THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 258, No. 7, Issue of April 10, pp. 45344538, 1983 Printed in U.S.A.

Histone H1 and H3 Phosphorylation during Premature Chromosome Condensation in a Temperature-sensitive Mutant (tsBN2) of Babv Hamster Kidney Cells*

- "

(Received for publication, September 3, 1982)

Kozo Ajiro, Takeharu NishimotoS, and Taijo Takahashi From the Aichi Cancer Center, Research Institute. Laboratory of Cell Biology and Laboratory of Biochemistry, Chikusa-ku, Nagoya 464, Japan

The histone phosphorylations of temperature-sensi- tive mutant cells (tsBN2) were investigated during the induction of premature chromosome condensation (PCC). At the permissive temperature (33.5 "C), the histones of the cells were phosphorylated typically as in any other mammalian cell. However, at the nonper- missive temperature (40.5 "C), both histone H1 and H3 were phosphorylated extensively as in mitotic cells, and the increase in these phosphorylations throughout S to Gz phase was closely correlated to the frequency of cells showing PCC. The pattern of H1 subtype phos- phorylations was quite similar, and the sites of H1 phosphorylation from PCC were the same as those from mitotic cells. Although the degree of phosphorylation was low, H1 and H3 phosphorylations were observed even in GI phase at the nonpermissive temperature. The effects of metabolic inhibitors on the induction of PCC were parallel in H1 and H3 phosphorylations; actinomycin D failed to inhibit either PCC induction or these phosphorylations, whereas cyclohexamide did, completely inhibiting H3 phosphorylation.

The phosphorylation of histone H1 is known to be very dynamic during the cell cycle and is maximum in mitosis (1- 6). The phosphorylation of H3' was observed specifically only in mitosis (2, 7, 8). H1 phosphorylation has been reported to control the initiation step of mitosis through chromosome condensation (3, 9, 10). Gurley et al. (11) suggested that H ~ M and H3 phosphorylation occurred during the final organiza- tion and maintenance of chromosomes because of the quan- titative agreement between the phosphorylated histone data and chromatin morphological data. However, a causal rela- tionship between histone phosphorylation and mitotic-chro- mosome condensation has not been established.

TsBN2 cell, a temperature-sensitive mutant of BHK21/13, shows PCC at high temperature (12). Recently, we examined the mechanism of PCC induction in tsBN2 at the microscop- ical level (13). The PCC observed in tsBN2 at high tempera- ture was found to be the same as the PCC induced in inter- phase cells by fusion with mitotic cells (14). TsBN2 cells

* This work was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture, and by The Ishida Foundation 57-244, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Present address, Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan.

The abbreviations used are: HI and H3, histone 1 and histone 3; PCC, premature chromosome condensation; HU, hydroxyurea.

showed PCC even in the absence of DNA synthesis. The PCC induction was blocked by cyclohexamide but not by actino- mycin D (13). Thus, it was considered necessary to study H1 and H3 together during PCC induction in tsBN2 and to compare with mitotic cells, because H3 phosphorylation was mitosis specific and could be a more clear index of chromatin condensation. Also, more extensive studies with the subtypes and sites of H1 phosphorylation (5, 6 ) may facilitate analysis of the relationship between PCC and mitosis.

MATERIALS AND METHODS

Cell Culture and Synchronization-BHK21/13, a continuous line of Syrian hamster fibroblasts, and tsBN2, a temperature-sensitive mutant derived from BHK21/13, were used. Cells were maintained in Dulbecco's modified Eagle's medium containing 10% calf serum at 33.5 "C. TD buffer (Tris-buffer saline minus CaZ+ and Mg2+ by Kimura et al. (15)) was used to wash the cells. As the nonpermissive temper- ature of tsBN2,40.5 "C was used. Cells were synchronized at GI and the G1/S boundary as described previously (16).

Mitotic and PCC Index-Growing cells were treated with 0.3 pg/ ml of colcemid for the indicated periods, and the mitotic cells were counted as described (16). For the PCC index, cultures of tsBN2 were incubated for the indicated periods at 40.5 "C and then whole cells, including floating cells, were collected by centrifugation after trypsin- king cells and then futed with Carnoy's solution.

32P Labeling-Cells grown in 15-cm diameter dishes were labeled with [32P]orthophosphate at a concentration of 40-60 pCi/ml in phosphate-free medium (5) either at permissive (33.5 "C) or nonper- missive (40.5 "C) temperature for 3 or 4 h. After washing with cold medium, the cells were harvested with a rubber-coated nodule.

Histone Isolation-Cell pellets (containing approximately 5 X I d cells) were harvested by centrifugation at 600 X g for 5 min, then lysed by washing three times with 10 ml of 80 mM NaC1, 20 mM EDTA, and 1% Triton X-100 containing 0.05 M sodium bisulfite (pH 7.2). Protease inhibitors, phenylmethylsulfonyl fluoride, Ne-p-tosyl- L-lysine chloromethyl ketone, and tosylphenylalanyl chloromethyl ketone in concentrations of 0.1 mM were added throughout the experiment. Sodium bisulfite was added as a phosphate inhibitor. Total and HI histone preparations were by the methods described previously (5). For the analysis of 32P-labeled phosphopeptides by TLC plate, 32P-labeled H1 was further purified by CG-50 ion exchange column chromatography as described (6).

