J. MOE. Biol. (1975) 93, 405413
Fractionation
of Chromatin by Thermal Precipitation in Phosphate Buffer
GEORGEG. MARKOV, IVAN G. IVANOV AND ILIYA G. PASKEV Institute of Biochemistry Bulgarian Academy of Sciences Sofia 13, Bulgaria (Received 5 August 1974, and in revised form 31 December 1974) The stability of soniceted rat liver chromatin in sodium phosphate buffer, pH 6.8 was studied &s a function of buffer concentration (O-012 to 0.16 M) and temperature (20 to 98°C). It was found that as the temperature was increased a stepwise precipitation of chromatin took place which was revealed by the presence of three plateaux (20 to 5O”C, 70 to 75°C and above 90°C) and two transitional zones (55 to 70°C and 75 to 90°C) on the &,, curves and on the percentage precipitated nucleoprotein versus temperature curves as well. This permitted the fractionation of ohromatin in 0.08 M-phosphate buffer into three fractions by a stepwise heating at 50°C (SO%-pellet) and 98°C (50-98°C pellet and post 98Y%supernatant). DNA isolated from these fractions was characterized in respect to sedimentation velocity and hybridization with studies showed a different ability heterogeneous nuclear RNA. The hybridization of these three DNA preparations in binding nuclear heterogeneous RNA: IS%, 8% and 30% for DNA isolated from BO’C-pellet, 50-98%pellet and post 98”Csupernatant, respectively. The results are discussed in terms of chromatin structure and function.
1. Introduction The study of the genome of eukaryotic cells requires methods for fractionation of chromatin based on differences in its structure and function. Several approaches have been applied: differential centrifugation (Duerksen & McCarthy, 1971; Frenster et al., 1963; Frenster, 1965; Yasmineh & Yunis, 1969; Yunis & Yasmineh, 1971), enzymic digestion (Billing & Bonner, 1972), gradient centrifugation in non-ionic median (Hossaini et al., 1973; Rickwood et al., 1973), thermal chromatography on hydroxylapatite (McConaughy $ McCarthy, 1972), chromatography on ECTHAMcellulose (Reeck et al., 1972) and chromatography in agarose gel columns (Janowski et al., 1972). Although some progress has been achieved, a further development of meaningful fractionation methods is of particular interest. In the course of our attempts to apply chromatography on hydroxylapatite for separation of active and inactive chromatin it became necessary to study in more detail the stability of sonicated chromatin in phosphate buffer. It was found that using different buffer concentrations and temperatures, conditions could be found for separation of chromatin into several fractions. The reproducibility of the results as well as the characteristics of the fractions obtained suggested that they represented structurally and functionally different nucleoproteins. 27
405
406
G.
G.
SIARKOV,
I.
G.
IVANOV
AND
I.
G.
PASHEV
2. Materials and Methods Hydroxylapatite BioGelHTP was obtained from Bio-Rad Laboratories, Richmond, California. Deoxyribonuclease I (EC 3.1.4.5), electrophoretically purified, free from ribonuclease was purchased from Worthington. [6-i%]orotic acid and [3H]thymidine were products of the Institute of Isotopes, Budapest, Hungary. Nitrocellulose Millipore filters HAWP, 0.45 pm (Millipore) were used for hybridization studies. Pronase and pancreatic ribonuclease (EC 2.7.7.16) were obtained from Sigma (London) and Reanal (Hungary). respectively. (a) Preparation
of chromatin
Rat liver chromatin was isolated as described by Tsanev & Russev (1974) using Nonidet P40 and salt extractions with NaCl up to 0.3 M. The preparations were dialyzed overnight against deionized water. The swollen gels were solubilized by homogenization in deionized water. The viscous solutions were clarified by centrifugation at 20,000 g for 15 min. The preparations had the following characteristics: Asso/&,,, 1.75&0*05; Aaso/Az~o, 1.45* 2.0f 0.1; hi&one/DNA, 1.0 f 0.1. 0.05; Asz,,, less than 5% of Ass,,; total protein/DNA, The total chromatin was sheared in an MSE ultrasonic power unit for 30 s at 1 A, centrifuged for 15 min at 20,000 g and the superantant was stored at 4°C. Chromatin preparations were used not later than 3 days after isolation. (b) Preparation
of DNA from
total chronaatin
and from chronaatinfractions
DNA was isolated from total ohromatin and from chromatin fractions aa described previously (Markov & Ivanov, 1974), procedure II, with the following modifications. (i) Ieobtion
of DNA from total chromatin
The chromatin solution was made 1 M in NaCl and 0.1 M in EDTA; sodium dodecyl sulphate was added to a tial concentration of 1% and the sample was heated at 60°C for 10 min with vigorous shaking, then cooled quickly and deproteinized with watersaturated phenol, pH 7.0. The aqueous layer was extracted 3 times with ether to remove the phenol, EDTA was neutralized with CaCl, and PO., buffer (sodium phosphate buffer, was loaded on a hydroxylapatite column pH 6.8) was added to 0.18 M. The solution equilibrated with 0.18 M-PO, buffer. After loading, the column was washed with the same buffer until Age0 of the effluent was less than 0.005 and DNA was eluted with 0.48 M-PO, buffer. (ii) Isolation
of DNA
from precipitated
chromatin
The pellets (see Results, (c)) were suspended in 0.05 M-Tris*HCI, pH 7.6 containing 200 pg Pronase/ml and incubated at 37°C for 2 h. The hydrolysate was made 1 M in NaCl or NaC104 and 1% in sodium dodecyl sulphate and deproteinized twice with an equal volume of phenol/chloroform/isoamylalcohol mixture (26/24/l). The aqueous phase was extracted 3 times with ether and PO4 buffer was added to 0.06 M. The hydroxylapatite chromatography was performed as described above except that 0.06 M-PO, buffer was used instead of 0.18 M for equilibration and washing the column to avoid loss of partially denatured single-stranded DNA. (iii)
The same procedure wae applied to ieokate DNA from poet 98”C-8upernutant the incubation with Pronase was omitted
except that
The 0.48 M-PO, buffer eluates containing DNA were dialysed overnight against distilled water at 4”C, solid NaCl was added to 0.2 M and DNA was precipitated with 2 vol. ethanol at -20°C. After 24 h the precipitates were collected by centrifugation, dissolved in 0.03 M-PO* buffer and passed through a Sephadex SE-C26 column (Na+ form) to eliminate traces of bivalent cations. The eluates in 0.03 M-PO, buffer were dialysed overnight against distilled water and stored at 4°C.
