones distribution, more especially as the main structural features are maintained (vide infra). After precipitation by hexamminecobalt(III) (Figure 3), similar differences appear in the supernatant and precipitate patterns, although somewhat attenuated as regards the enrichment in HMG proteins 1 and 2. The situation is quite different in the case of precipitation by Mg2+ ions. Here the proportions ofhistones and HMG proteins are the same in both fractions, whereas disappearing of a few LMG proteins occurs in the precipitate. In conclusion, precipitation ofchromatin by the tetra- and trivalent cations assayed by us leads to a complete redistribution of proteins whereas Mi+ ions do not bring about any important separation.
Fluorescence The only tryptophan-containing proteins of chromatin are HMG proteins 1 and 2 which are responsible for the fluorescence band observed with a maximum around 340-350 nm when excited at 290 nm. These proteins contain each two tryptophan residues per molecule (34). Rat liver chromatin is particularly rich in NHPs and consequently well appropriate for such fluorescence studies. The tryptophan fluorescence can be distinguished from that of tyrosine which is brought about by excitation at 280 nm and gives a very faint band with maximum at 310 nm only detectable by high-sensitivity spectrometers. We observed that, with increasing degree of precipitation by spermine, spermidine and to a lesser extent hexamminecobalt(III), the fluorescence of the supernatant increases very much whereas that of the precipitate remains negligible. Values of specific fluorescence, i.e., fluorescence intensities at 356 nm in arbitrary units divided by the absorbance of the supernatant, are given in Figure 4 for different degrees of precipitation. For spermine and spermidine, initial values are multiplied by a factor of4, for hexamminecobalt(III) by a factor of2. With Mg2+ ions, the variation is negligible. This behaviour is to be related to the increase in HMG proteins 1 and 2 brought
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F/A
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oj.
I
I .
/, I I
I
I .
I
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lo I I
/ I
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0 0
0----------~----------_.
100
50
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Figure 4: Specific fluorescence (F/A where F is the fluorescence intensity in arbitrary units and A the absorbance at 260 nm) of chromatin-cation complexes (soluble fraction) as a function of the degree of precipitation, i.e., solubility (see Figure I). Precipitating cations: spermine (0), spermidine (8.), hexammine cobalt(III), Mg2+ (D).
about by the tetra- and trivalent cations. It is difficult to make a precise quantitative correlation because we lack a reliable method for determining the content in these fluorescent proteins. Taking into account the data of Table I, we can estimate the NHP content by subtracting the histone/DNA weight ratio, i.e., around 1, from the total protein/DNA ratio. This gives 1.3 for chromatin. In the supernatant described in Table I, since there are no histones left, the protein/DNA ratio is identical to the NHP/DNA ratio, i.e. 4. The multiplication of the NHP content by a factor higher than 3 may reasonably explain the increase in fluorescence. Modifications of fluorescence could be brought about by structural changes occurring in the environment of the chromophores during the precipitation process and the redistribution of proteins. This point was investigated by the determination of the fluorescence lifetimes oftryptophan in the supernatant resulting from the precipitation of chromatin by spermine. For chromatin the decay curve of fluorescence was very satisfactorily fitted by a two components exponential, the major one representing 66% with a lifetime equal to 5.4 ns and the minor one (34%) with a lifetime of 1.6 ns. These lifetimes are in the range ordinarily found in proteins containing two kinds of tryptophan residues (35). In all the supernatants (Table II), despite the important enhancement of fluorescence reported above, the lifetimes remain identical within
855
Chromatin Condensation and Cations Table II Fluorescence Lifetimes of Supernatants After Precipitation of Chromatin By Spermine
1/P
F!A"
t Ib
%tl
~c
% t2
0 0.08 0.18 0.24
2.0 2.3 12 16
5.4 4.8 5 5.4
66 68
1.6 1.0 1.5 1.5
34 32 36 38
64
62
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•Fluorescence intensity over absorbance. bFirst lifetime in ns. csecond lifetime in ns.
experimental error. This is in line with the assumption that the modification of fluorescence intensity is entirely due to an enrichment of the sample in the tryptophancontaining proteins HMG 1 and 2, without any structural change in the environment of these residues. It is likely that the tryptophan residues in HMG proteins 1 and 2 are buried inside the folded part of these molecules (34).
