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Non-histone proteins
Experimental
Cell Research 82 (1973) 175-191
NON-HISTONE The Effect
of Nuclear
of Medical
of Metaphase
Washes and Comparison D. E. COMINGS
Department
PROTEINS
Genefics,
City
of Hope
and Interphase
Chromatin
and LOIS 0. TACK National
Medical
Center,
Duarte,
Calif.
91010,
USA
SUMMARY SDS Gel electrophoresis of mouse nuclei washed with various salt solutions and of Chinese hamster metaphase and interphase chromatin led to the following conclusions. 1. The pattern of non-histone proteins was the same after nuclei were washed with solutions varying markedly in ionic strength and cation content. 2. The pattern of the nuclear sap and the non-histone proteins showed many similar bands suggesting they are not totally distinct entities. The nuclear sap appears to be composed of several classes of proteins varying in their affinity for DNA. 3. Some of even the most tightly binding non-histone proteins could be removed from the DNA by low ionic strength salts simply by repeated washing of the chromatin. By contrast the histones were not removed by repeated washing. 4. When nuclei were washed with a salt solution that closely mimicked normal intranuclear conditions the lysine-rich histone was removed from the chromatin suggesting this histone is loosely bound to DNA in vivo. 5. It is pointed out that even though some nuclear sap proteins appear to be readily washed off the chromatin this does not mean they have no gene regulatory function. 6. The acid-soluble non-histone proteins reported to be unique to metaphase chromosomes are shown to be the result of the adsorption of cytoplasmic proteins onto the chromosomes during the isolation procedure. 7. Electrophoresis of well washed metaphase and interphase chromatin indicated their nonhistone proteins were quite similar.
Although the non-histone proteins appear to be an important component of chromatin, and have been implicated in many functions, they remain somewhat elusive as far as their precise definition is concerned. Some of the difficulties were demonstrated by Johns & Forrester [l] who showed that much of what had formerly been accepted as acidic nonhistone proteins were cytoplasmic or nucleoplasmic contaminants which could be washed off in 0.35 M NaCl. A second, rarely discussedproblem, concerns the abnormal conditions under which chromatin is usually isolated. Even though 12 - 731807
intranuclear chromatin exists in a milieu that is high in potassium, high in ionic strength, and contains Ca and Mg [2] the first thing that is usually done to chromatin during isolation is to expose it to low ionic strength solutions which contain no divalent cations. While a great number of studies have been done with this type of preparation it should be kept in mind that these are far removed from normal intranuclear conditions. To be more specific, studies by Langendorf et al. [2] on rat liver nuclei isolated in non-aqueous media indicate that the electrolyte concentration in the nucleoplasm in moles is Na+ Exptl
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176 D. E. Comings & Lois 0. Tack
0.131 and K+ 0.265, Ca2+ 0.004 and Mg2+ 0.022 for a total cation cont. of approx. 0.4 M. By contrast, chromatin is usually washed and prepared in saline-EDTA (0.075 M NaCl, 0.024 M EDTA) or 0.01 M Tris [3, 41. The present study examines the question: What proteins remain associated with chromatin after repeated washing with (1) SalineEDTA; (2) 0.35 M NaCl; (3) physiologic intranuclear salts (Langendorf salts); (4) 0.265 M KCl; (5) Saline-Mg2+-Ca2+? Are the tightly bound non-histone proteins distinct from nuclear sap proteins? Metaphase chromatin, which has been reported to contain many acid soluble nonhistone proteins which are absent from interphase chromatin [5], provides a good example of the problems encountered in distinguishing non-histone proteins from cytoplasmic contaminants. The present studies indicate that the non-histones that appeared to be unique to metaphase chromosomes are all cytoplasmic in origin. METHODS Nuclear washes Mouse liver nuclei were isolated by the Chaveau technique [6]. The livers of white Swiss mice were cut into small pieces in TMNC (0.01 M Tris, pH 8.0, 2.5 mM MgCl,, 10d8 M NaHSO,, and 10m4M CdSOJ. The NaHSO, and CdSO, were added to inhibit proteolysis [7, S]. They were then homogenized in a Teflon glass homogenizer in 2.4 M sucrose containing 3.3 mM CaCI, and lOMa M NaHSO,, filtered through cheesecloth, adjusted to a density of 12” 10’ with an Abbe refractometer, and centrifuged at 16 000 rpm for 1 h in a Beckman SW 41 Ti at 4°C. The pellets were washed 5 times in one of the following solutions: (1) Saline-EDTA (0.075 M NaCI, 0.024 M EDTA); (2) 0.35 M NaCl; (3) Langendorf salts (0.131 M NaCI, 0.265 M KCI, 0.022 M MgCl,, and 0.004 M CaCI,, in 0.01 M Tris, pH 7.4, with 1OmsM NaHSO, and 1O-4 M CdSO,: (4) 0.265 KCl: (5) Saline-Mg-Ca (0.075 M Na&O.i)22 M McCI,, 6.004 M CaCl&The nuclei were pelleted each time by centrifugation at 10 000 g for 10 min. After each wash the supematant and an aliquot of the pellet were saved and brought to 1 % with SDS. After the last wash the nuclear pellet was resuspended and sonicated until the nuclei were ruptured. This chromatin was washed two more times in the appropriate salt and again an aliquot of the chromatin pellets and supematants brought to 1 % with SDS. The final Exptl Cell Res 82 (1973)
pellet was resuspended in 0.4 M HzSOB for 30 min and pelleted. The pellet represented acid-washed chromatin, and the supernatant acid-soluble proteins. For the saline-EDTA washes, a portion of the liver was homogenized in TMNC until the cytoplasmic membranes were iust ruptured. This was then centrifuged at 100 000 g for 1-h and the supematant saved as cytoplasm. The nuclei were isolated from the rest of the liver, the purified nuclei washed once in salineEDTA, then once in 0.01 M Tris, pH 8.0, and resuspended in the Tris. The nuclei were homogenized with a Teflon glass homogenizer to release the chromatin and this was layered over 1.7 M sucrose and centrifuged in a Spinco 41 Ti rotor at 20 000 rpm for 3 h. The pellet was then washed 5 times in saline-EDTA and aliquots of each supernatant and each pellet brought to 1 % with SDS. All operations were performed at 4°C. The amount of protein extracted in each wash was determined by a turbidimetric assay [9]. Protein/DNA ratios were determined on the chromatin of the first nuclear pellet and the final chromatin nellet. An aliquot of the pellet was suspended in l:O ml of 0.5 N perchloric acid, incubated at 70°C for 20 min and centrifuged at 10000 g for 10 min. DNA in the supematant was determined by the diphenylamine assay [lo]. The pellet was solubilized in 1 N NaOH and protein determined by the Lowry assay.
Mitotic cells Chinese hamster cells (Don) were grown in 250 ml plastic Falcon flasks in McCoy’s media containing 10 % fetal calf serum, 1 % non-essential amino acids, and penicillin and streptomycin. The cells were reseeded into fresh flasks daily. To obtain metaphase cells, colcemid, 0.05 pg/ml, was added to 15 to 20 flasks for 3 h, and the cells in mitosis shaken off. This resulted in a suspension of 98 to 100 % metaphase cells.
Preparation of metaphaseand interphase chromatin Interphase cells were scraped off the flasks with a rubber policeman. The metaphase and interphase cells were washed twice in a balanced salt solution, and once in TMNC. The cells were then resuspended in 2 to 5 ml of this buffer and the interphase cells were sonicated until the cytoplasm was ruptured. The lysate was centrifuged at 10 000 g for 10 min and the sunernatant taken as the cytoplasm + nucleoplasm. The pellet was resuspended and-again centrifuged to pellet the crude chromatin. This was then centrifuged through 1.7 M sucrose, 0.01 M Tris, pH 8.0 at 26 &0 rpm in a Beckman SW Ti rotor (50000 g) for 3 h. The pellet was used as chromatin. It was treated in various ways, which for clarity and brevity of presentation are detailed in the legends of the figures.
Nuclear isolation in tissueculture cells Tissue cell nuclei were isolated without detergents by an osmotic shock technique [ll]. The cells were
Electrophoresis
of nuclear-+i>ashed chromatin
I77
placed in a small Waring blender and 30 ml of shocking media (5 mM MgCl*, 1 mM CaCl,, 50 mM glycine adjusted to pH 7.8 with KOH) was added and the cells blended at 60 V for 30 sec. Five ml of hypotonic buffer (0.54 M NaCl, 0.012 M CaCl,, 0.04 M Tris, pH 7.6) was added and blending continued at 25 V for 60 sec. The nuclei were pelleted at 500 g for 10 min, resuspended in 0.14 M NaCI, 3 mM CaCl,, 0.01 M Tris, pH 7.6, then washed twice in this buffer.
SDS gel electrophoresis The respective samples of protein on chromatin were solubilized by bringing them to between 1 and 3 % with sodium-dodecyl sulfate (SDS). After solubilization they were dialysed overnight against 0.01 M phosphate buffer, pH 7.1, 0.1 %-/Lm&aptoethanol, 0.1 % SDS. In some cases the chromatin samples were centrifuged at 100000 g for 18 h to remove DNA. SDS gel electrophoresis was by the technique of Weber & Osborn [12]. The protein cont. was determined bv TCA turbidity 191. Fifty to 100 PR of protein were measured out for- each sample and-the sample made about 20% with respect to sucrose. Ten ~‘1 of p-mercaptoethanol was added to each sample immediately before application to each gel to ensure reduction of S-S linkages during electrophoresis. After layering on the gel, enough Sephadex G-200 was added to each tube to give the sample a slurry consistency. This acted as a stacking gel [13] and resulted in flatter, sharper bands, particularly in the higher mol. wt range. Electrophoresis was carried out at 8 to 10 mA/tube for 6.5 h. The gels were stained in Coomassie brilliant blue [12] and destained in a Canalco lateral destainer. The following standards were used for estimation of mol. wt: Bovine serum albumin (A).,, 68 000 D: ovalbumin (0), 43 000 D; chymotrypsinogen (T); 25 000 D; and cytochrome c (C), 12 400 D.
RESULTS Terminology For reference purposes a typical SDS gel electrophoresis pattern of a well washed chromatin preparation is shown in fig. 1, along with a densitometry scan of the photograph. The most prominent bands are the histones. These show up as two bands near the middle of the gels and two bands at the ends of the gels. The two middle bands are composed of lysine-rich histones (histone I, LAP [14]). Some histone III in dimer form may also occur in this region [14]. Of the two bands at the end of the gels, the more slowly migrating one is composed of histone 111monomers (ARE) + histone IIb2 (KAS) +
ID+Eb2
+ IIbl
It?