(c) Determination The analytical band centrifugation 0.9 M-NaCl) was used to determine
of 820,~ of DNA
(Studier, 1966) in alkaline solution (O-1 M-NaOH, the sedimentation coefficients of the DNA isolated
CHROMATIN
FRACTIONATION
BY
PRECIPITATION
407
from the different chrometin fractions. The centrifugation was performed in a Beckman model E analytical ultracentrifuge (rotor AN-D) at 20°C at 44,000 revs/min (DNA from 50°Cpellet) and 56,000 revs/min (DNA from 50-98’C-pellet and post 98“~-supernatant). Pictures were taken at 8 min intervals and scanned in a Joyce-Loebl double-beam microdensit,ometer. (d) Preparation of 14C-labelled HnRNA Male albino rats (180 g body weight) were injected with 40 &i [6-14C]orotic acid each. The animals were killed 1 h later and the nuclear RNAs were extracted from the liver by the method of thermal phenol fractionation (Markov & Arion, 1973). Nuclear pre-rRNA was extracted at 55% and the nuclear pre-mRNA (HnRNA) at 85°C. The latter fraction was treated with DNAase, deproteinized, purified from low molecular weight contaminants (Markov & Arion, 1973) and used in the hybridization experiments. (e) Hybridizatio?b DNA/RNA hybridization (1!)65) adapted to sheared
of RNA with Jilter bound DNA
was performed by the method DNA (Ivanov & Markov, 1975). (f) Analytical
of Gillespie
& Spiegelman
methods
The absorbance of the solutions was measured in a Zeiss VSU-2 spectrophotometer. Changes in the absorbance at 320 nm with temperature were followed in a Unicam SP1800 spectrophotometer with thermostatic cell-holder. In some experiments temperature was increased linearly at 1.5 deg. C/min, while in other cases the samples were heated for 10 min at 2 deg. C intervals. Radioactivity was measured in a Packard Tricarb scintillation spectrometer, model 3320. The filters were counted in 3 ml toluene-based scintillant. For scintillation counting of aqueous solutions, 10 ml of a dioxane-based scintillant (naphthalene 100 g, PPO 7 g, POPQP 300 mg and dioxane to make 11) were added to 2 ml of the sample. DNA was determined by the method of Burton (1968) and proteins by the procedure of Lowry et al. (1951).
3. Results (a) Factors affecting (i) Effect of phosphate
chromatin
stability
buffer concentration
In studies on fractionation of sheared chromatin on hydroxylapatite we have found that at the concentrations of PO4 buffer used (0.12, O-16 and 0.40 M) the chromatin solutions became turbid at room temperature, which increased the absorbance at 260 nm and 320 nm. This phenomenon, a result of chromatin aggregation, was studied at room temperature in a broader range of buffer concentration (0.012 to O-16 M), the changes in turbidity being followed by measuring A,,,. As shown in Figure 1, upon increasing the buffer concentration from zero to 20 mM-PO, buffer, no change in the turbidity takes place. However, a significant increase is observed in t’he region 40 to 120 mm-PO4 buffer. No further changes in the turbidity occur at PO, buffer concentration O-12 to 0.16 M. (ii) Effect of temperature at different phosphate buffer concentration Heating chromatin solutions in 0.012 M-PO, buffer up to 98°C does not affect the absorbance at 320 nm (Fig. 2). At all higher concentrations (0.024 to 0.08 M), however, the raising of the temperature causes a stepwise increase in turbidity. AS shown in Figure 2, all A,,, curves have a similar shape with three plateaux and two transitional zones, located in the same temperature intervals. The first plateau is due to initial aggregation of the chromatin which occurs at room temperature upon
408
G. G. MARKOV,
I. G. IVANOV
AND
I. G. PASHEV
addition of buffer (cf. Fig. 1) and continues up to 52°C. The second plateau is locatetl at 70 to 76°C and the third one above 90°C. At lower concentrations (O-024 to 0.04 M) the increase in turbidity occurs mainly at temperatures above 75°C (second transitSion), whereas at higher concentrations (0.06 to O-08 M) a drastic change in A,,, takes place during the first transition (60 to 70°C). 30
4
0
PO, buffer x 10Tz
FIQ. 1. Effect of phosphate buffer concentration on aggregation of chromatin at room tempera. ture. Chromatin samples (l-0 Aaso unit in deionized water) were mixed with PO4 buffer (pH 6.8) to obtain different 6nal concentrations in a fkal vol. of 2 ml. A 310was measuredand related to A,,, of a oontrol sample of ohromatin containing 1.0 A aso unit in 2 ml of deionized water. I/
nw-POo
buffer
I
,
0.5
0.4
: 0.3 :: Trn 0.2
0.1
(22’
I 50
I
I 60
I
I I I 70 80 Temperature (“C)
, 90
3
FIN. 2. Effect of temperature on aggregation of chrometin at different phosphate buffer oonoentration. 1.6 Aaeo units of ohromatin (measured in distilled water) were mixed with PO, buffer (pH 6.8) to obtain 6x4 oonoentrations from 12 maa to 80 mM-P04 buffer in a final vol. of 2 ml. The temperature of the samples was increased at l-5 deg. C/min and Aaao was recorded.
