F1-histone modification at metaphase in Chinese hamster cells

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Experimental Cell Research 73 (1972) 113-121

F I-HISTONE

MODIFICATION AT METAPHASE IN CHINESE HAMSTER CELLS

R. S. LAKE,

JO ALENE

GOIDL

and N. P. SALZMAN

Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md 20014, USA

SUMMARY When the histones of metaphase and interphase Chines: hamster. HeLa S 3 and rat nephroma cells are compared by polyacrylamide gel elecrophoresis, a shift to’slower mobility of metaphase F 1-histone is observed. This change in mobility of the F 1-histone results from enhanced nhosphorylation since it is not observed after treatment of the extracted proteins with alkaline phosphatase. The phosphorylation is found in mitotic cells collected without use of a metaphase arrest agent. Further, the extent of phosphorylation of F 1 is independent of the elapsed time of metaphase arrest with vinblastine. The phosphoryl-rich F 1 subcomponents are dephosphorylated upon allowing the metaphase cell to enter the G 1 phase. The observed protein phosphorylation may be related to the unique physical state of metaphase chromatin. It has a pronounced influence on the chemical extraction of lysine-rich histone during isolation of metaphase chromosomes.

Histones of interphase and metaphase chromosomes have previously been compared in several cultured cell types. Comings [5] reported experiments which failed to show differences in the histone electrophoretic patterns of metaphase and interphase human amnion cells. Maio & Schildkraut [ 171 showed that acid-soluble proteins from nuclei and metaphase chromosomes of three different cell lines were qualitatively the same. Similarly, Hancock [8] observed no detectable changes in the quantity or types of histones at mitosis of HeLa cells. More recently, Sadgopal & Bonner [26] have presented evidence that HeLa cell interphase and metaphase chromosomes contain a similar spectrum of histones, but that metaphase chromosomes are slightly deficient in lysine-rich histones. This report presents evidence which indi8 - 721897

cates that enhanced phosphorylation of the lysine-rich (F 1) histone is characteristic of Chinese hamster metaphase cells. The experimental approach has been to prepare metaphase and interphase chromatin in parallel from the same cell population and analyze the identifiable acid-soluble proteins in the refined analytical polyacrylamide gel system of Panyim & Chalkley [23]. MATERIALS

AND

METHODS

Cell cultures Chinese hamster cells (V79-589FR [7]; male Chinese hamster; 23 chromosomes) were grown in Tricine [N-tris-(hydroxymethyl) methyl glycine] buffered Eagle’s no. 2 medium supplemented with 10% fetal calf serum. Metaphase and interphase cells were prepared from the same cell population. Each disposable roller bottle (680 cm2 area) was seeded with 5 x 10’ cells obtained from a suspension culture. After 18-24 h of incubation at 36”C, vinblastine sulfate at ExptI Cell Res 73 (1972)

114

R. S. Lake et al.

0.1 pg/ml (Velban; Eli Lilly & Co., Indianapolis) or Colcemid at 0.06 fig/ml were added for a period of 4 to 6 h. Cells in metaphase were selectively detached from the monolayer culture. About 1 x 10’ metaphase cells of 85 to 90 % purity were obtained from each bottle in 50 ml of warm serum free Eagle’s spinner no. 2 medium. The cells that were -not detached were vigorously rinsed to remove the remaining metaphase cells and the interphase cells were then detached by gently rolling 2 mm glass beads in the bottle. Use of trypsin, which invariably leads to altered protein patterns, was avoided at all stages of cell handling.

Nucleoprotein preparation A procedure for crude chromatin preparation utilizing a minimum number of steps and low ionic strength was used. This avoided possible alteration of proteins by proteolysis at the expense of having greater than normal non-histone proteins. It was found in control experiments that these proteins neither interfered with histone mobility on electrophoresis nor coelectrophoresed with the histone bands being studied. All steps were at 02°C. Washed cells were swollen for 10 min in hvnotonic buffer (HB) consisting of 5 mM NaCl, 015 mM M&l,, 5 mM Tris-HCI. aH 7.6. Swollen cells were gently-homogenized with an all glass Dounce homogenizer until the nuclei were free of cytoplasm. Nuclear fractions were pelleted by centrifugation at 2 500 g for 10 min. Under these conditions metaphase chromosome morphology is disrupted, but the recovery of DNA in the 2 500 g pellet is approx. 90 % from both metaphase and interphase cells. After two successive washes in hypotonic buffer with centrifugation at 10000 g the crude nucleoprotein was either extracted immediately or washed twice in 20 vol of 0.35 M NaCl in 10 mM Tris-HCI. 1 mM Na, ethylene diaminetetraacetate (EDTA); pH 7.6.