Circular Dichroism The CD spectrum of chromatin between 250 and 350 nm (Fi~re 5) displays a maximum near 282 nm with an ellipticity of 1900 ± 200 deg em dmoC 1 in agreement with the data of Marion et al. (36). The CD spectra obtained with the precipitates at various degrees of precipitation by tetra- and trivalent cations are similar to the chromatin spectrum (results given only for spermine). Only a slight enhancement of maximum ellipticity is observed but this may be due to the fact that the structure has been a little altered by the dissolution in 2M NaCl and recombination at low ionic strength. A blank of chromatin treated in the same way gives also a slightly higher ellipticity at 282 nm, i.e., 2100 deg cm2 dmol- 1• More important changes are observed in the supernatants at high degree of precipitation: the shape of the spectrum is shifted towards that of protein-free DNA (Figure 5) with a significant increase in maximum ellipticity. The lowering of ellipticity between 250 and 300 nm observed for chromatin compared to free DNA is attributed to the combination of DNA to histones (37). So it appears that, under the action of the tetra- and trivalent cations tested, the DNA is partly liberated from protein constraints. This is in line with the release of histones demonstrated by electrophoresis. We assume that the NHPs remaining in the supernatant are still, partly at least, bound to DNA (see discussion) explaining the lowering of ellipticity compared to that of free DNA
Melting Curves The melting curve of our chromatin samples presents the classical monotonic aspect (Figure 6A). The differential curve displays a slightly asymmetric peak, i.e., widened on the side of low temperature, the maximum laying at 80-81 oc. The
856
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8
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6
0~~-+,--------------~~-~-~-~------i I
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320 Figure 5: Ellipticity of various fractions of chromatin-cation complexes versus wavelength: native DNA (-), native chromatin (-----), soluble complex with spermine, 1/P = 0.10 or spermidine 1/P = 0.30 (······ ), precipitate with spermine, 1/P = 0.10 ( ·- ·- · ).
hyperchromism has the regular value close to 40%. At high degree of precipitation by spermine (Figure 6C), the supernatants curves present two parts: the high temperature peak with its maximum at 81 o C and a wide peak at low temperature whose
857
Chromatin Condensation and Cations
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Figure 6: Melting curves ofvarious fractions ofchromatin~ation complexes. Left ordinate: hyperchromism, right ordinate: normalized derivative. A: native chromatin, B: precipitate with spermine (1/P = 0.12), C: soluble complex with spermine (1/P = 0.09), D: soluble complex with Mg2+ (1/P = 0.9).
importance increases with 1/P value. Its maximum which in some cases is not clearly defined, lies in the range 55-60oC. The hyperchromism is decreased to 2530%. A similar behaviour is displayed in the case of precipitation by spermidine (results not shown in the figure). With Mg2+ the peak is unique and better defined although still rather wide (Figure 6D). Its maximum lies near60°C for high values of I/P. The curves of the precipitates are all much more regular (Figure 6B). They remain similar to that of chromatin, with a maximum near 81 oc and a hyperchromicity near 40%. The peak is somewhat widened but this is probably due to the dissociation and reconstitution of the precipitates, as may be seen on a curve obtained with chromatin after the same treatment (results not shown). From our experiments it appears that, in the supernatants, there is a slight quantity of the original chromatin giving the peak at 81 oC, progressively disappearing as precipitation is increased. The remaining soluble part contains only DNA and proteins,
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forming at least in part, heterogeneous complexes, where a small portion of DNA is more or less denatured in view of the reduced hyperchromicity. It is difficult to decide whether the wide low temperature peak is due to complexes DNA-NHP or to complexes polyamine-DNA which present also the type ofheterogeneous curves in the case of spermidine at least (38). In the precipitates there is a chromatin which is deprived of the greater part of its NHPs but has its full content of DNA and histones and keeps its original structural features.
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Electric Dichroism The curve of electric dichroism vs field strength (Figure 7) for rat liver chromatin is similar to that previously found for chicken erythrocyte chromatin (17). Addition of spermine or spermidine brings about a strong decrease of the negative dichroism in the supernatants, already at the beginning of the precipitation (Table III). This is in
0
10 E (kV/cm)
15
Figure 7: Specific electric dichroism of various fractions of chromatin-cation complexes versus field strength: native chromatin (e), reconstituted chromatin (lt.), soluble complex with spermine, 1/P = 0.08 (0), precipitate with spermine, 1/P = 0.07 (V), precipitate with spermine, 1/P = 0.09 (~).soluble complex with spermidine, 1/P = 0.065 (0), precipitate with spermidine, 1/P = 0.12 (()).