CHROMATIN
FRACTIONATION
BY PRECIPITATION
409
Temperature PC)
ourve of the ohromatin residue after removing the material FIG. 3. A320 WXWM temperature precipitated at 50°C and 73°C. Chrometin samples (0.8 Aaao units/ml) in 0.06 M-PO, buffer were heated at 60°C or 73°C for 15 min and the precipitated material was removed by centrifugstion for 16 min at 20,000 g. A :120of the supernatant was recorded as a function of temperature and comparedto that of the total chromatin. ( ) Total ohromatin; (-----) post SO’Wsupernatant; (-*-.--) post 73”Csupernatant.
Additional experiments were made to see whether and to what extent the material already precipitated at a given temperature contributed to the further increase of A 320 at higher temperatures. In Figure 3 the A,,, curve of the total chrometin in 0.06 M-PO, buffer is compared with the curves after removing the nucleoproteins precipitated at 50°C. (1st plateau) and 73°C (2nd plateau). As seen, the curve of the post 50”C-supernatant has the same shape as that of the total chrometin. This indicated that the 50%pellet, which at this buffer concentration amounts to about 50% of the total material, is not responsible for the biphasic character of the A,,, upon curve. On the other hand, the A,,, changes of the post 73”Csupernatant further heating are negligible. Therefore, the biphasic character of the AazO curve of the t’otal chromatin is due mainly to the nucleoprotein which aggregates between 50°C and 73°C (about 35% of the material). The relative heights of the three plateaux at a given buffer concentration are not strictly reproducible with different chromatin preparations. However, the position of the transition zones and the plateaux on the temperature scale remain constant independently of the ionic strength, the concentration of the chromatin (O-8 to 5-O A 260 units/ml), time of sonication (30 s to 4 min) and the mode of heating (continuous or stepwise increase of temperature). (iii) Effect of incubation time The effect of time of heating at a given temperature on the aggregation of chromatin was also studied. It was found that no further increase in Aszo occurred upon heating the chromatin in O-05 M and 0.08 M-PO~ buffer for 30 minutes at the typical plateau temperatures (5O”C, 73°C and 98°C). When the temperature was fixed on transition zone points (65°C and 84°C) A,,, continued to increase for about 20 minutes until reaching a constant level lower than that of the next plateau.
410
G. G. MARKOV,
I.
G. IVANOV
(b) Fractionation
AND
I.
G. PASHEV
of chromutin
The shape of the turbidity/temperature curves points to the existence of several discrete stages of aggregation which probably concern different nucleoprotjein fractions. Judging from the data in Figure 2, the most reasonable procedure to separate these fractions appeared to be the consecutive heating of the chromatin at the plateau temperatures and collection of the precipitated material by centrifugation. Such a procedure would be correct if the curve of percentage precipitated nucleoprotein versus temperature followed a similar course as the Aszo curve. The quantity of the precipitated material as a function of temperature was measured at two buffer concentrations (0.06 and 0.08 M). As shown in Figure 1, the shape of the precipitation curves is similar to that of the turbidity curves-plateaux from 50 to 60°C and above 70°C and a steep increase between 60°C and 70°C. The last transition (75°C to 9O’C) is not well expressed on the precipitation curves. This is not surprising if we consider the data in Figure 3 showing that the last transition is duo almost exclusively to the material aggregating up to 73°C.
0 id-. 50
- L--i60
70 80 90 Temperature PC)
-- .’ xi.
FIG 4. Effeot of temperature on the amount of precipitated ohromatin. Samples of ohromatin (6.0 ASa0 units/ml) in 0.06 M and 0.08 M-PO, buffer were heated for 16 min at different temperatures, cooled and oentrifuged at 4°C for 16 min at 20,000 g. Amount of protein in the supernatants was determined directly; amount of protein in the precipitates was calculated by subtxwtion of the values for the supernatants from those of the samples before heating. --O-O-, Chromatin in 0.00 a4-P04 buffer; -a----, ohromatin in 0.08 M-PO, buffer.
Since the aggregation process depends also on the buffer concentration, the effect of this factor on the amount of the material precipitating at the plateau temperatures was also investigated. Samples of chromatin in different, PO4 buffer concentrations (0.02 to 0.1 M) were heated for 15 minutes consecutively at 5O”C, 73°C and 98°C and the aggregated material for each temperature interval was collected by centrifugation for 15 minutes at 20,000 g. It was found that the amount of the precipitated nucleoprotein depended upon the buffer concentration, but only up to 0.06 M. At higher concentrations (0.07 to 0.1 M) the distribution of the nucleoprotein among the fractions remained the same. Therefore, for a standardized fractionation procedure a buffer concentration of 0.08 M seems to be preferable. Taking into account that under
CHROMATIN
FRACTIONATION
BY
PRECIPITATION
411
these conditions the amount of nucleoprotein precipitated between 73°C and 98°C was negligible, the 73”Cprecipitation step was omitted and the preparative procedure was performed by a consecutive heating of the chromatin in 0.08 M-PO, buffer at 50°C and 98°C giving 3 fractions: 50°Cpellet, 50-98°Cpellet and post-98°Csupernatant. Sixty to seventy percent of the DNA was recovered in the 50”C-pellet, 16 to 25% in the 50%98”C!-pellet and 10 to 15% in the post 98”CLsupernatant (data from 4 experiments). (c) Characterization of DNA in the chronmtin fractions (i) Sedimentation analysis The sedimentation analysis gave average sZO,Wvalues of 6.5 for DNA in the 50°C pellet, 4.8 for that in the 50-98°Cpellet and 4.2 for DNA isolated from the post 98”CLsupernatant. DNA of the 50°Cpellet was more homogeneous compared to DNA of the other two fractions. (ii) Hybridization with non-ribosonaal n2lclear RNA Hybridization experiments were carried out to see whether DNA isolated from the chromatin fractions differed in its ability to hybridize with RNA isolated from rat liver by the phenol fractionation method in the temperature interval 55°C to 85°C. This RNA contained the non-ribosomal nuclear RNA species (HnRNA, pre-mRNA). As shown in Figure 5, about 14% of the DNA of the total sonicated chromatin anneals with this RNA. 16% and 8% of DNA from the 50”C-pellet and 50-98°C pellet, respectively hybridized with the same RNA preparation, while 30% hybridization was reached with DNA from the post 98”Csupernatant.