Extraction of chromosomalproteins Nucleoprotein was dispersed in a volume of deionized water to give 10 OD,,, units. An equal volume of 0.5 N HCl was added to the stirred mixture. After 30 min the extract was separated from the residue by centrifugation at 12000 g in the Sorvall SS-34 head. A second extraction of 30 min was repeated with the same volume of 0.25 N HCI. Acid-soluble proteins (ASP) were precipitated from the combined supernatants by addition of 50 % trichloroacetic acid (TCA) until the final concentration was 1.1 M (18 %). After standing 2 h at 0°C the precipitate was collected by centrifugation and washed once in acetone containing 0.5 % HCl, twice in acetone and then air dried. For electrophoresis on polyacrylamide gels, the sample was dissolved in a loading buffer of 0.9 N acetic acid, 6.25 M urea and 0.5 M p-mercaptoethanol (HUM). Not more than 100 pg of ASP in 50 I were loaded onto 0.6 x 10 cm lo-15 % gels and electrophoresed according to Panyim & Chalkley [23]. Densitometer tracings of Amido-Schwartz stained gels were done in 30 % Exptl

Cell Res 73 (1972)

methanol 7.5 % acetic acid in a Gilford 2 400 spectrophotometer at 615 nm. Chart and scan speeds were not the same for each experiment but were uniform for comparison between gels in each experiment.

Treatment with alkaline phosphatase Acid-soluble protein samples in HUM were precipitated with 20 vol cold acid-acetone and washed twice with acetone to remove residual urea. The precipitate was redissolved in 0.01 M HCl and treated with l/5 (w/w) an amount of alkaline phosphatase (E.C. 3.1.3.1, Worthington Biochem., Freehold, NJ. BAPC) in 0.1 M Tris, pH 8.0 at 37°C for 4-6 h. The reaction was stopped by precipitation of total proteins in 10 vol acetone at -20°C overnight.

Salt extraction of histones Washed chromatin from metaphase or interphase cells was divided into 5 equal aliquots. Each was stirred at 0°C with 5 vol. NaCl in 10 mM Tris, 1 mM EDTA (pH 7.6) at the indicated NaCl molaritv for 1 h. Aggregated chromatin was pelleted at 2 500 g for 15 min and a wash in each salt solution reneated. Proteins from the combined supernatants were precipitated at 1.1 M TCA and prepared for electrophoresis as described above.

RESULTS For orientation, fig. 1 shows the electrophoretic profile of Chinese hamster cell histones obtained by 0.25 N HCl extraction of nucleohistone. The nucleohistone was washed with 0.35 M NaCl during its preparation to remove non-histone proteins [12]. With the exception of protein X, whose appearance is variable, these bands represent undegraded Chinese hamster histones which maintain a constant mass ratio, one to another, irrespective of the chromatin isolation technique employed. We observe a minor component F lc [19, 241 which has the same properties as F 1, i.e. extracted by 5 % TCA and unique color when stained with Amido-Schwartz. For the following series of experiments, attention was restricted to the F 1 region of the gel between X and F3. The initial observation which prompted a close examination of chromosomal protein modifications at metaphase was that when

Lysine-rich histones at metaphase metaphase chromosomes were isolated at pH 3 [27] there was a deficiency in the lysinerich histone. Since Murray [21] has shown that chromatographically separated histones I, and I, are not extracted from calf thymus chromatin above pH 2.60, it was expected that at pH 3.0 the F 1 should not be extracted. Acid-soluble proteins from a variety of Chinese hamster interphase and metaphase chromosomes prepared at neutral and acid pH were therefore examined to determine the basis for this unexpected loss of the Fl histone. Four equivalent aliquots of metaphase and interphase chromatin were resuspended in 20 vol of either pH 3 buffer or 0.01 Tris-HCI, 1 mM EDTA, pH 7.6 at 0°C. The suspensions were intermittently shaken on a vortex mixer

115

INTERPHASE

-

pH 7.6

METAPHASE F; la2 F; !b

X CD

FI

FIC -0

F3

Fig. 2. Electrophoreticprofile in the Fl region of

F3 F2al

acid-solubleproteinsof Chinesehamstermetaphase and interphasechromatin. Proteinswere extracted after washingthe chromatinwith either pH 3 buffer [27] or 0.01 M Tris-HCI, 1 mM EDTA, pH 7.6. In this and subsequent profilesa referenceline is drawn through the positionof the interphasepeak.