Fig. 1. Reference SDS gels. Mouse liver nuclei washed 5 times with saline-Mg, Ca. The histones in the hatched areas consist of histone I near the middle of the gel. and histones III -t Ilb, ‘~IIb, and histone IV, near the end of the gel. The non-histone proteins are divided into 6 zones (see text).
histone Ilbl (LAK). The more rapidly migrating band is histone IV (GRK) [14, 151. The non-histone proteins were divided into 6 regions. Region 1 represents a series of moderately well separated high mol. wt proteins of 71 000 D or greater. Region 2 represents 3 bands that migrate close together and are prominent in all of the washed chromatin samples. They have a mol. wt of 65 000 to 71 000 D. Region 3 is composed of approx. 6 bands ranging in mol. wt from 40 000 to 65 000 D. Region 4 is a single band by SDS gel electrophoresis with a mol. wt of 40 000 to 43 000 D. It is also prominent in all the washed chromatin samples. It is partially soluble in acid (fig. 2, gel 12). Region 5 usually contains two bands migrating between the major histone bands. As shown in fig. 2, gel 12, it is acid-soluble and probably a Exptl
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118 D. E. Comings & Lois 0. Tack
Zyta EDT& T&s ;;N
1 I
2
3 4 Pellets
Saline-EDTA washes. Cyto = 100 000 g supernatant of cytoplasm; EDTA = supernatant of first salineEDTA wash of nuclei; Tris = supernatant of 0.01 M Tris, pH 8.0 wash of nuclei. The nuclei were then homogenized in Tris and centrifuged through 1.7 M sucrose (see Methods). The Chrom sup = 1.7 M sucrose supernatant. Pellets l-6 = the chromatin pellets following successive saline-EDTA washes. The last pellet was washed once in 0.4 M H,SO,. The pellet of this wash = AC Chr. The supernatant = AC Ext. Supernatants l-5 = the supernatants corresponding to the pellets l-5. Gel no. 18 = standards albumin, ovalbumin, chymotrypsinogen, and cytochrome c. Fig. 2.
basic protein. On the basis of migration it has a mol. wt of 21000 to 23 000 D.
rate
Saline-EDTA washes (fig. 2) When the Chaveau technique of nuclear isolation is used the yield is good but the nuclei are sometimes contaminated with cytoplasmic proteins. This is shown in fig. 2 where the cytoplasmic proteins (gel no. 1) and the proteins in the first saline-EDTA wash (gel no. 2) show some similar bands. The subsequent tris wash (gel no. 3) began to produce nucleoplasmic proteins, most of which were unique to the nucleoplasm. While some of the cytoplasmic proteins presumably occur at lower concentrations in the nucleus, a combination of non-aqueous methods of nuclear isolation and electrophoresis in both SDS and non-SDS gels will be necessary to definitively examine these relationships. In most preparations the cytoplasmic and nucleoplasmic patterns were quite distinct. Exptl
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The results of successive washes of chromatin in saline-EDTA are shown in gels 5-10. The histones are most prominent and the bands in regions 2 and 4 are the most prominent non-histone proteins. Successive washing does not change this pattern but does remove additional proteins (fig. 7). The non-histones are relatively enriched in the acid-washed chromatin (gel no. 11) and the histones as well as some of regions 4 and 6 are present in the acid wash (gel no. 12). The relative amounts of protein removed were as follows: EDTA-saline wash of nuclei, 35 200 ,ug; Tris wash of nuclei, 7 730 ,ug; chromatin supernatant, 3 000 rug; saline-EDTA chromatin wash no. 1, 816 ,ug; no. 2, 273 ,ug; no. 3, 152 lug; no. 4, 35 pg; no. 5,21 ,ug. In the first chromatin pellet (gel no. 5) the protein:DNA ratio was 3.66, and in the last (gel no. 10) it was 2.17. The supernatants are shown in gels 13-17. In the first supernatant (gel no. 13) all mol. wt classes of non-histones are present and
Electrophoresis
WI
1
2 3 Pellets
4’51Ch
1
211
of nuclear-washedchromatin
2 3 Supernants
4
5
I19
I Son
Fig. 3. 0.35 M NaCl washes. W N = aliquot of whole nuclei before washes. Pellets l-5 7 pellets after successive washes with 0.35 M NaCI. The last pellet was resuspended in 0.35 M NaCl and gently sonicated to break open the nuclei. An aliquot of this =Ch. This was then washed twice more with 0.35 M NaCl -m1 and 2 (gels 8 and 9). Supernatants l-5 mzthe supernatants corresponding to the pellets 1-5. Son the supernatant after centrifugation of the nuclear sonicate at 10 000 g for 10 min.
these probably represent poorly bound nucleoplasmic proteins. In the successive washes the high mol. wt proteins in regions I, 2 and 3 are most prominent. No histones appeared in the supernatants. Two major factors affect what proteins appear in the supernatant: (1) the amount of protein remaining on the chromatin; (2) how tightly the proteins are bound to DNA. These results indicate that following successivewasheswith saline-EDTA large amounts of all classesof histones remain associatedwith the DNA and they are very tightly bound. Lesser amounts of ‘non-histone’ proteins remain associated with the DNA. Some are loosely bound and come off in the first wash. Others are less tightly bound and come off in the successive washes but at rapidly decreasing yields. A final group is tightly bound and relatively resistant to successivewashes.This is especially true of proteins in regions 2 and 4.
0.35 M saline washes(fig. 3) The nuclear pellet, after centrifugation through heavy sucrose, contains many bands (gel no. 1) which represent the proteins of both the nuclear sap and the chromatin. The first 0.35 M saline wash of these nuclei removed 24 mg of protein compared to only 4 mg in the second wash. With successive washes(gels 2-9) the proteins in regions 2 and 4 became the most prominent. The bands in the supernatants (gels 10-14) tended to mimic the ‘non-histone’ bands of the comparable pellets. This suggeststhat all of the ‘nonhistone’ proteins can come off in the washes but those present in the nuclei washed 4 or 5 times represent proteins that are much more tightly bound to the DNA. After the nuclei were broken up by sonication and the chromatin further washed with 0.35 M NaCI. the major remaining non-histone proteins were in regions 2 and 4 (gels 7-9). Exptl
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180 D. E. Comings & Lois 0. Tack
Wash
PeJlets
Fig. 4. “Langendorf” washes (0.131 M NaCl, 0.265 M KCl, 0.022 M MgCI,, and 0.004 M CaCl,). Cyto = 100 000 g cytoplasmic supernatant. Wash supernatants 14 correspond to the wash pellets l-4. Whole nuclei = aliquot of nuclei before washing. Wash pellets l-4 =pellets after successive washing with Langendorf salts. The last pellet was resuspended in Langendorf salts and sonicated until the nuclei were ruptured. Chrom = pellet after centrifugation of sonicate at 10 000 g for 10 min. This pellet was then washed twice more with Langendorf buffer and once with 0.4 M HzS04. The final pellet = Acid Chrom. In standards (gel 13) A, albumin; 0, ovalbumin; T, chymotrypsinogen; C, cytochrome c.
The amount of protein removed in the successive washes was as follows: 0.35 M NaCl wash 1, 23 800 ,ug; wash 2, 4 363 ,ug; wash 3, 1 029 ,ug; wash 4, 465 ,ug; wash 5, 236 pg. The protein : DNA ratio of the first nuclear pellet (gel no. 2) was 2.07, and that of the second chromatin pellet (gel. no. 9) was 1.62. ‘Langendorf’ washes(fig. 4)
The results of successive washing of nuclei in ‘Langendorf’ salts are shown in fig. 4. The cytoplasm and first nuclear wash show a few bands that could be in common but most are distinct (gels 1 and 2). The whole nuclei (gel no. 6) show many bands, most of which have disappeared after the first nuclear wash (gel no. 7). The lysine-rich histones which are Exptl
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extracted under these conditions (0.43) show up in the supernatants (gels 2-4) and disappear from the pellets (gels 7-10). The major non-histone proteins remaining after the Langendorf washes are those in regions 1 and 2 and have mol. wt of 60 000 D or greater (gels 7-10). The proteins in regions 4 and 6 are also prominent (fig. 5). The relative amounts of protein in the different washeswere as follows: nuclear wash 1,27 400 ,ug; wash 2, 6 300 pg; wash 3, 1 740 ,ug; wash 4, 400 ,ug. We feel the pattern in gel no. 10 (figs 4, 5) is of particular interest since this represents those proteins which are most tightly bound to DNA under physiological intranuclear conditions. This suggests that in vivo the lysine-rich histones are poorly bound to DNA, the rest of the histones are tightly
Electrophoresis
of nuclear-Isashed
chromatin
1K I
bound, and that most of the non-histone proteins which are tightly bound in vivo belong to a dass of high mol. wt proteins of 60 000 D or greater. The major proteins that are not in this category are those in regions 4 and 6. 0.265 M KCI washes Since KC1 rather than NaCl is the major salt in the nucleoplasm, nuclei were washed successively in 0.265 M KC1 to determine its effect on the pattern of non-histone proteins. The results were similar to those with the 0.35 M NaCl washes. The protein:DNA ratio for the first nuclear pellet was 3.56, and for the last chromatin pellet was 1.35. Saline-magnesium,
calcium washes
Since there are some proteins that remain tightly bound to DNA only in the presence of divalent cations [16] nuclei were washed in salts in which the EDTA of saline-EDTA was replaced with 0.022 M MgCl, and 0.004 M CaCI,. Under these conditions the initial washes produced only minor changes over the pattern of proteins in the whole nuclei. Nevertheless, the pattern after the final nuclear wash was basically the same as with the other salts (fig. 6). The relative rate of removal of proteins by the successive washes iongendorf
Wash
iB+U,+
Ub2
Unwashed
Washed
5. Densitometric tracing of gel no. 6 (unwashed) and gel no. 10 (washed) representing the chromatin before and after washing with ‘Langendorf salts’. The stippled areas represent the non-histone proteins which remain tightly bound to the DNA under physiological intranuclear conditions. The arrows represent the non-histone and nuclear sap proteins which are washed off. Crosshatched areas represent the histones.
Fig.
6. Comparison of chromatin washed with salt solutions. See text. Fig.
~various
was also similar to that with the other salts (fig. 7). The protein:DNA ratio of the first nuclear pellet was 2.75 and that of the last chromatin pellet was 2.05. Similarity of non-histoneproteins after different washes Fig. 6 shows the final nuclear pellets after washing with each of the five different salts. Except for the Langendorf washes, which also removed histone I, the protein patterns in all the final pellets are quite similar. The relative rate of removal of nuclear proteins was also quite similar for all give different salt solutions (fig. 7), with each showing an exponential rate of extraction. Metaphase versusinterphase chromatin Since the nuclear membrane in mammals begins to break down at prophase, the isolaExptl
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182 D. E. Comings h Lois 0. Tack sented the crude metaphase and interphase chromatin. SDS gel electrophoresis of these fractions showed that except for the histones present in the chromatin, the protein patterns for the cytoplasm fnucleoplasm were identical to those of the chromatin, and there was no difference between metaphase and interphase cells (fig. 8). This suggeststhat most of the ‘non-histone’ proteins were the result of non-specific adsorption of cytoplasmic and nucleoplasmic proteins onto the chromatin. Further evidence for this was provided by microdensitometric tracings of the gel photographs which indicated that the ratio of ‘non-
Fig. 7. Abscissa: wash supernatants 1-5; ordinate: relative amount of protein removed by successive washes expressed as % of first wash. l , saline-EDTA; n , 0.35 N NaCl; 0, Langendorf salts; 0, 0.265 M KCl; A, saline-Mg-Ca.
tion of metaphase chromatin will result in its exposure to cytoplasmic proteins. This is not the case with interphase chromatin since it remains sequesteredbehind the intact nuclear membrane during the initial steps which remove most of the cytoplasmic proteins. In order to obtain comparable chromatin preparations, our initial step in the isolation of both metaphase and interphase chromatin involved the brief sonication of the cells until both the cytoplasmic and nuclear membranes were ruptured. This sonicate was centrifuged at 10 000 g for 10 min and the supernatant taken as the cytoplasm + nucleoplasm. After an additional wash, the chromatin pellets were layered over 1.7 M sucrose and centrifuged at 50 000 g for 3 h. The pellets repreExptl
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Fig. 8. SDS gel electrophoresis of Chinese hamster metaphase (M) and interphase (I) chromatin and cytoplasmic + nucleoplasmic proteins. Calf thymus histone control. Metaphase and interphase cells were washed and sonicated in TMNC and centrifuged at 10 000 g for 10 min. The supematants are cytoplasmic + nucleoplasmic proteins. The pellets were washed again at 10 000 g for 10 min, resuspended in TMNC and centrifuged through 1.7 M sucrose 50 000 g for 3 h. These pellets were washed in 0.15 N NaCl and solubilized in SDS.