Input
RNA (mg/ml)
Pro. 6. Hybridization of 14C-labelled rat liver HnRNA with filter bound DNA from different chromatin fix&ions. Rat liver heterogeneous nuclear RNA wa.s isolated after 1 h labelling with [‘*C]orotic acid and purified as desoribed in Materials and Methods. Its specif?c radioactivity was 1000 ote/min per pg. Hybridization with increasing conaentrations of RNA was carried out in 2 x SSC (SSC is 0.16 MNaCl, 0.016 ~-sodium citrate, pH 7.0) at 66°C for 18 h. 13 mm HAWP Millipore filters oontaining 6 to 10 pg DNA were used. To determine the amount of DNA on the filters at the end of the ljrocedure, control filters with 3H-labelled DNA isolated from sheared chromatin were incubated Ilnder the 8-e conditions (Ivanov 85 Markov, 1976). -O-O-, DNA from total ohromatin; ---I-J--•--, DNA from 60°Cpellet; ---A--A---, DNA from SO-98%pellet; -+--+-, DNA Jiom post 98°C-supernatant.
412
G. G. MARKOV,
I.
G. IVANOV
If we calculate the hybridization ability from the percent hybridization values of their relative amount (see above) a value close to 14 “/o hybridization experimentally
AND
I.
G. PASHEV
of the total DNA in rat liver chromatin the different DNA fractions (Fig. 5) and of 15 to 16% is obtained, which is very found for DNA of the total chromat,in.
4. Discussion The results presented in this paper show that the aggregation of sonicated chromatin as a function of temperature and buffer concentration is a complex but specific and reproducible process. Taking advantage of the existence of discrete precipitation steps the chromatin can be fractionated into several fractions: 50°Cpellet, 50-98°C pellet and post 98”C-supernatant. The size of the DNA fragments in the three chromatin fractions is slightly different. Larger sizes are found in the fractions precipitating at lower temperatures and vice versa. Therefore, one of the factors determining the behaviour of the chromatin in the process of aggregation might be the size of its fragments. Differences in the protein components may also contribute to the fractionation. This is in accordance with the finding that the non-precipitable fraction (post 98”C-supernatant) contains much less protein than the total chromatin. Do the thermal fractions differ only by physicochemical properties or do they contain fragments of different biological function 1 The hybridization data suggest the existence of functional differences between the fractions. The post 98%supernatant fraction shows a DNA/RNA hybridization ability much higher than that of the other fractions and than that of total DNA as well. It is noteworthy that this chromatin fraction, which does not precipitate upon heating has the lowest protein content and DNA with smallest fragment size. All these features could be explained assuming that the chromatin fragments in this fraction originate from highly decondensed, transcriptionally active chromatin regions, having a lower protein content (Shih & Bonner, 1970; Li & Bonner, 1971; Clark & Felsenfeld, 1971; Spelsberg et al., 1971). Such regions may be expected to be more “fragile” under the conditions of sonication. This would mean that the different sizes of fragments obtained by sonication would reflect in vivo existing structural features as association with protein and supercoiling with a definite biological significance. We thank Mrs I. Dimitrova for excellent technical assistance. One of us (I. P.) is very grateful to Dr W. Upholt from Carnegie Institution of Washington, Dept. of Embryology, Baltimore, U.S.A., for his help in sedimentation velocity analysis. REFERENCES Billing, R. J. & Bonner, J. (1972). Biochim. Biophys. Acta, 281, 453-462. Burton, K. (1968). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 12, part B, pp. 163-166, Academic Press, New York. Clark, R. J. & Felsenfeld, G. (1971). Nature New Biol. 229, 101-106. Duerksen, J. D. & McCarthy, B. J. (1971). Biochemistry, 10, 1471-1478. Frenster, J. H. (1965). Nature (London), 206, 680-683. Frenster, J. H., Allfrey, V. G. & Mirsky, A. E. (1963). Proc. Nat. .4cad. Sci., U.S.A. 50, 102&1032. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829-842.
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FRACTIONATION
BY PRECIPITATION
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Hossaini, E., Zweidler, A. & Bloch, D. P. (1973). J. Mol. Biol. 74, 283-289. Ivanov, I. G. & Markov, G. G. (1975). Biochim. Biophye. Acta, in the press. Janowski, M., Nasser, D. S. & McCarthy, B. J. (1972). In 5th Karolinska Symposium uu Research Methods in Reproductive Endocrinology, Gene Transcription. in Reproductive Tissue (Diczfalusy, E., ed.), Acta Endocrinologica, suppl. 168, pp. 112-129. Li, H. & Bonner, J. (1971). Biochemietry, 10, 1461-1470. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265-275. Markov, G. G. & Arion, V. J. (1973). Eur. J. Biochem. 35, 186-200. Markov, G. G. & Ivanov, I. G. (1974). An&. Biochem. 59, 555563. McConaughy, B. L. & McCarthy, B. J. (1972). Biochemistry, 11, 998-1003. Reeck, G., Simpson, R. & Sober, H. A. (1972). Proc. Nat. AcadS&, U.S.A. 69, 2317--2321. Rickwood, D., Hell, A. & Birnie, G. D. (1973). FEBS Letters, 33, 221-224. Shih, T. & Bonner, J. (1970). J. Mol. Biol. 48, 469-487. Spelsberg, T., Hnilica, L. & Anseven, A. (1971). Biochim. Biophys. Acta, 228, 550-568. Stndier, F. W. (1965). J. Mol. Biol. 11, 373-390. Tsanev, R. & Russev, G. (1974). Eur. J. Biochem. 43, 257-263. Yasmineh, W. & Yunis, J. (1969). Biochem. Biophys. Res. Commun,. 35, 779-782. Yiuiis, J. $ Yasmineh, W. (1971). Science, 174, 1200~~1209.