FI

for 1 min and centrifuged at 20 000 g for 15 min. Proteins were then extracted from each pellet. By loading the gels with ASP extracted from nearly equivalent amounts (on a DNA basis) of each chromatin it is Fig. 1. Electrophoreticprofileof acid-soluble proteins possible to get some idea of the relative from 0.35 M NaCl washedChinesehamstercell amounts of Fl remaining bound in the nucleoprotein. ExptI Cell Res 73 (1972)

116 R. S. Lake et al.

Fig. 3. Electropherogram of the proteins recovered from salt washings of Chinese hamster cell metaphase and interphase chromatin.

Exptl Cell Res 73 (1972)

Lysine-rich histones at metaphase chromatins. It is seen from fig. 2 that exposure to pH 3 buffer [27] completely extracts F 1 from metaphase chromatin but not as extensively from interphase chromatin. Also, there is a heterogeneity in the Fl peak such that pH 3 buffer extracts the trailing F 1 components from the interphase chromatin. The FI peak from metaphase chromatin is greatly enriched in these slower migrating subcomponents. Also noteworthy is the fact that total recovery of Fl from metaphase chromatin is reduced even when the wash is done at pH 7.6. This indicated that loss of lysine-rich histone is not a function of pH alone, but also of some state of the histone peculiar to arrested-metaphase chromatin. Salt extraction of Fl Loss of the Fl of metaphase chromatin at low pH should also occur during other treatBefore Alk. PO, 0%

After

Before Alk PO4 ase

After

I FI

I X 0

FIC

I F3

-0

Fig. 4. Comparison of the effect of alkaline phosphatase (Alk. PO,ase) on the electrophoretic profile of Chinese hamster cell metaphase and interphase acid-soluble proteins.

x

FI

FIC

117

F3

O--o

Fig. 5. Electrophoretic profile in the Fl region of non-arrested metaphase and interphase acid-soluble proteins from Chinese hamster cells.

ments which dissociate protein from DNA. To test this, equivalent sized aliquots of metaphase and interphase chromatin were extracted twice for 1 h each at 0°C with NaCl concentrations of 0.3 to 0.45 M. F l-h&tone begins to be extracted from calf-thymus chromatin at 0.4-0.5 M NaCl [22]. Shown in the electropherograms of fig. 3 are the proteins of supernatant fluids from such salt extractions. DNA was not detected in these supernatants spectrophotometrically or by direct assay with the diphenylamine test. In contrast to the interphase histones, Fl, Flc, and small amounts of F3 are seen to extract readily at 0.4 M NaCl and 0.45 M NaCl from metaphase chromatin. Apparent also, is an enrichment in the slower migrating subcomponent of the Fl band from metaphase chromatin. Treatment with alkaline phosphatase To verify that the observed mobility shift in Fl was due to phosphorylation, samples of metaphase and interphase ASP were treated Exptl Cell Res 73 (1972)

118 R. S. Lake et al. early G 1 cells contaminate such preparations, but their combined contribution is no more than 30 %. Fig. 5 shows tracings of the F lregion of gels from non-arrested metaphase (84 % mitotic, 65 % metaphase) in comparison with the interphase counterpart from the same cell population. A similar mobility shift is seen.

Interphase

Independence of time of arrest

Fl

FIC

o--o

Fig. 6. Influence of vinblastine metaphase-arrest time on the mobility shift in the Fl peak. The interphase chromatin proteins were prepared from the 7 h arrest Chinese hamster cell population. Gels were 15 % acrylamide and run 8 h at 2 mA/gel. Note that on 15 % gels and long running times that the Fl peak is beginning to separate into two components.

with alkaline phosphatase [3, 281. Enzymatic removal of phosphate should return the slower migrating components of Fl to a higher mobility. Densitometer tracings of fig. 4 show that this is the case; metaphase Fl and the phosphoryl-rich interphase components of Fl shift to a faster mobility after enzyme treatment. Shifts in F3 mobility are also noted, but are difficult to quantitate with assurance.