859
Chromatin Condensation and Cations Table III Reduced Electric Dichroism at 12.5 kV/em of Supernatants and Precipitates After Precipitation By Spermine and Spermidine 1/P
Supernatants
Precipitates•
0
-0.065h
-0.08c
Spermine
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0.06 0.07 0.08 0.09 0.12
-O.ol
-0.01 +0.02 0 0
-0.11 -0.115 -0.115 -0.115
Spermidine 0.065 0.12 0.16
-0.06 0 0
-0.095 -0.085
"Redissolved in 2M NaCl and dialysed against 0.5 mM EDTA, pH 7. ~ative chromatin. ~ative chromatin treated as in".
line with the effect of various cations on chicken erythrocyte chromatin in the range of solubility of the complexes ( 11, 16,17,21 ). These observations were interpreted on the basis of a transition from the 10 nm to the 30 nm fibre. At a degree of precipitation of 20%, we found that the dichroism becomes slightly positive at intermediate field strength and is no longer measurable at higher I/P ratios. We do not think that the reversal of sign occurring at higher field strength indicates a field-induced stretching, since we observed the same curve after application of several pulses. In fact the pulse signals for that liP value become complex at high field strength, revealing the presence of entities with different dichroism signs and relaxation times. This is in line with the heterogeneity of the samples as evidenced by our other measurements (vide supra). On the contrary, the precipitates obtained by spermine or spermidine action display a more negative dichroism than a blank of reconstituted chromatin (Table III). Although the recombination process may have altered the structure of these precipitates to some extent, it seems likely that here again the two complexes obtained by spermine and spermidine differ considerably in the soluble and insoluble phases: our experimental results show that the former is a much more condensed assembly and that the latter remains closer to the original structure of chromatin. It is interesting to correlate our findings with previous reports: a positive linear
dichroism was found for chromatin at low ionic strength (40) which was attributed to the presence of spermine and spermidine in the preparation (20). On the other hand, Matsuoka eta!. (41) found that under the action of nucleases, two fractions of chromatin may be obtained,one released with a weak positive linear dichroism and one non-released with a negative dichroism.
Fredericq eta/.
860 2
As regards Mg + ions, numerous studies have shown that they bring about a strong
decrease of the negative dichroism and even in some cases a reversal of its sign, i.e., they have an effect similar to that of tetra- and trivalent cations but at higher concentrations (11,17,19,20,39). Binding of Spermidine to Chromatin
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In view of the different properties displayed by the soluble and insoluble complexes of chromatin and tetra- or trivalent cations, it seemed important to determine the binding capacity of the two forms of complexes. This was done for spermidine since we disposed only of eH)spermidine required for binding determination. Bound and free quantities of the cation were measured by the classical equilibriumdialysis method, the titration being made by radioactive counting. A large range of 1/P ratios covering solubility and precipitation domains was examined. In the range of complete solubility, i.e., below 0.018 mM spermidine under our experimental conditions, a fairly regular Langmuir isotherm was obtained (Figure 8) which gave a reasonably straight line for the Scatchard plot, r/c vs r (Figure 9), considering an error estimated to ± 5% in the determination of rand c (r is the number of spermidine bound per chromatin phosphate and cis the free spermidine concen-
r PRECIPITATION RANGE
l
0.1
•
0
6
••
•• •
•
••
%~--------~,--------~2--------~3----~ c x 10 5 free spermidine Figure 8: Binding curve of spermidine by chromatin: r (moles of spermidine bound per mole of chromatin phosphate) as a function of c (free spermidine concentration): in the range of total solubility, soluble complex (0); in the range of partial solubility, soluble complex (e), insoluble complex (A).
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r/c x 10-4
0.