Fractionation
of Chromatin by Thermal Precipitation in Phosphate Buffer
GEORGEG. MARKOV, IVAN G. IVANOV AND ILIYA G. PASKEV Institute of Biochemistry Bulgarian Academy of Sciences Sofia 13, Bulgaria (Received 5 August 1974, and in revised form 31 December 1974) The stability of soniceted rat liver chromatin in sodium phosphate buffer, pH 6.8 was studied &s a function of buffer concentration (O-012 to 0.16 M) and temperature (20 to 98°C). It was found that as the temperature was increased a stepwise precipitation of chromatin took place which was revealed by the presence of three plateaux (20 to 5O”C, 70 to 75°C and above 90°C) and two transitional zones (55 to 70°C and 75 to 90°C) on the &,, curves and on the percentage precipitated nucleoprotein versus temperature curves as well. This permitted the fractionation of ohromatin in 0.08 M-phosphate buffer into three fractions by a stepwise heating at 50°C (SO%-pellet) and 98°C (50-98°C pellet and post 98Y%supernatant). DNA isolated from these fractions was characterized in respect to sedimentation velocity and hybridization with studies showed a different ability heterogeneous nuclear RNA. The hybridization of these three DNA preparations in binding nuclear heterogeneous RNA: IS%, 8% and 30% for DNA isolated from BO’C-pellet, 50-98%pellet and post 98”Csupernatant, respectively. The results are discussed in terms of chromatin structure and function.
1. Introduction The study of the genome of eukaryotic cells requires methods for fractionation of chromatin based on differences in its structure and function. Several approaches have been applied: differential centrifugation (Duerksen & McCarthy, 1971; Frenster et al., 1963; Frenster, 1965; Yasmineh & Yunis, 1969; Yunis & Yasmineh, 1971), enzymic digestion (Billing & Bonner, 1972), gradient centrifugation in non-ionic median (Hossaini et al., 1973; Rickwood et al., 1973), thermal chromatography on hydroxylapatite (McConaughy $ McCarthy, 1972), chromatography on ECTHAMcellulose (Reeck et al., 1972) and chromatography in agarose gel columns (Janowski et al., 1972). Although some progress has been achieved, a further development of meaningful fractionation methods is of particular interest. In the course of our attempts to apply chromatography on hydroxylapatite for separation of active and inactive chromatin it became necessary to study in more detail the stability of sonicated chromatin in phosphate buffer. It was found that using different buffer concentrations and temperatures, conditions could be found for separation of chromatin into several fractions. The reproducibility of the results as well as the characteristics of the fractions obtained suggested that they represented structurally and functionally different nucleoproteins. 27
405
406
G.
G.
SIARKOV,
I.
G.
IVANOV
AND
I.
G.
PASHEV
2. Materials and Methods Hydroxylapatite BioGelHTP was obtained from Bio-Rad Laboratories, Richmond, California. Deoxyribonuclease I (EC 3.1.4.5), electrophoretically purified, free from ribonuclease was purchased from Worthington. [6-i%]orotic acid and [3H]thymidine were products of the Institute of Isotopes, Budapest, Hungary. Nitrocellulose Millipore filters HAWP, 0.45 pm (Millipore) were used for hybridization studies. Pronase and pancreatic ribonuclease (EC 2.7.7.16) were obtained from Sigma (London) and Reanal (Hungary). respectively. (a) Preparation
of chromatin
Rat liver chromatin was isolated as described by Tsanev & Russev (1974) using Nonidet P40 and salt extractions with NaCl up to 0.3 M. The preparations were dialyzed overnight against deionized water. The swollen gels were solubilized by homogenization in deionized water. The viscous solutions were clarified by centrifugation at 20,000 g for 15 min. The preparations had the following characteristics: Asso/&,,, 1.75&0*05; Aaso/Az~o, 1.45* 2.0f 0.1; hi&one/DNA, 1.0 f 0.1. 0.05; Asz,,, less than 5% of Ass,,; total protein/DNA, The total chromatin was sheared in an MSE ultrasonic power unit for 30 s at 1 A, centrifuged for 15 min at 20,000 g and the superantant was stored at 4°C. Chromatin preparations were used not later than 3 days after isolation. (b) Preparation
of DNA from
total chronaatin
and from chronaatinfractions
DNA was isolated from total ohromatin and from chromatin fractions aa described previously (Markov & Ivanov, 1974), procedure II, with the following modifications. (i) Ieobtion
of DNA from total chromatin
The chromatin solution was made 1 M in NaCl and 0.1 M in EDTA; sodium dodecyl sulphate was added to a tial concentration of 1% and the sample was heated at 60°C for 10 min with vigorous shaking, then cooled quickly and deproteinized with watersaturated phenol, pH 7.0. The aqueous layer was extracted 3 times with ether to remove the phenol, EDTA was neutralized with CaCl, and PO., buffer (sodium phosphate buffer, was loaded on a hydroxylapatite column pH 6.8) was added to 0.18 M. The solution equilibrated with 0.18 M-PO, buffer. After loading, the column was washed with the same buffer until Age0 of the effluent was less than 0.005 and DNA was eluted with 0.48 M-PO, buffer. (ii) Isolation
of DNA
from precipitated
chromatin
The pellets (see Results, (c)) were suspended in 0.05 M-Tris*HCI, pH 7.6 containing 200 pg Pronase/ml and incubated at 37°C for 2 h. The hydrolysate was made 1 M in NaCl or NaC104 and 1% in sodium dodecyl sulphate and deproteinized twice with an equal volume of phenol/chloroform/isoamylalcohol mixture (26/24/l). The aqueous phase was extracted 3 times with ether and PO4 buffer was added to 0.06 M. The hydroxylapatite chromatography was performed as described above except that 0.06 M-PO, buffer was used instead of 0.18 M for equilibration and washing the column to avoid loss of partially denatured single-stranded DNA. (iii)
The same procedure wae applied to ieokate DNA from poet 98”C-8upernutant the incubation with Pronase was omitted
except that
The 0.48 M-PO, buffer eluates containing DNA were dialysed overnight against distilled water at 4”C, solid NaCl was added to 0.2 M and DNA was precipitated with 2 vol. ethanol at -20°C. After 24 h the precipitates were collected by centrifugation, dissolved in 0.03 M-PO* buffer and passed through a Sephadex SE-C26 column (Na+ form) to eliminate traces of bivalent cations. The eluates in 0.03 M-PO, buffer were dialysed overnight against distilled water and stored at 4°C.