To further rule out the possibility that the altered Fl is due to prolonged arrest, the shift in mobility of the Fl histone was examined at various elapsed times of vinblastine-metaphase arrest. If the slower migrating component of the F 1 peak were being generated as an artifact of arrest, an enrichment at longer arrest times would be expected. Fig. 6 shows patterns obtained from cells arrested in metaphase for 1, 3, 5 and 7h. The overall shape of the Fl peak seems independent of arrest time. Reversal upon removal of Colcemid

Metaphase and interphase cells which had been treated with Colcemid for 4 h were split

Occurrence in non-arrested metaphases

To exclude the possibility that protein alteration at metaphase is due to artifacts arising during and because of the arrest period, it was necessary to examine non-arrested metaphases for similar properties. Non-arrested metaphase cells were obtained by selective detachment. Excess-thymidine synchronized Chinese hamster cells were collected during half-hour intervals of the mitotic peak and pooled. Prophase, anaphase, telophase and Exptl CeN Res 73 (1972)

METAPHASE RELEASE METAPHASE INTERPHASE

_

I--x

I FI @

I F3

FIG 0

Fig. 7. Acid-soluble protein electrophoretic profile of Colcemid-arrested metaphase chromatin and cbromatin

obtained

from

from Colcemid arrest.

cells

1.0 h after

release

Lysine-rich

histones at metaphase

119

HELA-S3

RAT NEPHROMA

Mefophose

8. Electrophoretic profile in the Fl region of rat nephroma and HeLa-S3 acid-soluble proteins of metaphase and interphase chromatin.

Fig.

Interphase $4

x

FI

o--------o

into two aliquots. After being washed twice at room temperature in Tricine Eagle medium with or without Colcemid, they were seeded in plastic flasks in a humidified 37°C incubator. When all the metaphase cells incubated in medium without Colcemid had entered Cl as monitored by phase-microscopy (1.0 h), all cells were chilled and gently removed with glass beads and crude chromatin prepared. Fig. 7 shows that the slower migrating phosphorylated components of F 1 shift back to the interphase mobility within 1.0 h of reversal of metaphase arrest. It is difficult to determine how quickly dephosphorylation occurs after metaphase due to the limited degree of synchrony with which Colcemid-arrested cells enter G 1. Occurrence in other cell types

Two additional cell lines have been examined for Fl changes. Metaphase HeLa-S3 and

F3

rat nephroma [2] cells were prepared by selective detachment as with Chinese hamster cells. Although different, these cell types also exhibited a mobility shift in electrophoretitally separated Fl (fig. 8). This strongly suggests that the phenomenon is an event of general occurrence in all cell types at metaphase. DISCUSSION In the present study, a consistent change has been observed to occur as cultured cells enter metaphase. The lysine-rich histone Fl shows a slower electrophoretic mobility upon acrylamide gel electrophoresis which can be attributed to phosphorylation. How phosphorylation affects a shift of Fl to slower mobility is not certain [3, 251, but it is probably due to an increase in the molecule’s net negative charge. The reproducibility of Exptl

Cell Res 73 (1972)

120 R. S. Lake et al. this mobility shift has encouraged us to use it for initial characterization of F 1 changes at metaphase in Chinese hamster cells under the various conditions described. From the data presented we have established that: (a) the mobility shift is due to phosphorylation since it is reversed by alkaline phosphatase treatment; (b) F 1-phosphorylation occurs naturally at metaphase and is not due to an artifact of metaphase-arrest with either vinblastine or Colcemid; (c) in the presence of a metaphase arresting agent the degree of phosphorylation is independent of the elapsed time of arrest; (d) metaphase phosphorylation occurs in various cell types; (e) phosphoryl-rich F 1 is rapidly dephosphorylated as the cell enters G 1. The most pertinent question to raise is whether the F 1 molecules are transiently phosphorylated during mitosis because of participation in coarse genetic control and the super-folding of metaphase chromatin or whether this covalent group substitution is part of an unrelated cellular event. As suggested from the previous work of Littau [16], Johns & Forrester [13] and Jensen & Chalkley [lo], F 1-histone has an active influence on chromatin condensation in vitro by crosslinking under appropriate ionic conditions. On the other hand, histone phosphorylation is pictured as being associated with increased transcription and replication and as decreasing the binding of histone to DNA [6]. From this, it would be expected that during mitosis, when transcription is stopped and the chromatin is condensed, F 1 would exist in its dephosphorylated form. As shown in the present experiments, the opposite situation exists; F 1 is highly phosphorylated and tenuously associated with the DNA. This paradox points up the difficulty arising from examining simple bilateral interaction between protein and DNA. Exptl

Cell Res 73 (1972)