• 00
0.05
,. '' 0.10
'r
Figure 9: Scatchard plot of the binding of spermidine by chromatin: r/c versus r. Same symbols as in Figure 8.
tration). An intrinsic binding constant of(2 ± 0.2) .105 mol- 1 was deduced. Tentative extrapolation of the curve gave a maximum amount of binding sites around 0.12, i.e., one spermidine trivalent cation per eight nucleotides. This represents a neutralization of3/8 of DNA negative charges. Accepting a value of60% ofDNAcharges neutralized by histones in chromatin, this would indicate that the saturation of the binding sites occurs with a complete neutralization of the net negative charge. In the range of partial precipitation of the complexes, results were a little more irregular (Figure 8), due to additional errors introduced by the determination of the amounts of soluble and insoluble fractions and to more variability in the binding process in a heterogeneous medium. Nevertheless the data clearly indicate that the amount of spermidine bound by the insoluble complex was close to that found in the soluble fraction, being in excess of 10 to 20%. Scatchard plots did not allow any significant estimation of the binding parameters in that range. We have also investigated by radioactive counting what happens to spermidine bound to precipitates after their dissolution in 2M NaCl and dialysis against 0.5 mM
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EDTA. We found that radioactivity was completely lost and consequently that spermidine had been dialyzed out of dissociated chromatin during its recombination. These results are in agreement with those of Colson and Houssier ( 19) who found that polyamines are dissociated from chromatin at relatively high ionic strength.
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Discussion
As has been already reported in various works, the solubility of chromatin varies along an S-shape curve with the relative concentration of multivalent cations (1/P ratio) in a range which is well defined for a given type of the nucleoprotein at a specified concentration (Figures 1 and 2). A comparison between the cations used here may be done by considering the point of 50% precipitation. These are respectively, in 1/P values, 0.075 (spermine), 0.135 (spermidine), 0.165 (hexamminecobalt(III)), 0.55 (Mg2+). These values are valid only for an initial concentration of chromatin equal to 0.6 mM phosphate (A260 = 4). There is certainly a concentration dependence for the solubility as well as for other parameters studied in this work. The onset of compaction and aggregation in particular must be submitted to the influence of the total concentration of the particles involved. It appears that the cation charge is the determining factor but that s~ecific effects add up, when considering the higher precipitation power of spermidine3 compared to hexamminecobalt(III)3+ ions. However the difference is small and does not account for the important physiological action specifically displayed by the polyamines.
The main objective of this work was to investigate the composition and structural features of the cation-chromatin complexes in the soluble and precipitated phases. The chemical composition revealed in the case of tetra- and trivalent cations an overall enrichment in proteins with the increase ofi/P in the soluble complexes, due to a strong increase in NHPs, whereas histones progressively disappear, the contrary happening in the precipitates. At high precipitation degree by spermine and spermidine, the NHPs are totally concentrated in the soluble complex (except for a very slight amount of some HMG proteins, 14 or 17), a little less by hexamminecobalt(III); at the same time all histones are excluded. The concentration of HMG proteins 1 and 2 is particularly evidenced by tryptophan fluorescence. The strong enhancement of fluorescence in the supernatants occurs without any change in the two tryptophan lifetimes, which indicates that the presence of two classes of residues differing in their environment is maintained even in complexes almost entirely constituted of DNA and NHPs. In this respect the action ofMg2+ ions is quite different: compared to intact chromatin, the soluble Mg 2+-chromatin complexes do not display any change in composition; there is only a slight depletion of LMG proteins in the precipitates. The circular dichroism in the 250-300 nm positive band of chromatin confirms the histone depletion of the soluble complexes, i.e., it is becoming gradually closer to that offree DNA when 1/P increases whereas that of precipitated complexes redissolved and recombined remains practically unaltered.
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From the electric dichroism and the melting curves, we conclude that spermine and spermidine deeply affect the structure of the soluble phase: strong condensation and replacement of the native particles by a heterogeneous mixture ofDNA-NHP complexes, as a result of histone depletion. The insoluble phase, on the contrary, which consists of"nucleohistone" seems to retain the main features of the original chromatin. However it must be recognized that they have undergone a dissociation and recombination for their characterization and that we have no guarantee for an exact reconstitution. Moreover the polyamines have been eliminated by the dialysis process. Are these differences in protein distribution and structural features in relation with a different binding of the precipitating cations in the soluble and insoluble phases? Binding studies made with spermidine at least, reveal that this trivalent cation is bound in similar amount by the soluble and insoluble complexes. Consequently charge effects could not account for the observed differences. Let us now examine to what extent the NHPs found in the supernatants are still bound to DNA or are liberated during the precipitation. We think that at least a part of them remains bound in a protein-DNA complex for the following reasons: 1. The negative electric dichroism at high 1/P values of spermine and spermidine in the supernatant tends to 0 and can even be positive. A solution of protein-free DNA should always display a high negative dichroism even at high spermine to DNA ratio: Marquetet al. (12) found a dichroism equal to -0.3 at 1/P = 0.28. The zero value indicates that DNA has lost its native elongated structure to become a highly condensed particle.