Gel Electrophoresis-Samples containing 30 pg of histones were resolved by electrophoresis on 30-cm long 12% polyacrylamide slab gels containing 7.5 M urea, 5% acetic acid, and 6 m~ Triton X-100 (17, 18). For the analysis of H I subtypes, 3-4 pg of proteins were resolved by electrophoresis at 60 V for 72 h on slab gels containing 12% acrylamide and 1% sodium dodecyl sulfate (19, 20) in a Hoeffer SE- 500 apparatus. The other procedures, gel staining and scanning, were described earlier (18).

Resolution of Phosphopeptides by Electrophoresis and Chroma- tography on Cellulose TLC Plates-The trypsinization method and the tryptic mapping of phosphopeptides of HI were presented in detail in an earlier study (6). However, a glass plate (20 X 20 cm) with a 250-pm coating of cellulose (D-3354 Dassel, Schleicher and Schull, Inc.) was used in the present procedure.

Resolution in the first dimension was at 500 V for 50 min in a

4534

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Chromatin Structure 4535

chamber that permitted simultaneous electrophoresis of two TLC plates under Isoper (Chukyo-yushi Co.).

Autoradiography-For detection of "*P-labeled phosphorylated histones and HI phosphopeptides, gels were dried on Whatman 3MM paper, and TLC plates containing tryptic maps of H1 were exposed to x-ray film (DuPont Cornex 4). The film was developed in a Kodak RP-OMAT automatic x-ray film developer.

RESULTS

Analysis of Acid-soluble Nuclear Protein to tsBN2"Elec- trophoretic patterns of the proteins were the same in both extracts from permissive (33.5 "C) and nonpermissive (40.5 "C) cultures (Fig. 1, a and b); H3 has three distinct variants (H3.1, H3.2, and H3.3) resolved in this gel system (17). Since H3.1 has a rather low electrophoretic mobility, only H3.2 and H3.3 were followed in later experiments.

By autoradiographic analysis, the two kinds of histones (H2A and H1) were principally phosphorylated throughout the cell cycle at 33.E 'C (Fig. la). A traced 32P labeling observed in the gel region between H3 and H4 was possibly phosphorylated H4. The gel electrophoresis revealed a little gap between the protein and its '"P autoradiography, because

a b

H2A.1 e i .2

H3.1 *

H3.i c

.L

H2E

H4 0

A P FIG. 1. Analysis of acid-soluble nuclear protein from expo-

nentially growing tsBN2 cells resolved in 5% acetic acid, 7.5 m urea, 6 m~ Triton X-100-containing polyacrylamide gel elec- trophoresis. Half of the exponentially growing cultures of tsBN2 at 33.5 "C were shifted to 40.5 "C and labeled by '"P from the 1st to the 4th h after the temperature shift. As a control, the other half were labeled by =P for 4 h at 33.5 "C. Thirty pg of histone from these cells was loaded on slab gels and run for 48 h at 150 V. Protein was stained with 0.2% Amido black. a, permissive temperature, 33.5 "C; b, non- permissive temperature, 40.5 "C. A, Amido black-staining proteins, P, "P-labeled autoradioeranhv.

phosphorylated histone molecules have lower electrophoretic mobilities.

At 40.5 "C (Fig. lb), H3 was phosphorylated and H1 was highly so. H3 phosphorylation was observed to be specific in the mitotic phase at the permissive temperature. In this experiment, PCC was observed in 30% of the cells cultured (data not shown).

Phosphorylation of Histone HI and H3 during PCC In- duction and Cell Cycle Progression-Phosphorylation of his- tone H1 and H3 was followed during the cell cycle progression from S to M phase and compared with the pattern of histone H1 and H3 phosphorylation during PCC induction in tsBN2 (Fig. 2).

The progression of the cell cycle was followed by cytofluo- rography (data not shown) and the mitotic and PCC index (Table I). PCC induction was determined microscopically by counting the number of cells showing PCC (Table I).

At 33.5 "C, the incorporation of :12P into H1 was very low and that into H3 was not observed in GI phase (Fig. 2a). Histone H1 started to be phosphorylated during S phase, and the level increased from Gz to M phase (Fig. 2,b-d). However, H3 was phosphorylated only in M phase (Fig. 2d). These patterns of H1 and H3 phosphorylation in the tsBN2 cell were consistent with those of S and M of wild BHK21/13 (data not shown).