The weak forces involved in the regulation of chromosome superstructure and movements at mitosis are surely a collection of multilateral interactions between DNA, protein, RNA and lipid, where the dominant forces may be protein-protein interactions. Thus, despite the great amount of information available from model systems [6], it is yet difficult to predict which if any of the unique properties of the mitotic cell is attributable to Fl modification. Recent findings of tissue specific patterns of F 1-phosphorylation [l 1, 25, 281 are in accord with the possibility that metaphase F l-phosphorylation is a general case of the more specific process of gene repression in differentiated tissues, the metaphase cell being representative of a completely repressed genome [14]. Reversibility of F 1-phosphorylation is of particular interest. In the typical cell, F 1 exists as 3 to 5 chromatographic subcomponents [4, 151 in which the level of phosphoryl groups is maintained in a tissue and species specific manner by a cyclic 3’,5’-adenosine monophosphate sensitive histone kinase and a counteracting phosphatase activity [20]. Evidence from the present experiments indicates that, once phosphorylated, metaphase Fl is not dephosphorylated until the cell is allowed to progress into G 1. As was shown, the enhanced phosphorylation cannot be due to the direct action of vinblastine or Colcemid on these enzymatic activities since interphase cells do not show phosphorylated F 1. Rather, this is a unique case where metaphase-arrest coincides with arrest of a phosphatase activity. In addition to a possible role in mitotic cycle events, the observed metaphase phosphorylation explains why F 1 is readily lost during isolation of metaphase chromosomes [9, 261. The phosphorylated slowly-migrating subcomponents are more readily extracted

Lysine-rich

at low pH and moderately high salt (0.350.4 M). This observation is contradistinct from observations by Marushige et al. [18] which showed that salt or detergent dissociation of trout testis histones from DNA was uninfluenced by phosphorylation. This discrepancy from the present results could be reconciled if metaphase phosphorylation were either multiple or at a unique site in the F I-histone molecule. The burden of future experiments is to determine in what ways metaphase-F 1 phosphorylation is unique. REFERENCES 1. Adler, A J, Schaffhausen, B, Langan, T A & Fasman, G D, Biochemistry 10 (1971) 908. 2. Babcock, V I & Southam, C M, Proc sot exptl biol med 124 (1967) 217. 3. Balhorn, R, Rieke, W 0 & Chalkley, R, Biochemistry 10 (1971) 3952. 4. Bustin, M & Cole, R D, J biol them 244 (1969) 5286. 5. Comings, D E, J cell biol 35 (1967) 699. 6. DeLange, R J & Smith, E L, Ann rev biochem 40 (1971) 279. 7. Elkind, M M, Kano, E & Sutton-Gilbert, H, J cell biol 42 (1969) 366. 8. Hancock, R, J mol biol 40 (1969) 457. 9. Huberman, J A & Attardi, G, J cell biol 31 (1966) 95.

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10. Jensen, R H & Chalkley, R, Biochemistry 7 (1968) 4388. 11. Jergil, B, Sung, M & Dixon, G H, J biol them 245 (1970) 5867. 12. Johns, E W & Forrester, S, Eur j biochem 8 (1969) 547. 13. - Biochem j 111 (1969) 371. 14. Johnston, T C & Holland, J J, J cell biol 27 (1965) 565. 15. Kinkade. J M & Cole. R D. J biol them 241 (1966) 5790. ’ ’ 16. Littau, V C, Burdick, C J, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 54 (1965) 1204. 17. Maio, J J & Schildkraut, C L, J mol biol 24 (1967) 29. 18. Marushige, K, Ling, V & Dixon, G H, J biol them 244 (1969) 5953. 19. Marzluff, W F’ & McCarty, K S, J biol them 245 (1970) 5635. 20. Meisler, M H & Langan, T A, J biol them 244 (1969) 4961. 21. Murray, K, J mol biol 15 (1966) 409. 22. Ohlenbusch, H H, Olivera, B M, Tuan, D Davidson, N, J mol biol 25 (1967) 299. 23. Panyim, S & Chalkley, R, Arch biochem biophys 130 (1969) 337. 24. - Biochem biophys res commun 37 (1969) 1942. 25. Panyim, S, Bilek, D & Chalkley, R, J biol them 246 (1971) 4206. 26. Sadgopal, A & Bonner, J, Biochim biophys acta 207 (1970) 227. 27. Salzman, N P, Moore, D E & Mendelsohn, J, Proc natl acad sci US 56 (1966) 1449. 28. Sherod, D, Johnston, G & Chalkley, R, Biochemistry 9 (1970) 461 I. Received October 5, 1971 Revised version received December 6, 1971

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