(c) Determination The analytical band centrifugation 0.9 M-NaCl) was used to determine
of 820,~ of DNA
(Studier, 1966) in alkaline solution (O-1 M-NaOH, the sedimentation coefficients of the DNA isolated
CHROMATIN
FRACTIONATION
BY
PRECIPITATION
407
from the different chrometin fractions. The centrifugation was performed in a Beckman model E analytical ultracentrifuge (rotor AN-D) at 20°C at 44,000 revs/min (DNA from 50°Cpellet) and 56,000 revs/min (DNA from 50-98’C-pellet and post 98“~-supernatant). Pictures were taken at 8 min intervals and scanned in a Joyce-Loebl double-beam microdensit,ometer. (d) Preparation of 14C-labelled HnRNA Male albino rats (180 g body weight) were injected with 40 &i [6-14C]orotic acid each. The animals were killed 1 h later and the nuclear RNAs were extracted from the liver by the method of thermal phenol fractionation (Markov & Arion, 1973). Nuclear pre-rRNA was extracted at 55% and the nuclear pre-mRNA (HnRNA) at 85°C. The latter fraction was treated with DNAase, deproteinized, purified from low molecular weight contaminants (Markov & Arion, 1973) and used in the hybridization experiments. (e) Hybridizatio?b DNA/RNA hybridization (1!)65) adapted to sheared
of RNA with Jilter bound DNA
was performed by the method DNA (Ivanov & Markov, 1975). (f) Analytical
of Gillespie
& Spiegelman
methods
The absorbance of the solutions was measured in a Zeiss VSU-2 spectrophotometer. Changes in the absorbance at 320 nm with temperature were followed in a Unicam SP1800 spectrophotometer with thermostatic cell-holder. In some experiments temperature was increased linearly at 1.5 deg. C/min, while in other cases the samples were heated for 10 min at 2 deg. C intervals. Radioactivity was measured in a Packard Tricarb scintillation spectrometer, model 3320. The filters were counted in 3 ml toluene-based scintillant. For scintillation counting of aqueous solutions, 10 ml of a dioxane-based scintillant (naphthalene 100 g, PPO 7 g, POPQP 300 mg and dioxane to make 11) were added to 2 ml of the sample. DNA was determined by the method of Burton (1968) and proteins by the procedure of Lowry et al. (1951).
3. Results (a) Factors affecting (i) Effect of phosphate
chromatin
stability
buffer concentration
In studies on fractionation of sheared chromatin on hydroxylapatite we have found that at the concentrations of PO4 buffer used (0.12, O-16 and 0.40 M) the chromatin solutions became turbid at room temperature, which increased the absorbance at 260 nm and 320 nm. This phenomenon, a result of chromatin aggregation, was studied at room temperature in a broader range of buffer concentration (0.012 to O-16 M), the changes in turbidity being followed by measuring A,,,. As shown in Figure 1, upon increasing the buffer concentration from zero to 20 mM-PO, buffer, no change in the turbidity takes place. However, a significant increase is observed in t’he region 40 to 120 mm-PO4 buffer. No further changes in the turbidity occur at PO, buffer concentration O-12 to 0.16 M. (ii) Effect of temperature at different phosphate buffer concentration Heating chromatin solutions in 0.012 M-PO, buffer up to 98°C does not affect the absorbance at 320 nm (Fig. 2). At all higher concentrations (0.024 to 0.08 M), however, the raising of the temperature causes a stepwise increase in turbidity. AS shown in Figure 2, all A,,, curves have a similar shape with three plateaux and two transitional zones, located in the same temperature intervals. The first plateau is due to initial aggregation of the chromatin which occurs at room temperature upon
408
G. G. MARKOV,
I. G. IVANOV
AND
I. G. PASHEV
addition of buffer (cf. Fig. 1) and continues up to 52°C. The second plateau is locatetl at 70 to 76°C and the third one above 90°C. At lower concentrations (O-024 to 0.04 M) the increase in turbidity occurs mainly at temperatures above 75°C (second transitSion), whereas at higher concentrations (0.06 to O-08 M) a drastic change in A,,, takes place during the first transition (60 to 70°C). 30
4
0
PO, buffer x 10Tz
FIQ. 1. Effect of phosphate buffer concentration on aggregation of chromatin at room tempera. ture. Chromatin samples (l-0 Aaso unit in deionized water) were mixed with PO4 buffer (pH 6.8) to obtain different 6nal concentrations in a fkal vol. of 2 ml. A 310was measuredand related to A,,, of a oontrol sample of ohromatin containing 1.0 A aso unit in 2 ml of deionized water. I/
nw-POo
buffer
I
,
0.5
0.4
: 0.3 :: Trn 0.2
0.1
(22’
I 50
I
I 60
I
I I I 70 80 Temperature (“C)
, 90
3
FIN. 2. Effect of temperature on aggregation of chrometin at different phosphate buffer oonoentration. 1.6 Aaeo units of ohromatin (measured in distilled water) were mixed with PO, buffer (pH 6.8) to obtain 6x4 oonoentrations from 12 maa to 80 mM-P04 buffer in a final vol. of 2 ml. The temperature of the samples was increased at l-5 deg. C/min and Aaao was recorded.