2. The circular dichroism of the supernatants although closer to that of free DNA than to that of native chromatin reaches half the ellipticity of protein-free DNA and this must be due to a binding of proteins since polyamines themselves do not modify the CD of pure DNA at low ionic strength (42,43). 3. Various studies have demonstrated the binding ofHMG proteins to DNA at low and moderate ionic strength (44). In conclusion, spermine and spermidine bind to chromatin free phosphate-groups in a first step, gradually neutralizing the charge and leading to a condensation of the particles from the 10 nm to the 30 nm fibre without disrupting the general assembly. These complexes remain soluble at low ionic strength. In a second step, the addition of higher amounts of these cations brings about precipitation at I/P values where DNA binding sites for cations are close to saturation and the net charge approaching zero value. For spermidine this would happen at a ratio corresponding to one molecule per eight nucleotides where 40% of phosphates are neutralized by the polyamines and the rest by histones. Under such conditions, a further addition of cation brings about a displacement of his tones which are concentrated in the soluble phase whereas part of the particles consisting finally of an
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assembly of DNA and NHPs remains soluble, in a very condensed state, presenting an important heterogeneity. The insoluble particles, after dissociation in 2M NaCl, dialysis and recombination, recover structural features close to those of native chromatin.
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The precipitation of chromatin by Mg2+ ions differs in many respects from these lines. There is no redistribution of proteins and the soluble and insoluble Mg2+chromatin complexes are similar to each other and to native chromatin. Numerous works have already reported the effectiveness of Mg2+ ions in the formation of higher structure and condensation of chromatin (45,46). This condensation is preliminary to aggregation (17,21). Finally the accumulation ofNHPs and the liberation ofhistones in some regions of chromatin under the influence of high amounts of polyamines could contribute to their physiological action since it is well established that histones maintain DNA in a condensed state and that NHPs and in particular HMG proteins play an important role in transcription and regulation (44,47).
Acknowledgments The financial support of the "Services de Programmation de la Politi que Scientifique, Actions de Recherches Concertees, ARC contract no. 80/85-90" and of the "Fonds National de la Recherche Scientifique, IISN contract no. 4.9004.88" is gratefully acknowledged. Thanks are due to Dr. Claire Duyckaerts for her help in the determination of labelled spermidine and to Dr. Francoise Huriaux for electrophoreses of chromatin proteins. References and Footnotes
Abbreviations: PMSF Phenylmethylsulfonyl fluoride, 1/P number of moles of cation over number of chromatin phosphate, NHP non-histone protein, HMG high mobility group, LMG low mobility group, CD circular dichroism, SDS sodium dodecylsulfate. I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16.
Bachrach, U., Functions of Naturally Occurring Polyamines, Academic Press, New York (1983). Tabor, C.W. and Tabor, T.,Ann. Rev. Biochem. 53,749-790 (1984). Nizamuddin, A, Biochem. Educ. 15, 106-110 (1987). Pommier, Y., Kerrigan, D. and Kohn, K., Biochemistry 28,995-1002 (1989). Heby, 0. and Person, L., TTBS 15, 153-158 (1990). Jerzmanowski, A, Staron, K., Tyniec-Kroenke, B. and Toczko, K., Biochim. Biophys. Acta 565, 356364 (1979). Billett, M.A. and Hall, T.J., Nucleic Acids Res. 6, 2929-2945 (1979). Widom, J. and Klug, A., Cell43, 207-213 (1985). Jin, Y.J. and Cole, R.D.,J. Bioi. Chern. 261, 15805-15812 (1986). Wid om, J., J. Mol. Bioi. 190, 411-424 (1986). Sen, D. and Crothers, D.M., Biochemistry 25, 1495-1503 (1986). Marquet, R., Houssier, C. and Fredericq, E., Biochim. Biophys. Acta 825, 365-374 (1985). Marquet, R., Colson, P. and Houssier, C.,J. Biomol. Struct. Dyn. 4, 205-218 (1986). Makarov, V.L., Smirnov, I. and Dimitrov, S.I.,FEBS Lett. 212,263-266 (1987). Fredericq, E., Hacha, R. and Houssier, C.,Int. J. Bioi. Macromol. 10, 51-54 (1988). Koch, M.H.J., Sayers, Z., Michon, AM., Marquet, R., Houssier, C. and Wilffiihr, J., Eur. Biophys. J. 16, 177-185 (1988).