Electrophoresis
of nuclear-washed
chromatin
I X3
Fig. 9. SDS gel electrophoresis of HCI and H,SO, extracts of metaphase (M) and interphase (I) chromatin. Histone, calf thymus histone; A, albumin, mol. wt 68 000; 0, ovalbumin 43 000; T, chymotrypsinogen, 27 500; C, cytochrome c, 12 400. Cytoplasmic+nucleoplasmic proteins were prepared as in fig. 8. The I .7 M sucrose pellet was washed in TMNC and divided into 2 parts. The first part was extracted with0.2 N H&SO, for 30 min, and centrifuged at 10 000 g for 10 min. The second part was extracted with 0.2 N HCI in the same manner. The supernatants are H,SO, and HCl sol. The pellets were solubilized in 0.01 M phosphate buffer pH 7.1 containing 1 Y SDS, dialysed against 0.01 M phosphate, pH 7.1, 0. I % /I-mercaptoethanol, 0. I (I,,SDS. and centrifuged at 100000 g for 18 h to remove DNA. This supernatant is H,SO, and HCI insol.
histone’ to histone proteins for both metaphase and interphase cells was 3.5. The ratio for most interphase chromatin is less than I [17, 181. This experiment was repeated 4 times with the sameresult. HCI versus H,SO, extracts Sadgopal & Bonner [5] found the non-h&tone proteins present in metaphase chromosomes to be more soluble in HCl than H,SO,. To further investigate this, the chromatin from metaphase and interphase cells was extracted with 0.2 N HCl or H,SO, and the soluble and
insoluble portions compared by SDS gel electrophoresis. This showed that when both interphase and metaphase chromatin were isolated in a similar fashion, they both possessedthe same non-histone proteins that were preferentially extracted with HCl (fig. 9). Most of the sameproteins were also present in the H,SO, extracts but to a lesser degree. The electrophoretic patterns of these acid extracts were similar to those of the acidinsoluble proteins and to the pattern of the cytoplasmic -t nucleoplasmic proteins. This indicated that the HCl acid-soluble proteins were not unique to metaphase chromosomes Exptl
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Res 82 (1973)
184 D. E. Comings h Lois 0. Tack Are the non-histone chromosomalproteins of metaphaseand interphase cells different?
I
‘*_,. I
CVTO
c
CV+TO Ch
‘Cy+TO AcCh
Ch
AC Ch
Fig. 10. Adsorption of mouse cytoplasmic proteins onto mouse chromatin and acid washed chromatin. Arrow, 50000 mol. wt cytoplasmic protein (P-50) which was markedly concentrated by binding to chromatin. Lines. other cytoplasmic proteins that also bind to chromatin. Mouse liver- nuclei were washed 3 times in saline-EDTA, one time in 0.34 M sucrose, homogenized to a viscous gel and centrifuged through 1.7 M sucrose at 50 000 g for 3 h. The pellet was resuspended in 0.01 M Tris, pH 8.0 and== chromatin (CH). An aliauot was washed 2 x in 0.2 N H&SO,, then ‘2 x in Tris = acid washed chromatin (AC Ch). The 100 000 g supernatant of cytoplasm in TMNC=cytoplasm (cyto). One ml of chromatin in 0.01 M Tris, pH 8.0 and containing 43 ,ug of DNA, and 1 ml of acid-washed chromatin containing 30 pg of DNA, were each mixed with 2.5 ml of cytoplasmic proteins in TMNC containing 35.5 mg of protein, and incubated at 4°C for 30 min. The chromatin was then centrifuged through 1.7 M sucrose at 50 000 g for 3 h and the pellets taken up in SDS represent cytoplasm + chromatin (Cyto -t- Ch), and cytoplasm + acid-washed chromatin (Cyto + AC Ch). Standards A, albumin; 0, ovalbumin; T, chymotrypsinogen; C, cytochrome c; Hist, calf thymus histones.
but were in fact cytoplasmic and nucleoplasmic proteins that had adhered to the chromatin during its isolation, and remained adherent after centrifugation through 1.7 M sucrose and washing in dilute Tris buffer. When chromatin from well washed nuclei was extracted with 0.4 M H$O, very few non-histone proteins were removed (fig. 2, gel no. 12). Expti Celt Res 82 (19731
Since the large amount of non-histone protein present in these chromatin preparations was primarily contaminating proteins, the question of whether the true chromosomal non-histone proteins of metaphase and interphase cells are different remained unanswered. To investigate this the chromatin samples were (a) washed 3 times with saline-EDTA; (6) washed 5 times with 0.35 M NaCl; or (c) the ‘non-histones’ were extracted by solubilizing the chromatin in 1.0 M NaCl then reducing the molarity to 0.4 M [19, 201. By all of these procedures the non-histone proteins of metaphase and interphase chromatin showed the same electrophoretic pattern. Chromatin from interphase nuclei
If the ‘non-histone’ proteins present in metaphase chromatin are due to cytoplasmic contamination, these proteins should be absent when chromatin is prepared from purified nuclei. This proved to be the case. Nuclei were prepared from Chinese hamster tissue culture cells and the proteins of the cytoplasm, nucleoplasm, crude chromatin, and chromatin were dialysed overnight, and compared by SDS gel electrophoresis (not shown). Now the chromosomal proteins of crude chromatin resembled those of the nuclear sap rather than those of the cytoplasm. The ratio of ‘non-histone’ to histone proteins is also lower, 1.3. Thus, when chromatin was prepared from nuclei after the cytoplasmic proteins had been removed, the amount of ‘non-histone’ protein was decreased and the pattern no longer resembled that of cytoplasmic proteins. Adsorption of proteins onto chromatin
To further investigate this adsorption phenomenon, chromatin and acid-washed chromatin isolated from mouse liver nuclei was
Electrophoresis suspended in a solution of mouse liver cytoplasmic proteins, then centrifuged through sucroseand the chromatin pellet electrophoresed (fig. 10). Many, but not all, of the cytoplasmic proteins were adsorbed onto the chromatin. There was one cytoplasmic band, with a mol. wt of about 50 000 D, that was avidly bound to the chromatin, resulting in a significant enrichment over and above its concentration in the cytoplasm (fig. IO, arrow). When most of the histones were removed from the chromatin by an acid wash, and this acid chromatin exposed to cytoplasm, even greater amounts of protein were bound, especially the 50 000 mol. wt protein. As a further check on whether all or only some proteins are adsorbed, acid chromatin was exposed to a solution containing 1 mg/ml of albumin, ovalbumin and cytochrome c, then centrifuged through 1.7 M sucrose and electrophoresed (fig. 11). Albumin bound very poorly, ovalbumin slightly, and cytochrome c avidly. This indicated, as expected, that different proteins vary considerably in their ability to bind to chromatin and DNA, and that molecular weight per se is not a major factor in the binding.
of nuclear-washed
chromatin
18.5
Fig. II. Adsorption of albumin (A), ovalbumin to), and cytochrome c (C) onto acid washed chromatin (Acid Chrome; A, 0, C). Acid washed chromatin alone (Acid Chrom). Chromatin alone (C’hrom). Histones extracted by the acid wash m.(Acid Ext). One ml of acid-washed chromatin containing 30 1’8 of DNA was incubated for 30 min at 4 C with 1.5 ml of 0.01 M Tris, pH 8.0, containing 2 mg each of albumin, ovalbumin, and cytochrome c. The chromatin was then centrifuged through 1.7 M sucrose at 50 000 g for 3 h and the pellets taken up in SDS.
DISCUSSION The definition of non-histone proteins has been complicated by the observation that in many preparations a large fraction of the socalled non-histone proteins are actually cytoplasmic or nucleoplasmic proteins that have remained adherent to the chromatin during the isolation procedure. For example, Johns & Forrester [I] studied calf thymus nucleoprotein which had been prepared by repeatedly washing minced calf thymus with 0.14 M NaCl, a treatment which is supposed to remove the cytoplasm and nucleoplasm. They found that the amount of ‘non-histone’
protein present on the chromatin could be reduced 66 “/bby further washing with 0.35 M NaCl and concluded that many of the ‘nonhistone’ proteins in the usual nucleoprotein preparations were actually contaminating cytoplasmic or nucleoplasmic proteins. Although they compared the amino acid composition of the proteins in the different washes they did not examine them by electrophoresis. In further studies, Goodwin & Johns [21] found that if calf thymus chromatin was thoroughly washed with 0.35 M NaCl no further ‘alkali’ soluble non-histone protein Exptl
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186 D. E. Comings h Lois 0. Tack
could be removed by dissolving the chromatin in 2 M NaCl and then precipitating the DNA and histone by dilution. These experiments raise a number of questions concerning the definition and function of non-histone proteins. They suggest that the only way to be certain of what is cytoplasm, what is nucleoplasm, and what is DNA bound non-histone proteins, is to repeatedly wash purified nuclei with various salt solutions and monitor the fractions by SDS gel electrophoresis. This is what we have done. One question was whether the nucleoplasm and cytoplasm have similar bands. Although some appeared similar, in general the proteins from these two sources were quite distinct. While this conclusion is easy to arrive at, the converse question of whether there are some cytoplasmic proteins which are present in the nucleoplasm in other than trace amounts is more difficult to answer, especially since some cytoplasmic proteins may pass into the nucleus during the isolation procedures. Further studies of this question using nonaqueous methods of nuclear isolation and longer gels are in progress. Effect of nuclear washes on ‘non-histone’ proteins
A more immediate question was whether some of the proteins in the nucleoplasm are similar to those remaining on the chromatin after extensive washing. This could be determined by comparing the gels of the whole nuclei (nucleoplasm + non-histone proteins + histones) to the gels of the washed chromatin (nonhistone proteins + histones) to the gels of the first supernatants (nucleoplasm). This showed that in general the nucleoplasm tended to have a fairly even distribution of proteins of all molecular weight classes (fig. 2, gel 3; fig. 3, gels 10-11; fig. 4, gel 2). By contrast, the non-histone proteins remaining after Exptl
Cell Res 82 (1973)
multiple washes tended to show a disproportionate number of proteins in regions 2,4 and 6 (fig. 6). When most of the histones were removed with an acid wash, additional bands in regions 1 and 3 could be seen (fig. 2, gel 11). Some of the non-histone proteins appeared to be well represented in the nucleoplasm (wash supernatants) suggesting that the nucleoplasmic proteins and the non-histone proteins are not totally distinct classes of proteins. It appears more likely that in a genetically active nucleus the nucleoplasm is composed of numerous families of proteins differing in their affinity for DNA. Since there is an excess of protein compared to DNA the proteins are in a competitive flux and at one time not all can bind to DNA or chromatin. Those with a low affinity for DNA are easily washed out of the nucleus at both low and high salt concentrations. Those with a higher affinity for DNA can be washed out at low salt concentrations but come out more easily at higher molarities. Those with the greatest affinity for DNA, can also be washed off with repeated washing in low ionic strength salt, come off a little easier in higher ionic strength, but a significant proportion remain after even multiple washings and these constitute the non-histone chromosomal proteins. If one is sufficiently persistent even these can be almost completely washed off the chromatin with low ionic strength salts, leaving the histones unaffected (see fig. 3, gel 9). This agrees with the findings of Goodwin & Johns [21] that negligible amounts of ‘non-histone’ protein were left after thorough washing with 0.35 M NaCl. In general, the electrophoretic pattern was the same regardless of the type of wash used. This suggests that there are few non-histone proteins which bind to DNA only in the presence of divalent cations. The washes also indicated there were no marked differences in the extractability of nuclear proteins when
Electrophoresis
potassium instead of sodium salts are used. Also, since the electrophoretic patterns of chromatin washed in saline-EDTA and 0.35 M NaCl were the same,the effect of increasing the molarity from 0.1 to 0.35 was lessimportant than the simple physical effect of multiple washings. With all the washes,except the Langendorf salts, there was a gradual change from the protein patterns seen with the whole nuclei (nucleoplasm + non-histones + histones) to the pattern seen for well washed nuclei and chromatin (non-histones + histones). The Langendorf salts gave a sudden shift from nucleoplasm i- non-histones to the non-histone pattern. This was the buffer with the highest molarity and the buffer which most closely approximates in vivo conditions. The studies with Langendorf salts imply that in vivo, lysine-rich histones and non-histones in regions 3 and 5 are loosely bound to DNA, while the moderately lysine-rich and argininerich histones and non-histones in regions 1 and 2 are tightly bound to DNA. The nonhistone proteins in regions 1 and 2 are of high mol. wt (60 000 D or greater) and in this respect are similar to the cytoplasmic proteins which bind to DNA [22]. It is possible to visualize that under in vivo conditions of relatively high molarity, the competition for DNA sites is relaxed and only those proteins with a moderate or high affinity for DNA are ‘interested’ in making the association. However, when the investigator comes along and exposes the nuclei and chromatin to low ionic strength conditions there is suddenly a mad rush for all the nuclear proteins to bind to the DNA and chromatin. He is now faced with the problem of sorting out what is normally bound to the chromatin and what is not. Perhaps examining the function of chromatin under more physiological conditions would help to avoid some of these problems. In this regard
of nuclear-washed
chromatin
I X7
it is of interest to note that mammalian RNA polymerase functions with maximum efficiency at 0.45 M KC1 [23]; the conformation of chromatin is not altered by the removal of Type I histones [24-261, and the template activity of chromatin does not begin to revert toward that of free DNA until the salt concentration exceeds0.45 M [27]. The observation by Harlow et al. [28] that much of the non-histone proteins of avian erythroid cells were associated with the nuclear membrane, raisesthe question of how many of the tightly binding proteins in our study represent membrane proteins. ‘These authors found that following the centrifugation of solubilized chromatin through 1.7 M sucrose the membrane proteins tended to remain in the supernate while the “pure chromatin” was in the pellet. Since the chromatin pellets in fig. 2 (gels 5-10) were prepared in this manner, and their protein pattern are identical to those of the washed whole nuclear pellets (fig. 6), the membrane proteins apparently represent a negligible portion of these non-histone proteins. The nucleoplasm, gene regulation
non-h&one
proteins,
urld
Several further points seemworth emphasizing. It could be argued that the nuclear sap and non-histone proteins have little to do with gene regulation since most of them can be washed off the chromatin, even with low ionic strength salts. This is probably an unwarranted conclusion for the following reasons. (1) It seems likely that there may be several classesof regulatory proteins, those which bind to specific sequenceswith a high affinity and which are present in the nucleus in tiny amounts, and those which bind to specific sequences with a lower degree of affinity but compensate by being present in larger amounts. The former may represent Exprl
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Res 82 ( 1973)
188 D. E. Comings & Lois 0. Tack
those non-histone proteins which remain bound to DNA even after multiple washings, while the latter may represent those proteins which are washed off the DNA more readily. (2) Lin & Riggs [29,30] have shown that the lac repressor binds to the lac operator DNA with a dissociation constant of IO-l3 M and binds to non-operator DNA withadissociation constant of about 1O-6 M. There is, however, far more non-operator than operator DNA. If a mammalian regulatory protein with the same mol. wt as lac operator were present at the same molarity (lO-s M) there would be two molecules bound with a high affinity to operator DNA for every 5 000 molecules bound with low affinity to non-operator DNA. Examination of this protein would suggest that all of it was poorly bound to DNA. The resulting implication that it had no role in gene regulation would obviously be incorrect. This type of mechanism could explain why many of the proteins that remained tightly bound to DNA (wash pellets) could also be easily washed off and show up in the supernatants (fig. 2, gels 10 versus 17; fig. 3, gels 8 versus 14).