CHROMATIN
FRACTIONATION
BY PRECIPITATION
409
Temperature PC)
ourve of the ohromatin residue after removing the material FIG. 3. A320 WXWM temperature precipitated at 50°C and 73°C. Chrometin samples (0.8 Aaao units/ml) in 0.06 M-PO, buffer were heated at 60°C or 73°C for 15 min and the precipitated material was removed by centrifugstion for 16 min at 20,000 g. A :120of the supernatant was recorded as a function of temperature and comparedto that of the total chromatin. ( ) Total ohromatin; (-----) post SO’Wsupernatant; (-*-.--) post 73”Csupernatant.
Additional experiments were made to see whether and to what extent the material already precipitated at a given temperature contributed to the further increase of A 320 at higher temperatures. In Figure 3 the A,,, curve of the total chrometin in 0.06 M-PO, buffer is compared with the curves after removing the nucleoproteins precipitated at 50°C. (1st plateau) and 73°C (2nd plateau). As seen, the curve of the post 50”C-supernatant has the same shape as that of the total chrometin. This indicated that the 50%pellet, which at this buffer concentration amounts to about 50% of the total material, is not responsible for the biphasic character of the A,,, upon curve. On the other hand, the A,,, changes of the post 73”Csupernatant further heating are negligible. Therefore, the biphasic character of the AazO curve of the t’otal chromatin is due mainly to the nucleoprotein which aggregates between 50°C and 73°C (about 35% of the material). The relative heights of the three plateaux at a given buffer concentration are not strictly reproducible with different chromatin preparations. However, the position of the transition zones and the plateaux on the temperature scale remain constant independently of the ionic strength, the concentration of the chromatin (O-8 to 5-O A 260 units/ml), time of sonication (30 s to 4 min) and the mode of heating (continuous or stepwise increase of temperature). (iii) Effect of incubation time The effect of time of heating at a given temperature on the aggregation of chromatin was also studied. It was found that no further increase in Aszo occurred upon heating the chromatin in O-05 M and 0.08 M-PO~ buffer for 30 minutes at the typical plateau temperatures (5O”C, 73°C and 98°C). When the temperature was fixed on transition zone points (65°C and 84°C) A,,, continued to increase for about 20 minutes until reaching a constant level lower than that of the next plateau.
410
G. G. MARKOV,
I.
G. IVANOV
(b) Fractionation
AND
I.
G. PASHEV
of chromutin
The shape of the turbidity/temperature curves points to the existence of several discrete stages of aggregation which probably concern different nucleoprotjein fractions. Judging from the data in Figure 2, the most reasonable procedure to separate these fractions appeared to be the consecutive heating of the chromatin at the plateau temperatures and collection of the precipitated material by centrifugation. Such a procedure would be correct if the curve of percentage precipitated nucleoprotein versus temperature followed a similar course as the Aszo curve. The quantity of the precipitated material as a function of temperature was measured at two buffer concentrations (0.06 and 0.08 M). As shown in Figure 1, the shape of the precipitation curves is similar to that of the turbidity curves-plateaux from 50 to 60°C and above 70°C and a steep increase between 60°C and 70°C. The last transition (75°C to 9O’C) is not well expressed on the precipitation curves. This is not surprising if we consider the data in Figure 3 showing that the last transition is duo almost exclusively to the material aggregating up to 73°C.
0 id-. 50
- L--i60
70 80 90 Temperature PC)
-- .’ xi.
FIG 4. Effeot of temperature on the amount of precipitated ohromatin. Samples of ohromatin (6.0 ASa0 units/ml) in 0.06 M and 0.08 M-PO, buffer were heated for 16 min at different temperatures, cooled and oentrifuged at 4°C for 16 min at 20,000 g. Amount of protein in the supernatants was determined directly; amount of protein in the precipitates was calculated by subtxwtion of the values for the supernatants from those of the samples before heating. --O-O-, Chromatin in 0.00 a4-P04 buffer; -a----, ohromatin in 0.08 M-PO, buffer.
Since the aggregation process depends also on the buffer concentration, the effect of this factor on the amount of the material precipitating at the plateau temperatures was also investigated. Samples of chromatin in different, PO4 buffer concentrations (0.02 to 0.1 M) were heated for 15 minutes consecutively at 5O”C, 73°C and 98°C and the aggregated material for each temperature interval was collected by centrifugation for 15 minutes at 20,000 g. It was found that the amount of the precipitated nucleoprotein depended upon the buffer concentration, but only up to 0.06 M. At higher concentrations (0.07 to 0.1 M) the distribution of the nucleoprotein among the fractions remained the same. Therefore, for a standardized fractionation procedure a buffer concentration of 0.08 M seems to be preferable. Taking into account that under
CHROMATIN
FRACTIONATION
BY
PRECIPITATION
411
these conditions the amount of nucleoprotein precipitated between 73°C and 98°C was negligible, the 73”Cprecipitation step was omitted and the preparative procedure was performed by a consecutive heating of the chromatin in 0.08 M-PO, buffer at 50°C and 98°C giving 3 fractions: 50°Cpellet, 50-98°Cpellet and post-98°Csupernatant. Sixty to seventy percent of the DNA was recovered in the 50”C-pellet, 16 to 25% in the 50%98”C!-pellet and 10 to 15% in the post 98”CLsupernatant (data from 4 experiments). (c) Characterization of DNA in the chronmtin fractions (i) Sedimentation analysis The sedimentation analysis gave average sZO,Wvalues of 6.5 for DNA in the 50°C pellet, 4.8 for that in the 50-98°Cpellet and 4.2 for DNA isolated from the post 98”CLsupernatant. DNA of the 50°Cpellet was more homogeneous compared to DNA of the other two fractions. (ii) Hybridization with non-ribosonaal n2lclear RNA Hybridization experiments were carried out to see whether DNA isolated from the chromatin fractions differed in its ability to hybridize with RNA isolated from rat liver by the phenol fractionation method in the temperature interval 55°C to 85°C. This RNA contained the non-ribosomal nuclear RNA species (HnRNA, pre-mRNA). As shown in Figure 5, about 14% of the DNA of the total sonicated chromatin anneals with this RNA. 16% and 8% of DNA from the 50”C-pellet and 50-98°C pellet, respectively hybridized with the same RNA preparation, while 30% hybridization was reached with DNA from the post 98”Csupernatant.