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17. Marquet, R., Colson, P., Matton, AM., Houssier, C., Thiry, M. and Goessens, G.,J. Biomol. Struct. Dyn. 5, 839-857 (1988). 18. Smimov, I.V., Dimitrov, S.l. and Makarov, V.L.,J. Biomol. Struct. Dyn. 5, 1149-1161 (1988). 19. Colson, P. and Houssier, C., FEBS Lett. 257, 141-144 (1989). 20. Hagmar, P., Marquet, R., Colson, P.,Kubista, M., Nielsen, P.E., Norden, B. and Houssier, C., J Biomol. Struct. Dyn. 7, 19-33 (1989). 21. Marquet, R., Thesis, University of Liege (1989). 22. Koch, M.H.J., Sayers, Z., Michon, AM., Sicre, P., Marquet R. and Houssier, C., Eur. Biophys. J 17, 245-255 (1989). 23. Chauveau, J., Maule, Y. and Rouiller, C., Exptl Cell Res. II, 317-321 (1956). 24. Thibodeau, L. and Verly, W., Eur. J Biochem. 107, 555-563 (1980). 25. Mandel, R. and Fasman, G.D., Nucleic Acids Res. 3, 1839-1855 (1976). 26. Houssier, C., Depauw-Gillet, M.C., Hacha, R. and Fredericq, E., Biochim. Biophys. Acta 739, 317325 (1983). 27. Sharp, P.A, Sugden, B. and Sambroek, J., Biochemistry 12, 3055-3063 (1973). 28. Laemmli, U.K., Nature 227, 680-685 (1970). 29. Fredericq, E. and Houssier, C., Electric Dichroism and Electric Birefringence, Clarendon Press, Oxford (1973). 30. Houssier, C. and O'Konski, C.T., in Molecular Electro-Optics (Krause, S., Ed.) NATO ASI Ser. B64, 309-339 (1981). 31. Seyedin, S.M. and Kistler, W.S.,J. Bioi. Chem. 254, 11264-11271 (1979). 32. Craddock, V.M. and Henderson, AR., Carcinogenesis I, 445-450 (1980). 33. Butler, AP., Mardian, J.K.W. and Olins, D.E.,J Bioi. Chem. 260, 10613-10620 (1985). 34. Baker, C., Isenberg, 1., Goodwin, G.H. and Johns, E.W., Biochemistry 15, 1645-1649 (1976). 35. Beechem, J.M. and Brand, L., Ann. Rev. Biochem. 54, 43-71 (1985). 36. Marion, C., Roux, B. and Coulet, P.R., FEBS Lett. 157, 317-321 (1983). 37. Watanabe, K. and lso, K,J Mol. Bioi. 151, 143-163 (1981). 38. Morgan, J.E., Blankenship, J.W. and Matthews, H.R.,Arch. Biochem. Biophys. 246,225-232 (1986). 39. Me Ghee, J.D., Rau, D.C., Charney, E. and Felsenfeld, G., Cell22, 87-96 (1980). 40. Tjemeld, F., Norden, B. and Wallin, H., Biopolymers 21, 343-358 (1982). 41. Matsuoka, Y., Nielsen, P.E. and Norden, B., FEBS Letters 169, 309-312 (1984). 42. Gosule, L.C. and Schellman, J.A,J Mol. Bioi. 121,311-326 (1978). 43. Wilson, R.W. and Bloomfield, V.A, Biochemistry 18,2192-2196 (1979). 44. Johns, E.W., The HMG Chromosomal Proteins, Academic Press, London (1982). 45. Thoma, F., Koller, Th. and Klug, A, J Cell. Bioi. 83, 403-427 ( 1979). 46. Watanabe, K. and Iso, K., Biochemistry 23, 1376-1383 (1984). 47. lgo-Kemenes, T., Harz, W. and Zachau, H.G.,Ann. Rev. Biochem. 51, 89-121 (1982). 48. Hubbard, R.W., Matthew, W.T. and Dubovik, D.A,Anal. Biochem. 38, 190-201 (1970). 49. Lowry, O.H., Rosebrough, N.J., Farr, AL. and Randall, R.J.,J Bioi. Chem. 193,265-275 (1951). Date Received: August 10, 1990
Communicated by the Editor C. W. Hilbers