Metaphase
versus interphase chromatin
The nuclear membrane breaks down during mitosis and when metaphase chromosomes are isolated they are exposed to cytoplasmic proteins. The protein:DNA ratio of such chromosomes is high, ranging from 3.3 to 5.2 [17]. When interphase chromatin is prepared, the isolation is usually preceded by a step that results in at least partial purification of the nuclei, and there is less exposure of the chromatin to cytoplasmic proteins. The protein : DNA ratio of such interphase chromatin ranges between 1 and 3 [17, 181. In a comparative study of metaphase and interphase chromatin, Sadgopal & Bonner [19] found that the protein : DNA ratio of Exptl
Cell
Res 82 (1973)
interphase HeLa cell chromatin was 2, while that of metaphase chromatin was almost 5. The histone : DNA ratio of these two chromatins was comparable and all of the excess proteins in metaphase chromatin were composed of ‘non-histones’. A portion of these were soluble in 0.2 M HCl while such HCl soluble ‘non-histones’ were absent from interphase chromatin. They proposed that metaphase chromatin contains acid-soluble proteins which are absent from interphase chromatin. The possibility that these might be the result of adventitious binding of ribosomes to the chromosomes was suggested [5]. The studies reported here indicate that the large amount of ‘non-histone’ protein present in metaphase chromosomes is due to the adherence of cytoplasmic proteins onto the chromosomes during their isolation. This conclusion is based on the following observations. (1) When the cells are disrupted so that both the metaphase and interphase chromatin is exposed to cytoplasmic proteins during isolation, the histone : ‘non-histone’ protein ratios of both chromatin preparations are the same, and the electrophoretic pattern of the ‘nonhistone’ proteins is the same as that of the cytoplasmic + nucleoplasmic proteins. (2) Under these conditions both the metaphase and interphase chromatin contain a large amount of HCl-soluble ‘non-histone’ protein, and much less H,SO,-soluble ‘nonhistone’ protein. In all fractions, no differences between metaphase and interphase chromatin were found. (3) When the interphase nuclei are freed of cytopiasmic proteins before isolation of the chromatin, the protein:DNA ratio is considerably less and the electrophoretic pattern of the chromosomal proteins no longer resembles that of the cytoplasmic proteins. (4) When chromatin isolated from purified nuclei is exposed to cytoplasmic proteins,
Electrophoresis
significant amounts of proteins are adsorbed onto the chromatin. While some of these adhering proteins may be derived from ribosomes, the presenceof all mol. wt classes,and not just those characteristic of ribosomes, indicates that non-ribosomal cytoplasmic proteins are also bound to the chromatin. There are apparently at least two ways in which proteins can bind to chromatin. When purified chromatin, isolated from interphase nuclei, is exposed to cytoplasmic proteins, or to a mixture of albumin, ovalbumin and cytochrome c, only certain classesof protein are bound. Since this binding is accentuated by removing the histones from the chromatin, this binding must be of protein to DNA. This is further demonstrated by the use of DNApolyacrylamide columns which also selectively extract DNA-binding proteins from Chinese hamster liver cytoplasm [22]. In contrast to this, when the cells were disrupted and the chromatin then isolated from the cytoplasm and nucleoplasm by centrifugation through 1.7 M sucrose, all of the cytoplasmic proteins were found to adhere to the chromatin and they were bound in the same ratio as they existed in the cytoplasm (i.e., the electrophoretic pattern of the cytoplasmic proteins and the chromosomal proteins were similar). This non-specific adsorption is probably the result of cytoplasmic proteins binding to chromosomal proteins. Theseremain adherent after sucrose washes but are removed with 0.35 M NaCl washes. These results suggest that there is protein-DNA binding which can be relatively specific (only some proteins involved), and protein-protein binding which is non-specific (all proteins involved). The adsorption of proteins onto chromatin can also be observed at the ultrastructural level. Solari [31] noted that hemoglobin free, EDTA washed, chicken red cell nuclei spread on water and fixed in alcohol and amyl
of nuclear-ttsashed
chromatin
189
acetate, had chromatin fibers with a mean diameter of 138.2 A. When the nuclei were floated on water containing hemoglobin,, the diameter of the chromatin fiber increased to 313.5 A. A related phenomenon was also observed by Bahr and co-workers [32, 331. By quantitative electron microscopy of human chromosomes, they found a broad weight distribution of chromosomes within a given group. This is probably due to the variable adsorption of cyroplasmic and nucleoplasmic proteins onto the chromosomes.This tendency has obvious relevance to any attempts to study chromosomal ‘non-histone’ proteins by autoradiographic techniques. In all probability the label will represent primarily the adhering cytoplasmic and nuclcoplasmic proteins. On the basis of the techniques used here. we found no quantitative or qualitative differences between the ‘non-histone’ proteins present on the chromatin of metaphase and interphase cells. It has also been observed that there are no significant quantitative differences in the histones of metaphase and interphase chromatin, although the histone III of metaphase cells may be enriched in S-S linkages [5], and histone I and III 134. 34a] more phosphorylated. These simple alterations could provide a mechanism for chromosome condensation. It has also been suggested that repressor-like proteins which undergo allosteric changes after binding to DNA might also be involved in chromosome condensation [35]. The concentration of these would be too small to be detected by the present methods. Evidence that there arc apparently some differences between the nonhistone proteins of metaphase versus interphase chromatin has been provided by the demonstration OFStein & Farber [36, 371that DNA reconstituted with metaphase nonhistone proteins showsdecreasedtranscriptive ability compared with DNA reconstituted
190 D. E. Comings & Lois 0. Tack with interphase non-histone proteins. The proteins involved are either modified or present in concentrations too small to be detected by the electrophoretic technique used here. Alternatively, it is possible that some of their “metaphase non-histone proteins” may have been contaminated with cytopIasmic proteins. Precautions In addition to the above, these studies also suggest the following conclusions or precautions. (1) Centrifugation of chromatin through 1.7 M sucrose does not remove adventitiously adhering proteins (but may partially remove membrane contaminants [28]. (2) Attempts to study non-h&one chromosomal proteins by autoradiography are probably fruitless since the predominant protein that would contribute to the grains would be adhering nucleoplasmic rather than the more tightly bound non-histone proteins. This may be circumvented by using only isolated, washed chromosomes [38]. (3) In whole mount preparations a disproportionate amount of the weight of the chromosome will be due to non-specifically adhering cytoplasmic and nucleoplasmic proteins. (4) Unless all contaminating cytoplasmic and nucleoplasmic proteins are washed off of the chromatin, acid extraction will result in the removal of large amounts of proteins that are not histones. Under these circumstances, the acid-soluble proteins will not be equivalent to histone proteins. (5) When isolating and studying ‘nonhistone’ proteins the technique used should be monitered by comparing the cytoplasm, nuclear sap and ‘non-histones’, by SDS gel electrophoresis.
Exptl
Cell Res 82 (1973)
REFERENCES 1. Johns, E W & Forrester, S, Eur j biochem 8 (1969) 547. 2. Langendorf, H, Siebert, G, Lorenz, I, Hannover, R & Beyer, R, Biochem Z 335 (1961) 273. 3. Bonner, J, Chalkley, G R, Dahmus, M, Frambrough, D, Fujimura, F, Huang, R C, Huberman, J, Jensen, R, Marushige, K, Ohlenbusch, H, Olivera, B & Widholm, J, Methods in enzymology (ed L Grossman & K Moldave) vol. 12B, p. 3. Academic Press, New York (1968). 4. Zubay, G & Doty, P, J mol biol 1 (1959) 1. 5. Sadgopal, A & Bonner, J, Biochim biophys acta 207 (1970) 206. 6. Chauveau. J. MoulC. Y & Rouiller. Ch. Exotl _ cell res ll’(l956) 317: I. Dounce, Z A L & Ickowicz, R, Arch biochem biophys 137 (1970) 143. 8. Panyim, S, Jensen, R H & Chalkley, R, Biochim biophys acta 160 (1968) 252. 9. Comines. D E & Tack. L 0. Exotl cell res 75 (1972) 53,. 10. Burton, K, Biochem j 62 (1956) 315. 11. Zweidler, A. Personal communication. 12. Weber, K & Osborn, M, J biol them 244 (1969) 4406. 13. Broome, J, Nature 199 (1963) 179. 14. Hnilica, L S, The structure and biological function of histones. Chemical Rubber Company Press, Cleveland. Ohio (1972). 15. Wilhelm, J A, A&e&n, A T, Johnson, A W & Hnilica. L S. Biochim bionhvs _ _ acta 272 (1972) 220. ’ ’ 16. Hotta, Y & Stern, H, Dev biol 26 (1971) 87. 17. Hearst, J E & Botchan, M, Ann rev biochem 39 (1970) 151. 18. MacGillivray, A J, Cameron, A, Krauze, R J, Rickwood, D & Paul, J, Biochim biophys acta 277 (1972) 384. 19. Kleinsmith, L J & Allfrey, V G, Biochim biophys acta 175 (1969) 123. 20. Gershey, E L & Kleinsmith, L J, Biochim biophys acta 194 (1969) 331. 21. Goodwin, G H & Johns, E W, FEBS letters 21 (1972) 103. 22. Vaughan, S T & Comings, D E, Exptl cell res 80 (1973) 264. 23. Butterworth, P H W, Cox, R F & Chesterton, C J, Eur j biochem 23 (1971) 229. 24. Olins, D E, J mol biol 43 (1959) 439. 25. Richards, B M & Pardon, J F, Exptl cell res 62 (1970) 184. 26. Simpson, R T & Sober, H A, Biochemistry 9 (1970) 3103. 27. Moore, I A R & Paul, J, Exptl cell res 76 (1973) 19. 28. Hsrlow, R, Tolstoshev, P & Wells, J R E, Cell diff 1 (1972) 341. 29. Lin, S-Y & Riggs, A D, Nature 228 (1970) 1184. 30. - Personal communication. 31. Solari, A J, Exptl cell res 67 (1971) 161. 32. Bahr. G F & Golomb. H M, Proc natl acad sci US 68 (1971) 726. 33. DuPraw, E J & Bahr, G F, Acta cytol 13 (1969) 188. I
,.