Input
RNA (mg/ml)
Pro. 6. Hybridization of 14C-labelled rat liver HnRNA with filter bound DNA from different chromatin fix&ions. Rat liver heterogeneous nuclear RNA wa.s isolated after 1 h labelling with [‘*C]orotic acid and purified as desoribed in Materials and Methods. Its specif?c radioactivity was 1000 ote/min per pg. Hybridization with increasing conaentrations of RNA was carried out in 2 x SSC (SSC is 0.16 MNaCl, 0.016 ~-sodium citrate, pH 7.0) at 66°C for 18 h. 13 mm HAWP Millipore filters oontaining 6 to 10 pg DNA were used. To determine the amount of DNA on the filters at the end of the ljrocedure, control filters with 3H-labelled DNA isolated from sheared chromatin were incubated Ilnder the 8-e conditions (Ivanov 85 Markov, 1976). -O-O-, DNA from total ohromatin; ---I-J--•--, DNA from 60°Cpellet; ---A--A---, DNA from SO-98%pellet; -+--+-, DNA Jiom post 98°C-supernatant.
412
G. G. MARKOV,
I.
G. IVANOV
If we calculate the hybridization ability from the percent hybridization values of their relative amount (see above) a value close to 14 “/o hybridization experimentally
AND
I.
G. PASHEV
of the total DNA in rat liver chromatin the different DNA fractions (Fig. 5) and of 15 to 16% is obtained, which is very found for DNA of the total chromat,in.
4. Discussion The results presented in this paper show that the aggregation of sonicated chromatin as a function of temperature and buffer concentration is a complex but specific and reproducible process. Taking advantage of the existence of discrete precipitation steps the chromatin can be fractionated into several fractions: 50°Cpellet, 50-98°C pellet and post 98”C-supernatant. The size of the DNA fragments in the three chromatin fractions is slightly different. Larger sizes are found in the fractions precipitating at lower temperatures and vice versa. Therefore, one of the factors determining the behaviour of the chromatin in the process of aggregation might be the size of its fragments. Differences in the protein components may also contribute to the fractionation. This is in accordance with the finding that the non-precipitable fraction (post 98”C-supernatant) contains much less protein than the total chromatin. Do the thermal fractions differ only by physicochemical properties or do they contain fragments of different biological function 1 The hybridization data suggest the existence of functional differences between the fractions. The post 98%supernatant fraction shows a DNA/RNA hybridization ability much higher than that of the other fractions and than that of total DNA as well. It is noteworthy that this chromatin fraction, which does not precipitate upon heating has the lowest protein content and DNA with smallest fragment size. All these features could be explained assuming that the chromatin fragments in this fraction originate from highly decondensed, transcriptionally active chromatin regions, having a lower protein content (Shih & Bonner, 1970; Li & Bonner, 1971; Clark & Felsenfeld, 1971; Spelsberg et al., 1971). Such regions may be expected to be more “fragile” under the conditions of sonication. This would mean that the different sizes of fragments obtained by sonication would reflect in vivo existing structural features as association with protein and supercoiling with a definite biological significance. We thank Mrs I. Dimitrova for excellent technical assistance. One of us (I. P.) is very grateful to Dr W. Upholt from Carnegie Institution of Washington, Dept. of Embryology, Baltimore, U.S.A., for his help in sedimentation velocity analysis. REFERENCES Billing, R. J. & Bonner, J. (1972). Biochim. Biophys. Acta, 281, 453-462. Burton, K. (1968). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 12, part B, pp. 163-166, Academic Press, New York. Clark, R. J. & Felsenfeld, G. (1971). Nature New Biol. 229, 101-106. Duerksen, J. D. & McCarthy, B. J. (1971). Biochemistry, 10, 1471-1478. Frenster, J. H. (1965). Nature (London), 206, 680-683. Frenster, J. H., Allfrey, V. G. & Mirsky, A. E. (1963). Proc. Nat. .4cad. Sci., U.S.A. 50, 102&1032. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829-842.
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BY PRECIPITATION
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Hossaini, E., Zweidler, A. & Bloch, D. P. (1973). J. Mol. Biol. 74, 283-289. Ivanov, I. G. & Markov, G. G. (1975). Biochim. Biophye. Acta, in the press. Janowski, M., Nasser, D. S. & McCarthy, B. J. (1972). In 5th Karolinska Symposium uu Research Methods in Reproductive Endocrinology, Gene Transcription. in Reproductive Tissue (Diczfalusy, E., ed.), Acta Endocrinologica, suppl. 168, pp. 112-129. Li, H. & Bonner, J. (1971). Biochemietry, 10, 1461-1470. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265-275. Markov, G. G. & Arion, V. J. (1973). Eur. J. Biochem. 35, 186-200. Markov, G. G. & Ivanov, I. G. (1974). An&. Biochem. 59, 555563. McConaughy, B. L. & McCarthy, B. J. (1972). Biochemistry, 11, 998-1003. Reeck, G., Simpson, R. & Sober, H. A. (1972). Proc. Nat. AcadS&, U.S.A. 69, 2317--2321. Rickwood, D., Hell, A. & Birnie, G. D. (1973). FEBS Letters, 33, 221-224. Shih, T. & Bonner, J. (1970). J. Mol. Biol. 48, 469-487. Spelsberg, T., Hnilica, L. & Anseven, A. (1971). Biochim. Biophys. Acta, 228, 550-568. Stndier, F. W. (1965). J. Mol. Biol. 11, 373-390. Tsanev, R. & Russev, G. (1974). Eur. J. Biochem. 43, 257-263. Yasmineh, W. & Yunis, J. (1969). Biochem. Biophys. Res. Commun,. 35, 779-782. Yiuiis, J. $ Yasmineh, W. (1971). Science, 174, 1200~~1209.