Electrophoresis 34. Lake, R S, Goidl, J A & Salzman, N P, Exptl cell res 73 (1972) 113. 34a. Gurley, L R, Walters, R A & Tobey, R A Fed proc 32 (1973) 587Abs. 35. Comings, D E & Riggs, A D, Nature 233 (1971) 48. 36. Stein, G & Farber, J, Proc natl acad sci US 69 (1972) 2910.
13 --731807
of nuclear-washedchromatin
I9 1
37. Farber, J, Stein, G &Baserga, R, Biochem hiophys res commun 47 (1972) 790. 38. Djondjurov, L, Markov, G & Tsanev, R, Exptl cell res 75 (I 972) 442. Received March 23, 1973 Revised version received May 8, 1973
Exptl Cell Res 82 (1973)
Cell Research 82 (1973) 175-191
NON-HISTONE The Effect
of Nuclear
of Medical
of Metaphase
Washes and Comparison D. E. COMINGS
Department
PROTEINS
Genefics,
City
of Hope
and Interphase
Chromatin
and LOIS 0. TACK National
Medical
Center,
Duarte,
Calif.
91010,
USA
SUMMARY SDS Gel electrophoresis of mouse nuclei washed with various salt solutions and of Chinese hamster metaphase and interphase chromatin led to the following conclusions. 1. The pattern of non-histone proteins was the same after nuclei were washed with solutions varying markedly in ionic strength and cation content. 2. The pattern of the nuclear sap and the non-histone proteins showed many similar bands suggesting they are not totally distinct entities. The nuclear sap appears to be composed of several classes of proteins varying in their affinity for DNA. 3. Some of even the most tightly binding non-histone proteins could be removed from the DNA by low ionic strength salts simply by repeated washing of the chromatin. By contrast the histones were not removed by repeated washing. 4. When nuclei were washed with a salt solution that closely mimicked normal intranuclear conditions the lysine-rich histone was removed from the chromatin suggesting this histone is loosely bound to DNA in vivo. 5. It is pointed out that even though some nuclear sap proteins appear to be readily washed off the chromatin this does not mean they have no gene regulatory function. 6. The acid-soluble non-histone proteins reported to be unique to metaphase chromosomes are shown to be the result of the adsorption of cytoplasmic proteins onto the chromosomes during the isolation procedure. 7. Electrophoresis of well washed metaphase and interphase chromatin indicated their nonhistone proteins were quite similar.
Although the non-histone proteins appear to be an important component of chromatin, and have been implicated in many functions, they remain somewhat elusive as far as their precise definition is concerned. Some of the difficulties were demonstrated by Johns & Forrester [l] who showed that much of what had formerly been accepted as acidic nonhistone proteins were cytoplasmic or nucleoplasmic contaminants which could be washed off in 0.35 M NaCl. A second, rarely discussedproblem, concerns the abnormal conditions under which chromatin is usually isolated. Even though 12 - 731807
intranuclear chromatin exists in a milieu that is high in potassium, high in ionic strength, and contains Ca and Mg [2] the first thing that is usually done to chromatin during isolation is to expose it to low ionic strength solutions which contain no divalent cations. While a great number of studies have been done with this type of preparation it should be kept in mind that these are far removed from normal intranuclear conditions. To be more specific, studies by Langendorf et al. [2] on rat liver nuclei isolated in non-aqueous media indicate that the electrolyte concentration in the nucleoplasm in moles is Na+ Exptl
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Res 82 (1973)
176 D. E. Comings & Lois 0. Tack
0.131 and K+ 0.265, Ca2+ 0.004 and Mg2+ 0.022 for a total cation cont. of approx. 0.4 M. By contrast, chromatin is usually washed and prepared in saline-EDTA (0.075 M NaCl, 0.024 M EDTA) or 0.01 M Tris [3, 41. The present study examines the question: What proteins remain associated with chromatin after repeated washing with (1) SalineEDTA; (2) 0.35 M NaCl; (3) physiologic intranuclear salts (Langendorf salts); (4) 0.265 M KCl; (5) Saline-Mg2+-Ca2+? Are the tightly bound non-histone proteins distinct from nuclear sap proteins? Metaphase chromatin, which has been reported to contain many acid soluble nonhistone proteins which are absent from interphase chromatin [5], provides a good example of the problems encountered in distinguishing non-histone proteins from cytoplasmic contaminants. The present studies indicate that the non-histones that appeared to be unique to metaphase chromosomes are all cytoplasmic in origin. METHODS Nuclear washes Mouse liver nuclei were isolated by the Chaveau technique [6]. The livers of white Swiss mice were cut into small pieces in TMNC (0.01 M Tris, pH 8.0, 2.5 mM MgCl,, 10d8 M NaHSO,, and 10m4M CdSOJ. The NaHSO, and CdSO, were added to inhibit proteolysis [7, S]. They were then homogenized in a Teflon glass homogenizer in 2.4 M sucrose containing 3.3 mM CaCI, and lOMa M NaHSO,, filtered through cheesecloth, adjusted to a density of 12” 10’ with an Abbe refractometer, and centrifuged at 16 000 rpm for 1 h in a Beckman SW 41 Ti at 4°C. The pellets were washed 5 times in one of the following solutions: (1) Saline-EDTA (0.075 M NaCI, 0.024 M EDTA); (2) 0.35 M NaCl; (3) Langendorf salts (0.131 M NaCI, 0.265 M KCI, 0.022 M MgCl,, and 0.004 M CaCI,, in 0.01 M Tris, pH 7.4, with 1OmsM NaHSO, and 1O-4 M CdSO,: (4) 0.265 KCl: (5) Saline-Mg-Ca (0.075 M Na&O.i)22 M McCI,, 6.004 M CaCl&The nuclei were pelleted each time by centrifugation at 10 000 g for 10 min. After each wash the supematant and an aliquot of the pellet were saved and brought to 1 % with SDS. After the last wash the nuclear pellet was resuspended and sonicated until the nuclei were ruptured. This chromatin was washed two more times in the appropriate salt and again an aliquot of the chromatin pellets and supematants brought to 1 % with SDS. The final Exptl Cell Res 82 (1973)
pellet was resuspended in 0.4 M HzSOB for 30 min and pelleted. The pellet represented acid-washed chromatin, and the supernatant acid-soluble proteins. For the saline-EDTA washes, a portion of the liver was homogenized in TMNC until the cytoplasmic membranes were iust ruptured. This was then centrifuged at 100 000 g for 1-h and the supematant saved as cytoplasm. The nuclei were isolated from the rest of the liver, the purified nuclei washed once in salineEDTA, then once in 0.01 M Tris, pH 8.0, and resuspended in the Tris. The nuclei were homogenized with a Teflon glass homogenizer to release the chromatin and this was layered over 1.7 M sucrose and centrifuged in a Spinco 41 Ti rotor at 20 000 rpm for 3 h. The pellet was then washed 5 times in saline-EDTA and aliquots of each supernatant and each pellet brought to 1 % with SDS. All operations were performed at 4°C. The amount of protein extracted in each wash was determined by a turbidimetric assay [9]. Protein/DNA ratios were determined on the chromatin of the first nuclear pellet and the final chromatin nellet. An aliquot of the pellet was suspended in l:O ml of 0.5 N perchloric acid, incubated at 70°C for 20 min and centrifuged at 10000 g for 10 min. DNA in the supematant was determined by the diphenylamine assay [lo]. The pellet was solubilized in 1 N NaOH and protein determined by the Lowry assay.
Mitotic cells Chinese hamster cells (Don) were grown in 250 ml plastic Falcon flasks in McCoy’s media containing 10 % fetal calf serum, 1 % non-essential amino acids, and penicillin and streptomycin. The cells were reseeded into fresh flasks daily. To obtain metaphase cells, colcemid, 0.05 pg/ml, was added to 15 to 20 flasks for 3 h, and the cells in mitosis shaken off. This resulted in a suspension of 98 to 100 % metaphase cells.
Preparation of metaphaseand interphase chromatin Interphase cells were scraped off the flasks with a rubber policeman. The metaphase and interphase cells were washed twice in a balanced salt solution, and once in TMNC. The cells were then resuspended in 2 to 5 ml of this buffer and the interphase cells were sonicated until the cytoplasm was ruptured. The lysate was centrifuged at 10 000 g for 10 min and the sunernatant taken as the cytoplasm + nucleoplasm. The pellet was resuspended and-again centrifuged to pellet the crude chromatin. This was then centrifuged through 1.7 M sucrose, 0.01 M Tris, pH 8.0 at 26 &0 rpm in a Beckman SW Ti rotor (50000 g) for 3 h. The pellet was used as chromatin. It was treated in various ways, which for clarity and brevity of presentation are detailed in the legends of the figures.
Nuclear isolation in tissueculture cells Tissue cell nuclei were isolated without detergents by an osmotic shock technique [ll]. The cells were
Electrophoresis
of nuclear-+i>ashed chromatin
I77
placed in a small Waring blender and 30 ml of shocking media (5 mM MgCl*, 1 mM CaCl,, 50 mM glycine adjusted to pH 7.8 with KOH) was added and the cells blended at 60 V for 30 sec. Five ml of hypotonic buffer (0.54 M NaCl, 0.012 M CaCl,, 0.04 M Tris, pH 7.6) was added and blending continued at 25 V for 60 sec. The nuclei were pelleted at 500 g for 10 min, resuspended in 0.14 M NaCI, 3 mM CaCl,, 0.01 M Tris, pH 7.6, then washed twice in this buffer.
SDS gel electrophoresis The respective samples of protein on chromatin were solubilized by bringing them to between 1 and 3 % with sodium-dodecyl sulfate (SDS). After solubilization they were dialysed overnight against 0.01 M phosphate buffer, pH 7.1, 0.1 %-/Lm&aptoethanol, 0.1 % SDS. In some cases the chromatin samples were centrifuged at 100000 g for 18 h to remove DNA. SDS gel electrophoresis was by the technique of Weber & Osborn [12]. The protein cont. was determined bv TCA turbidity 191. Fifty to 100 PR of protein were measured out for- each sample and-the sample made about 20% with respect to sucrose. Ten ~‘1 of p-mercaptoethanol was added to each sample immediately before application to each gel to ensure reduction of S-S linkages during electrophoresis. After layering on the gel, enough Sephadex G-200 was added to each tube to give the sample a slurry consistency. This acted as a stacking gel [13] and resulted in flatter, sharper bands, particularly in the higher mol. wt range. Electrophoresis was carried out at 8 to 10 mA/tube for 6.5 h. The gels were stained in Coomassie brilliant blue [12] and destained in a Canalco lateral destainer. The following standards were used for estimation of mol. wt: Bovine serum albumin (A).,, 68 000 D: ovalbumin (0), 43 000 D; chymotrypsinogen (T); 25 000 D; and cytochrome c (C), 12 400 D.
RESULTS Terminology For reference purposes a typical SDS gel electrophoresis pattern of a well washed chromatin preparation is shown in fig. 1, along with a densitometry scan of the photograph. The most prominent bands are the histones. These show up as two bands near the middle of the gels and two bands at the ends of the gels. The two middle bands are composed of lysine-rich histones (histone I, LAP [14]). Some histone III in dimer form may also occur in this region [14]. Of the two bands at the end of the gels, the more slowly migrating one is composed of histone 111monomers (ARE) + histone IIb2 (KAS) +
ID+Eb2
+ IIbl
It?
Fig. 1. Reference SDS gels. Mouse liver nuclei washed 5 times with saline-Mg, Ca. The histones in the hatched areas consist of histone I near the middle of the gel. and histones III -t Ilb, ‘~IIb, and histone IV, near the end of the gel. The non-histone proteins are divided into 6 zones (see text).
histone Ilbl (LAK). The more rapidly migrating band is histone IV (GRK) [14, 151. The non-histone proteins were divided into 6 regions. Region 1 represents a series of moderately well separated high mol. wt proteins of 71 000 D or greater. Region 2 represents 3 bands that migrate close together and are prominent in all of the washed chromatin samples. They have a mol. wt of 65 000 to 71 000 D. Region 3 is composed of approx. 6 bands ranging in mol. wt from 40 000 to 65 000 D. Region 4 is a single band by SDS gel electrophoresis with a mol. wt of 40 000 to 43 000 D. It is also prominent in all the washed chromatin samples. It is partially soluble in acid (fig. 2, gel 12). Region 5 usually contains two bands migrating between the major histone bands. As shown in fig. 2, gel 12, it is acid-soluble and probably a Exptl
Cell
Res 82 (1973)
118 D. E. Comings & Lois 0. Tack
Zyta EDT& T&s ;;N
1 I
2
3 4 Pellets
Saline-EDTA washes. Cyto = 100 000 g supernatant of cytoplasm; EDTA = supernatant of first salineEDTA wash of nuclei; Tris = supernatant of 0.01 M Tris, pH 8.0 wash of nuclei. The nuclei were then homogenized in Tris and centrifuged through 1.7 M sucrose (see Methods). The Chrom sup = 1.7 M sucrose supernatant. Pellets l-6 = the chromatin pellets following successive saline-EDTA washes. The last pellet was washed once in 0.4 M H,SO,. The pellet of this wash = AC Chr. The supernatant = AC Ext. Supernatants l-5 = the supernatants corresponding to the pellets l-5. Gel no. 18 = standards albumin, ovalbumin, chymotrypsinogen, and cytochrome c. Fig. 2.
basic protein. On the basis of migration it has a mol. wt of 21000 to 23 000 D.
rate
Saline-EDTA washes (fig. 2) When the Chaveau technique of nuclear isolation is used the yield is good but the nuclei are sometimes contaminated with cytoplasmic proteins. This is shown in fig. 2 where the cytoplasmic proteins (gel no. 1) and the proteins in the first saline-EDTA wash (gel no. 2) show some similar bands. The subsequent tris wash (gel no. 3) began to produce nucleoplasmic proteins, most of which were unique to the nucleoplasm. While some of the cytoplasmic proteins presumably occur at lower concentrations in the nucleus, a combination of non-aqueous methods of nuclear isolation and electrophoresis in both SDS and non-SDS gels will be necessary to definitively examine these relationships. In most preparations the cytoplasmic and nucleoplasmic patterns were quite distinct. Exptl
Cell
Res 82 (1973)
The results of successive washes of chromatin in saline-EDTA are shown in gels 5-10. The histones are most prominent and the bands in regions 2 and 4 are the most prominent non-histone proteins. Successive washing does not change this pattern but does remove additional proteins (fig. 7). The non-histones are relatively enriched in the acid-washed chromatin (gel no. 11) and the histones as well as some of regions 4 and 6 are present in the acid wash (gel no. 12). The relative amounts of protein removed were as follows: EDTA-saline wash of nuclei, 35 200 ,ug; Tris wash of nuclei, 7 730 ,ug; chromatin supernatant, 3 000 rug; saline-EDTA chromatin wash no. 1, 816 ,ug; no. 2, 273 ,ug; no. 3, 152 lug; no. 4, 35 pg; no. 5,21 ,ug. In the first chromatin pellet (gel no. 5) the protein:DNA ratio was 3.66, and in the last (gel no. 10) it was 2.17. The supernatants are shown in gels 13-17. In the first supernatant (gel no. 13) all mol. wt classes of non-histones are present and
Electrophoresis
WI
1
2 3 Pellets
4’51Ch
1
211
of nuclear-washedchromatin
2 3 Supernants
4
5
I19
I Son
Fig. 3. 0.35 M NaCl washes. W N = aliquot of whole nuclei before washes. Pellets l-5 7 pellets after successive washes with 0.35 M NaCI. The last pellet was resuspended in 0.35 M NaCl and gently sonicated to break open the nuclei. An aliquot of this =Ch. This was then washed twice more with 0.35 M NaCl -m1 and 2 (gels 8 and 9). Supernatants l-5 mzthe supernatants corresponding to the pellets 1-5. Son the supernatant after centrifugation of the nuclear sonicate at 10 000 g for 10 min.
these probably represent poorly bound nucleoplasmic proteins. In the successive washes the high mol. wt proteins in regions I, 2 and 3 are most prominent. No histones appeared in the supernatants. Two major factors affect what proteins appear in the supernatant: (1) the amount of protein remaining on the chromatin; (2) how tightly the proteins are bound to DNA. These results indicate that following successivewasheswith saline-EDTA large amounts of all classesof histones remain associatedwith the DNA and they are very tightly bound. Lesser amounts of ‘non-histone’ proteins remain associated with the DNA. Some are loosely bound and come off in the first wash. Others are less tightly bound and come off in the successive washes but at rapidly decreasing yields. A final group is tightly bound and relatively resistant to successivewashes.This is especially true of proteins in regions 2 and 4.
0.35 M saline washes(fig. 3) The nuclear pellet, after centrifugation through heavy sucrose, contains many bands (gel no. 1) which represent the proteins of both the nuclear sap and the chromatin. The first 0.35 M saline wash of these nuclei removed 24 mg of protein compared to only 4 mg in the second wash. With successive washes(gels 2-9) the proteins in regions 2 and 4 became the most prominent. The bands in the supernatants (gels 10-14) tended to mimic the ‘non-histone’ bands of the comparable pellets. This suggeststhat all of the ‘nonhistone’ proteins can come off in the washes but those present in the nuclei washed 4 or 5 times represent proteins that are much more tightly bound to the DNA. After the nuclei were broken up by sonication and the chromatin further washed with 0.35 M NaCI. the major remaining non-histone proteins were in regions 2 and 4 (gels 7-9). Exptl
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180 D. E. Comings & Lois 0. Tack
Wash
PeJlets
Fig. 4. “Langendorf” washes (0.131 M NaCl, 0.265 M KCl, 0.022 M MgCI,, and 0.004 M CaCl,). Cyto = 100 000 g cytoplasmic supernatant. Wash supernatants 14 correspond to the wash pellets l-4. Whole nuclei = aliquot of nuclei before washing. Wash pellets l-4 =pellets after successive washing with Langendorf salts. The last pellet was resuspended in Langendorf salts and sonicated until the nuclei were ruptured. Chrom = pellet after centrifugation of sonicate at 10 000 g for 10 min. This pellet was then washed twice more with Langendorf buffer and once with 0.4 M HzS04. The final pellet = Acid Chrom. In standards (gel 13) A, albumin; 0, ovalbumin; T, chymotrypsinogen; C, cytochrome c.
The amount of protein removed in the successive washes was as follows: 0.35 M NaCl wash 1, 23 800 ,ug; wash 2, 4 363 ,ug; wash 3, 1 029 ,ug; wash 4, 465 ,ug; wash 5, 236 pg. The protein : DNA ratio of the first nuclear pellet (gel no. 2) was 2.07, and that of the second chromatin pellet (gel. no. 9) was 1.62. ‘Langendorf’ washes(fig. 4)
The results of successive washing of nuclei in ‘Langendorf’ salts are shown in fig. 4. The cytoplasm and first nuclear wash show a few bands that could be in common but most are distinct (gels 1 and 2). The whole nuclei (gel no. 6) show many bands, most of which have disappeared after the first nuclear wash (gel no. 7). The lysine-rich histones which are Exptl
CeN Res 82 (1973)
extracted under these conditions (0.43) show up in the supernatants (gels 2-4) and disappear from the pellets (gels 7-10). The major non-histone proteins remaining after the Langendorf washes are those in regions 1 and 2 and have mol. wt of 60 000 D or greater (gels 7-10). The proteins in regions 4 and 6 are also prominent (fig. 5). The relative amounts of protein in the different washeswere as follows: nuclear wash 1,27 400 ,ug; wash 2, 6 300 pg; wash 3, 1 740 ,ug; wash 4, 400 ,ug. We feel the pattern in gel no. 10 (figs 4, 5) is of particular interest since this represents those proteins which are most tightly bound to DNA under physiological intranuclear conditions. This suggests that in vivo the lysine-rich histones are poorly bound to DNA, the rest of the histones are tightly
Electrophoresis
of nuclear-Isashed
chromatin
1K I
bound, and that most of the non-histone proteins which are tightly bound in vivo belong to a dass of high mol. wt proteins of 60 000 D or greater. The major proteins that are not in this category are those in regions 4 and 6. 0.265 M KCI washes Since KC1 rather than NaCl is the major salt in the nucleoplasm, nuclei were washed successively in 0.265 M KC1 to determine its effect on the pattern of non-histone proteins. The results were similar to those with the 0.35 M NaCl washes. The protein:DNA ratio for the first nuclear pellet was 3.56, and for the last chromatin pellet was 1.35. Saline-magnesium,
calcium washes
Since there are some proteins that remain tightly bound to DNA only in the presence of divalent cations [16] nuclei were washed in salts in which the EDTA of saline-EDTA was replaced with 0.022 M MgCl, and 0.004 M CaCI,. Under these conditions the initial washes produced only minor changes over the pattern of proteins in the whole nuclei. Nevertheless, the pattern after the final nuclear wash was basically the same as with the other salts (fig. 6). The relative rate of removal of proteins by the successive washes iongendorf
Wash
iB+U,+
Ub2
Unwashed
Washed
5. Densitometric tracing of gel no. 6 (unwashed) and gel no. 10 (washed) representing the chromatin before and after washing with ‘Langendorf salts’. The stippled areas represent the non-histone proteins which remain tightly bound to the DNA under physiological intranuclear conditions. The arrows represent the non-histone and nuclear sap proteins which are washed off. Crosshatched areas represent the histones.
Fig.
6. Comparison of chromatin washed with salt solutions. See text. Fig.
~various
was also similar to that with the other salts (fig. 7). The protein:DNA ratio of the first nuclear pellet was 2.75 and that of the last chromatin pellet was 2.05. Similarity of non-histoneproteins after different washes Fig. 6 shows the final nuclear pellets after washing with each of the five different salts. Except for the Langendorf washes, which also removed histone I, the protein patterns in all the final pellets are quite similar. The relative rate of removal of nuclear proteins was also quite similar for all give different salt solutions (fig. 7), with each showing an exponential rate of extraction. Metaphase versusinterphase chromatin Since the nuclear membrane in mammals begins to break down at prophase, the isolaExptl
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182 D. E. Comings h Lois 0. Tack sented the crude metaphase and interphase chromatin. SDS gel electrophoresis of these fractions showed that except for the histones present in the chromatin, the protein patterns for the cytoplasm fnucleoplasm were identical to those of the chromatin, and there was no difference between metaphase and interphase cells (fig. 8). This suggeststhat most of the ‘non-histone’ proteins were the result of non-specific adsorption of cytoplasmic and nucleoplasmic proteins onto the chromatin. Further evidence for this was provided by microdensitometric tracings of the gel photographs which indicated that the ratio of ‘non-
Fig. 7. Abscissa: wash supernatants 1-5; ordinate: relative amount of protein removed by successive washes expressed as % of first wash. l , saline-EDTA; n , 0.35 N NaCl; 0, Langendorf salts; 0, 0.265 M KCl; A, saline-Mg-Ca.
tion of metaphase chromatin will result in its exposure to cytoplasmic proteins. This is not the case with interphase chromatin since it remains sequesteredbehind the intact nuclear membrane during the initial steps which remove most of the cytoplasmic proteins. In order to obtain comparable chromatin preparations, our initial step in the isolation of both metaphase and interphase chromatin involved the brief sonication of the cells until both the cytoplasmic and nuclear membranes were ruptured. This sonicate was centrifuged at 10 000 g for 10 min and the supernatant taken as the cytoplasm + nucleoplasm. After an additional wash, the chromatin pellets were layered over 1.7 M sucrose and centrifuged at 50 000 g for 3 h. The pellets repreExptl
Cell Res 82 (1973)
Fig. 8. SDS gel electrophoresis of Chinese hamster metaphase (M) and interphase (I) chromatin and cytoplasmic + nucleoplasmic proteins. Calf thymus histone control. Metaphase and interphase cells were washed and sonicated in TMNC and centrifuged at 10 000 g for 10 min. The supematants are cytoplasmic + nucleoplasmic proteins. The pellets were washed again at 10 000 g for 10 min, resuspended in TMNC and centrifuged through 1.7 M sucrose 50 000 g for 3 h. These pellets were washed in 0.15 N NaCl and solubilized in SDS.
Electrophoresis
of nuclear-washed
chromatin
I X3
Fig. 9. SDS gel electrophoresis of HCI and H,SO, extracts of metaphase (M) and interphase (I) chromatin. Histone, calf thymus histone; A, albumin, mol. wt 68 000; 0, ovalbumin 43 000; T, chymotrypsinogen, 27 500; C, cytochrome c, 12 400. Cytoplasmic+nucleoplasmic proteins were prepared as in fig. 8. The I .7 M sucrose pellet was washed in TMNC and divided into 2 parts. The first part was extracted with0.2 N H&SO, for 30 min, and centrifuged at 10 000 g for 10 min. The second part was extracted with 0.2 N HCI in the same manner. The supernatants are H,SO, and HCl sol. The pellets were solubilized in 0.01 M phosphate buffer pH 7.1 containing 1 Y SDS, dialysed against 0.01 M phosphate, pH 7.1, 0. I % /I-mercaptoethanol, 0. I (I,,SDS. and centrifuged at 100000 g for 18 h to remove DNA. This supernatant is H,SO, and HCI insol.
histone’ to histone proteins for both metaphase and interphase cells was 3.5. The ratio for most interphase chromatin is less than I [17, 181. This experiment was repeated 4 times with the sameresult. HCI versus H,SO, extracts Sadgopal & Bonner [5] found the non-h&tone proteins present in metaphase chromosomes to be more soluble in HCl than H,SO,. To further investigate this, the chromatin from metaphase and interphase cells was extracted with 0.2 N HCl or H,SO, and the soluble and
insoluble portions compared by SDS gel electrophoresis. This showed that when both interphase and metaphase chromatin were isolated in a similar fashion, they both possessedthe same non-histone proteins that were preferentially extracted with HCl (fig. 9). Most of the sameproteins were also present in the H,SO, extracts but to a lesser degree. The electrophoretic patterns of these acid extracts were similar to those of the acidinsoluble proteins and to the pattern of the cytoplasmic -t nucleoplasmic proteins. This indicated that the HCl acid-soluble proteins were not unique to metaphase chromosomes Exptl
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184 D. E. Comings h Lois 0. Tack Are the non-histone chromosomalproteins of metaphaseand interphase cells different?
I
‘*_,. I
CVTO
c
CV+TO Ch
‘Cy+TO AcCh
Ch
AC Ch
Fig. 10. Adsorption of mouse cytoplasmic proteins onto mouse chromatin and acid washed chromatin. Arrow, 50000 mol. wt cytoplasmic protein (P-50) which was markedly concentrated by binding to chromatin. Lines. other cytoplasmic proteins that also bind to chromatin. Mouse liver- nuclei were washed 3 times in saline-EDTA, one time in 0.34 M sucrose, homogenized to a viscous gel and centrifuged through 1.7 M sucrose at 50 000 g for 3 h. The pellet was resuspended in 0.01 M Tris, pH 8.0 and== chromatin (CH). An aliauot was washed 2 x in 0.2 N H&SO,, then ‘2 x in Tris = acid washed chromatin (AC Ch). The 100 000 g supernatant of cytoplasm in TMNC=cytoplasm (cyto). One ml of chromatin in 0.01 M Tris, pH 8.0 and containing 43 ,ug of DNA, and 1 ml of acid-washed chromatin containing 30 pg of DNA, were each mixed with 2.5 ml of cytoplasmic proteins in TMNC containing 35.5 mg of protein, and incubated at 4°C for 30 min. The chromatin was then centrifuged through 1.7 M sucrose at 50 000 g for 3 h and the pellets taken up in SDS represent cytoplasm + chromatin (Cyto -t- Ch), and cytoplasm + acid-washed chromatin (Cyto + AC Ch). Standards A, albumin; 0, ovalbumin; T, chymotrypsinogen; C, cytochrome c; Hist, calf thymus histones.
but were in fact cytoplasmic and nucleoplasmic proteins that had adhered to the chromatin during its isolation, and remained adherent after centrifugation through 1.7 M sucrose and washing in dilute Tris buffer. When chromatin from well washed nuclei was extracted with 0.4 M H$O, very few non-histone proteins were removed (fig. 2, gel no. 12). Expti Celt Res 82 (19731
Since the large amount of non-histone protein present in these chromatin preparations was primarily contaminating proteins, the question of whether the true chromosomal non-histone proteins of metaphase and interphase cells are different remained unanswered. To investigate this the chromatin samples were (a) washed 3 times with saline-EDTA; (6) washed 5 times with 0.35 M NaCl; or (c) the ‘non-histones’ were extracted by solubilizing the chromatin in 1.0 M NaCl then reducing the molarity to 0.4 M [19, 201. By all of these procedures the non-histone proteins of metaphase and interphase chromatin showed the same electrophoretic pattern. Chromatin from interphase nuclei
If the ‘non-histone’ proteins present in metaphase chromatin are due to cytoplasmic contamination, these proteins should be absent when chromatin is prepared from purified nuclei. This proved to be the case. Nuclei were prepared from Chinese hamster tissue culture cells and the proteins of the cytoplasm, nucleoplasm, crude chromatin, and chromatin were dialysed overnight, and compared by SDS gel electrophoresis (not shown). Now the chromosomal proteins of crude chromatin resembled those of the nuclear sap rather than those of the cytoplasm. The ratio of ‘non-histone’ to histone proteins is also lower, 1.3. Thus, when chromatin was prepared from nuclei after the cytoplasmic proteins had been removed, the amount of ‘non-histone’ protein was decreased and the pattern no longer resembled that of cytoplasmic proteins. Adsorption of proteins onto chromatin
To further investigate this adsorption phenomenon, chromatin and acid-washed chromatin isolated from mouse liver nuclei was
Electrophoresis suspended in a solution of mouse liver cytoplasmic proteins, then centrifuged through sucroseand the chromatin pellet electrophoresed (fig. 10). Many, but not all, of the cytoplasmic proteins were adsorbed onto the chromatin. There was one cytoplasmic band, with a mol. wt of about 50 000 D, that was avidly bound to the chromatin, resulting in a significant enrichment over and above its concentration in the cytoplasm (fig. IO, arrow). When most of the histones were removed from the chromatin by an acid wash, and this acid chromatin exposed to cytoplasm, even greater amounts of protein were bound, especially the 50 000 mol. wt protein. As a further check on whether all or only some proteins are adsorbed, acid chromatin was exposed to a solution containing 1 mg/ml of albumin, ovalbumin and cytochrome c, then centrifuged through 1.7 M sucrose and electrophoresed (fig. 11). Albumin bound very poorly, ovalbumin slightly, and cytochrome c avidly. This indicated, as expected, that different proteins vary considerably in their ability to bind to chromatin and DNA, and that molecular weight per se is not a major factor in the binding.
of nuclear-washed
chromatin
18.5
Fig. II. Adsorption of albumin (A), ovalbumin to), and cytochrome c (C) onto acid washed chromatin (Acid Chrome; A, 0, C). Acid washed chromatin alone (Acid Chrom). Chromatin alone (C’hrom). Histones extracted by the acid wash m.(Acid Ext). One ml of acid-washed chromatin containing 30 1’8 of DNA was incubated for 30 min at 4 C with 1.5 ml of 0.01 M Tris, pH 8.0, containing 2 mg each of albumin, ovalbumin, and cytochrome c. The chromatin was then centrifuged through 1.7 M sucrose at 50 000 g for 3 h and the pellets taken up in SDS.
DISCUSSION The definition of non-histone proteins has been complicated by the observation that in many preparations a large fraction of the socalled non-histone proteins are actually cytoplasmic or nucleoplasmic proteins that have remained adherent to the chromatin during the isolation procedure. For example, Johns & Forrester [I] studied calf thymus nucleoprotein which had been prepared by repeatedly washing minced calf thymus with 0.14 M NaCl, a treatment which is supposed to remove the cytoplasm and nucleoplasm. They found that the amount of ‘non-histone’
protein present on the chromatin could be reduced 66 “/bby further washing with 0.35 M NaCl and concluded that many of the ‘nonhistone’ proteins in the usual nucleoprotein preparations were actually contaminating cytoplasmic or nucleoplasmic proteins. Although they compared the amino acid composition of the proteins in the different washes they did not examine them by electrophoresis. In further studies, Goodwin & Johns [21] found that if calf thymus chromatin was thoroughly washed with 0.35 M NaCl no further ‘alkali’ soluble non-histone protein Exptl
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186 D. E. Comings h Lois 0. Tack
could be removed by dissolving the chromatin in 2 M NaCl and then precipitating the DNA and histone by dilution. These experiments raise a number of questions concerning the definition and function of non-histone proteins. They suggest that the only way to be certain of what is cytoplasm, what is nucleoplasm, and what is DNA bound non-histone proteins, is to repeatedly wash purified nuclei with various salt solutions and monitor the fractions by SDS gel electrophoresis. This is what we have done. One question was whether the nucleoplasm and cytoplasm have similar bands. Although some appeared similar, in general the proteins from these two sources were quite distinct. While this conclusion is easy to arrive at, the converse question of whether there are some cytoplasmic proteins which are present in the nucleoplasm in other than trace amounts is more difficult to answer, especially since some cytoplasmic proteins may pass into the nucleus during the isolation procedures. Further studies of this question using nonaqueous methods of nuclear isolation and longer gels are in progress. Effect of nuclear washes on ‘non-histone’ proteins
A more immediate question was whether some of the proteins in the nucleoplasm are similar to those remaining on the chromatin after extensive washing. This could be determined by comparing the gels of the whole nuclei (nucleoplasm + non-histone proteins + histones) to the gels of the washed chromatin (nonhistone proteins + histones) to the gels of the first supernatants (nucleoplasm). This showed that in general the nucleoplasm tended to have a fairly even distribution of proteins of all molecular weight classes (fig. 2, gel 3; fig. 3, gels 10-11; fig. 4, gel 2). By contrast, the non-histone proteins remaining after Exptl
Cell Res 82 (1973)
multiple washes tended to show a disproportionate number of proteins in regions 2,4 and 6 (fig. 6). When most of the histones were removed with an acid wash, additional bands in regions 1 and 3 could be seen (fig. 2, gel 11). Some of the non-histone proteins appeared to be well represented in the nucleoplasm (wash supernatants) suggesting that the nucleoplasmic proteins and the non-histone proteins are not totally distinct classes of proteins. It appears more likely that in a genetically active nucleus the nucleoplasm is composed of numerous families of proteins differing in their affinity for DNA. Since there is an excess of protein compared to DNA the proteins are in a competitive flux and at one time not all can bind to DNA or chromatin. Those with a low affinity for DNA are easily washed out of the nucleus at both low and high salt concentrations. Those with a higher affinity for DNA can be washed out at low salt concentrations but come out more easily at higher molarities. Those with the greatest affinity for DNA, can also be washed off with repeated washing in low ionic strength salt, come off a little easier in higher ionic strength, but a significant proportion remain after even multiple washings and these constitute the non-histone chromosomal proteins. If one is sufficiently persistent even these can be almost completely washed off the chromatin with low ionic strength salts, leaving the histones unaffected (see fig. 3, gel 9). This agrees with the findings of Goodwin & Johns [21] that negligible amounts of ‘non-histone’ protein were left after thorough washing with 0.35 M NaCl. In general, the electrophoretic pattern was the same regardless of the type of wash used. This suggests that there are few non-histone proteins which bind to DNA only in the presence of divalent cations. The washes also indicated there were no marked differences in the extractability of nuclear proteins when
Electrophoresis
potassium instead of sodium salts are used. Also, since the electrophoretic patterns of chromatin washed in saline-EDTA and 0.35 M NaCl were the same,the effect of increasing the molarity from 0.1 to 0.35 was lessimportant than the simple physical effect of multiple washings. With all the washes,except the Langendorf salts, there was a gradual change from the protein patterns seen with the whole nuclei (nucleoplasm + non-histones + histones) to the pattern seen for well washed nuclei and chromatin (non-histones + histones). The Langendorf salts gave a sudden shift from nucleoplasm i- non-histones to the non-histone pattern. This was the buffer with the highest molarity and the buffer which most closely approximates in vivo conditions. The studies with Langendorf salts imply that in vivo, lysine-rich histones and non-histones in regions 3 and 5 are loosely bound to DNA, while the moderately lysine-rich and argininerich histones and non-histones in regions 1 and 2 are tightly bound to DNA. The nonhistone proteins in regions 1 and 2 are of high mol. wt (60 000 D or greater) and in this respect are similar to the cytoplasmic proteins which bind to DNA [22]. It is possible to visualize that under in vivo conditions of relatively high molarity, the competition for DNA sites is relaxed and only those proteins with a moderate or high affinity for DNA are ‘interested’ in making the association. However, when the investigator comes along and exposes the nuclei and chromatin to low ionic strength conditions there is suddenly a mad rush for all the nuclear proteins to bind to the DNA and chromatin. He is now faced with the problem of sorting out what is normally bound to the chromatin and what is not. Perhaps examining the function of chromatin under more physiological conditions would help to avoid some of these problems. In this regard
of nuclear-washed
chromatin
I X7
it is of interest to note that mammalian RNA polymerase functions with maximum efficiency at 0.45 M KC1 [23]; the conformation of chromatin is not altered by the removal of Type I histones [24-261, and the template activity of chromatin does not begin to revert toward that of free DNA until the salt concentration exceeds0.45 M [27]. The observation by Harlow et al. [28] that much of the non-histone proteins of avian erythroid cells were associated with the nuclear membrane, raisesthe question of how many of the tightly binding proteins in our study represent membrane proteins. ‘These authors found that following the centrifugation of solubilized chromatin through 1.7 M sucrose the membrane proteins tended to remain in the supernate while the “pure chromatin” was in the pellet. Since the chromatin pellets in fig. 2 (gels 5-10) were prepared in this manner, and their protein pattern are identical to those of the washed whole nuclear pellets (fig. 6), the membrane proteins apparently represent a negligible portion of these non-histone proteins. The nucleoplasm, gene regulation
non-h&one
proteins,
urld
Several further points seemworth emphasizing. It could be argued that the nuclear sap and non-histone proteins have little to do with gene regulation since most of them can be washed off the chromatin, even with low ionic strength salts. This is probably an unwarranted conclusion for the following reasons. (1) It seems likely that there may be several classesof regulatory proteins, those which bind to specific sequenceswith a high affinity and which are present in the nucleus in tiny amounts, and those which bind to specific sequences with a lower degree of affinity but compensate by being present in larger amounts. The former may represent Exprl
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188 D. E. Comings & Lois 0. Tack
those non-histone proteins which remain bound to DNA even after multiple washings, while the latter may represent those proteins which are washed off the DNA more readily. (2) Lin & Riggs [29,30] have shown that the lac repressor binds to the lac operator DNA with a dissociation constant of IO-l3 M and binds to non-operator DNA withadissociation constant of about 1O-6 M. There is, however, far more non-operator than operator DNA. If a mammalian regulatory protein with the same mol. wt as lac operator were present at the same molarity (lO-s M) there would be two molecules bound with a high affinity to operator DNA for every 5 000 molecules bound with low affinity to non-operator DNA. Examination of this protein would suggest that all of it was poorly bound to DNA. The resulting implication that it had no role in gene regulation would obviously be incorrect. This type of mechanism could explain why many of the proteins that remained tightly bound to DNA (wash pellets) could also be easily washed off and show up in the supernatants (fig. 2, gels 10 versus 17; fig. 3, gels 8 versus 14).
Metaphase
versus interphase chromatin
The nuclear membrane breaks down during mitosis and when metaphase chromosomes are isolated they are exposed to cytoplasmic proteins. The protein:DNA ratio of such chromosomes is high, ranging from 3.3 to 5.2 [17]. When interphase chromatin is prepared, the isolation is usually preceded by a step that results in at least partial purification of the nuclei, and there is less exposure of the chromatin to cytoplasmic proteins. The protein : DNA ratio of such interphase chromatin ranges between 1 and 3 [17, 181. In a comparative study of metaphase and interphase chromatin, Sadgopal & Bonner [19] found that the protein : DNA ratio of Exptl
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Res 82 (1973)
interphase HeLa cell chromatin was 2, while that of metaphase chromatin was almost 5. The histone : DNA ratio of these two chromatins was comparable and all of the excess proteins in metaphase chromatin were composed of ‘non-histones’. A portion of these were soluble in 0.2 M HCl while such HCl soluble ‘non-histones’ were absent from interphase chromatin. They proposed that metaphase chromatin contains acid-soluble proteins which are absent from interphase chromatin. The possibility that these might be the result of adventitious binding of ribosomes to the chromosomes was suggested [5]. The studies reported here indicate that the large amount of ‘non-histone’ protein present in metaphase chromosomes is due to the adherence of cytoplasmic proteins onto the chromosomes during their isolation. This conclusion is based on the following observations. (1) When the cells are disrupted so that both the metaphase and interphase chromatin is exposed to cytoplasmic proteins during isolation, the histone : ‘non-histone’ protein ratios of both chromatin preparations are the same, and the electrophoretic pattern of the ‘nonhistone’ proteins is the same as that of the cytoplasmic + nucleoplasmic proteins. (2) Under these conditions both the metaphase and interphase chromatin contain a large amount of HCl-soluble ‘non-histone’ protein, and much less H,SO,-soluble ‘nonhistone’ protein. In all fractions, no differences between metaphase and interphase chromatin were found. (3) When the interphase nuclei are freed of cytopiasmic proteins before isolation of the chromatin, the protein:DNA ratio is considerably less and the electrophoretic pattern of the chromosomal proteins no longer resembles that of the cytoplasmic proteins. (4) When chromatin isolated from purified nuclei is exposed to cytoplasmic proteins,
Electrophoresis
significant amounts of proteins are adsorbed onto the chromatin. While some of these adhering proteins may be derived from ribosomes, the presenceof all mol. wt classes,and not just those characteristic of ribosomes, indicates that non-ribosomal cytoplasmic proteins are also bound to the chromatin. There are apparently at least two ways in which proteins can bind to chromatin. When purified chromatin, isolated from interphase nuclei, is exposed to cytoplasmic proteins, or to a mixture of albumin, ovalbumin and cytochrome c, only certain classesof protein are bound. Since this binding is accentuated by removing the histones from the chromatin, this binding must be of protein to DNA. This is further demonstrated by the use of DNApolyacrylamide columns which also selectively extract DNA-binding proteins from Chinese hamster liver cytoplasm [22]. In contrast to this, when the cells were disrupted and the chromatin then isolated from the cytoplasm and nucleoplasm by centrifugation through 1.7 M sucrose, all of the cytoplasmic proteins were found to adhere to the chromatin and they were bound in the same ratio as they existed in the cytoplasm (i.e., the electrophoretic pattern of the cytoplasmic proteins and the chromosomal proteins were similar). This non-specific adsorption is probably the result of cytoplasmic proteins binding to chromosomal proteins. Theseremain adherent after sucrose washes but are removed with 0.35 M NaCl washes. These results suggest that there is protein-DNA binding which can be relatively specific (only some proteins involved), and protein-protein binding which is non-specific (all proteins involved). The adsorption of proteins onto chromatin can also be observed at the ultrastructural level. Solari [31] noted that hemoglobin free, EDTA washed, chicken red cell nuclei spread on water and fixed in alcohol and amyl
of nuclear-ttsashed
chromatin
189
acetate, had chromatin fibers with a mean diameter of 138.2 A. When the nuclei were floated on water containing hemoglobin,, the diameter of the chromatin fiber increased to 313.5 A. A related phenomenon was also observed by Bahr and co-workers [32, 331. By quantitative electron microscopy of human chromosomes, they found a broad weight distribution of chromosomes within a given group. This is probably due to the variable adsorption of cyroplasmic and nucleoplasmic proteins onto the chromosomes.This tendency has obvious relevance to any attempts to study chromosomal ‘non-histone’ proteins by autoradiographic techniques. In all probability the label will represent primarily the adhering cytoplasmic and nuclcoplasmic proteins. On the basis of the techniques used here. we found no quantitative or qualitative differences between the ‘non-histone’ proteins present on the chromatin of metaphase and interphase cells. It has also been observed that there are no significant quantitative differences in the histones of metaphase and interphase chromatin, although the histone III of metaphase cells may be enriched in S-S linkages [5], and histone I and III 134. 34a] more phosphorylated. These simple alterations could provide a mechanism for chromosome condensation. It has also been suggested that repressor-like proteins which undergo allosteric changes after binding to DNA might also be involved in chromosome condensation [35]. The concentration of these would be too small to be detected by the present methods. Evidence that there arc apparently some differences between the nonhistone proteins of metaphase versus interphase chromatin has been provided by the demonstration OFStein & Farber [36, 371that DNA reconstituted with metaphase nonhistone proteins showsdecreasedtranscriptive ability compared with DNA reconstituted
190 D. E. Comings & Lois 0. Tack with interphase non-histone proteins. The proteins involved are either modified or present in concentrations too small to be detected by the electrophoretic technique used here. Alternatively, it is possible that some of their “metaphase non-histone proteins” may have been contaminated with cytopIasmic proteins. Precautions In addition to the above, these studies also suggest the following conclusions or precautions. (1) Centrifugation of chromatin through 1.7 M sucrose does not remove adventitiously adhering proteins (but may partially remove membrane contaminants [28]. (2) Attempts to study non-h&one chromosomal proteins by autoradiography are probably fruitless since the predominant protein that would contribute to the grains would be adhering nucleoplasmic rather than the more tightly bound non-histone proteins. This may be circumvented by using only isolated, washed chromosomes [38]. (3) In whole mount preparations a disproportionate amount of the weight of the chromosome will be due to non-specifically adhering cytoplasmic and nucleoplasmic proteins. (4) Unless all contaminating cytoplasmic and nucleoplasmic proteins are washed off of the chromatin, acid extraction will result in the removal of large amounts of proteins that are not histones. Under these circumstances, the acid-soluble proteins will not be equivalent to histone proteins. (5) When isolating and studying ‘nonhistone’ proteins the technique used should be monitered by comparing the cytoplasm, nuclear sap and ‘non-histones’, by SDS gel electrophoresis.
Exptl
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Electrophoresis 34. Lake, R S, Goidl, J A & Salzman, N P, Exptl cell res 73 (1972) 113. 34a. Gurley, L R, Walters, R A & Tobey, R A Fed proc 32 (1973) 587Abs. 35. Comings, D E & Riggs, A D, Nature 233 (1971) 48. 36. Stein, G & Farber, J, Proc natl acad sci US 69 (1972) 2910.
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of nuclear-washedchromatin
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37. Farber, J, Stein, G &Baserga, R, Biochem hiophys res commun 47 (1972) 790. 38. Djondjurov, L, Markov, G & Tsanev, R, Exptl cell res 75 (I 972) 442. Received March 23, 1973 Revised version received May 8, 1973
Exptl Cell Res 82 (1973)