At 40.5 "C, histone H1 was heavily phosphorylated in all observed cases (Fig. 2, e-h). Histone H3 was phosphorylated even at 3 h after the shift-up (Fig. 2g). This is consistent with the time at which PCC was first observed microscopically (Table I). This phosphorylation of histone H1 and H3 at 40.5 "C, which was observed only in the mitotic phase at 33.5 "C as described above, would not be caused by the rapid progression of cell cycle due to the high temperature, because a high level of H1 and H3 phosphorylation was observed at 40.5 "C even in the presence of HU (Fig. 2e), which was a strong inhibitor of DNA synthesis. H3 phosphorylation in the presence of HU was not observed in tsBN2 at 33.5 "C as described later (Fig. 6a) nor in BHK21/13 at 40.5 "C (data not shown), so the HU itself has no effect on H3 phosphoryl- ation. Thus, the phosphorylations of H1 and H3, like those in

H1

H 3

a b C d

e f a h

li A P

FIG. 2. Histone H1 and H3 phosphorylation of tsBN2 cells under permissive (a-d) and nonpermissive temperatures (e- h). Culture of tsBN2 was synchronized at the G d S boundary by the deprivation of isoleucine and HU treatment as described previously (16). At 33.5 "C, cells were labeled by ''lP for 4 h as follows: a, GI; b, 04th h; c, 4-8th h; d, 8-12th h. At 40.5 "C, cells were labeled by 32P for 3 h as foUows: e, 2-5th h in the presence of HU; f, 0-3rd h; g, 3-6th h, and h, 6-9th h after HU release and temperature shift. Mitotic and PCC indexes of these cells are shown in Table I. The figure shows only the H1 and H3 region of gels. Each panel shows both a stained image of a gel as well as i ts autoradiogram. A, Amido black-staining proteins, P, '*P-labeled autoradiography.

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4536 Chromatin Structure

TABLE I Mitotic and PCC indexes of tsBN2 cells

Cultures of tsBN2 were synchronized at the GI/S boundary by HU treatment as described previously (16). The periods of 0-4,4-8, and 8-12 h after HU release corresponded approximately to S, G2, and M phase of cell cycle, respectively, by cytofluorography (12). A series of cultures was shifted up to 40.5 "C immediately after release from the HU block. Then at the indicated time, whole cells were collected and the frequency of cells showing PCC was counted. At 33.5 "C, cultures were treated with 0.3 pg/ml of colcemide during the indicated period following the release from the HU block and mitotic index was counted as described under "Materials and Methods." As the reference, one of the cultures (HU) was shifted to 40.5 "C without the release from the HU block, and whole cells were collected 5 h later.

Mitotic index (33.5 "C) PCC index (40.5 "C)

Hours after HU release 0-4 (SI 4-8 (G2) 8-12 (M) 0 (HU) 3 6 9 Number of cells showing 1 23 218 217 10 195 214

Number of total counted 617 572 788 492 616 525 605

Per cent 0.2 4.0 27.7 44.1 1.6 37.1 35.4

mitosis or PCC

cells -

mitotic cells, could occur at high temperature in tsBN2 with- out the concomitant DNA synthesis.

Phosphorylation Pattern of HI Subtypes a n d Phosphopep- tides-As noted above, H3 was phosphorylated only in the mitotic phase and PCC of tsBN2, but H1 was phosphorylated in both interphase and M phase, and also in tsBN2 showing PCC. Therefore, we checked for a possible difference in the H1 phosphorylation pattern between the normal progression of cell cycle and the PCC induction. H1 was isolated from the above sample (Fig. 2, a-h) and resolved by sodium dodecyl sulfate-gel electrophoresis into four subtypes (Hla, Hlb, Hlc, and Hld in Fig. 3). The pattern of H1 subtypes was the same at both permissive and nonpermissive temperatures (Fig. 3). The incorporation of '"'P into each subtype was then examined by autoradiography. As shown in Fig. 3, the incorporation of :'*P into H1 subtypes was negligible in GI (Fig. 3a) and clearly increased from S to M phase (Fig. 3, b-d). At 40.5 "C (Fig. 3, e-h), the level of H1 subtype phosphorylation was quite high in all cases including HU-treated cells (Fig. 3e), just as with those in mitotic phase at 33.5 "C (Fig. 3d).

T o examine more closely the phosphorylation of H1 during the induction of PCC, the sites of H1 phosphorylation in PCC were analyzed and compared with those in M phase of the normal cell cycle. H1 was extracted from cells labeled by "'P during M phase a t 33.5 "C and during the PCC induction a t 40.5 "C and purified by chromatography on Amberlite CG-50 resin. The incorporation of "'P per pg of H1 eluted from the column was approximately 19.4 cpm for S phase, 72.5 cpm for PCC, and 85.4 cpm for M phase. The difference in :'lP incor- poration between PCC and M was less than 15% of M. The [:"P]phosphopeptide pattern was then investigated by TLC (Fig. 4). PCC and M samples evidenced no difference in the number and position of the "'P autoradiograph of the spots. The only significant difference was the lower intensity of spot 2 observed in PCC.

HI a n d H 3 Phosphorylation in GI Phase-Although PCC could not be observed microscopically in GI phase a t 40.5 "C, both RNA and protein synthesis in GI phase was found to be severely inhibited at 40.5 "C in tsBN2, suggesting that similar changes might occur even in the GI phase cells a t 40.5 "C (13). Thus, H1 and H3 phosphorylation was investigated at both permissive and nonpermissive temperatures.

In GI phase at the normal growth condition, only a very small amount of '"'P incorporation into H1 was observed (Fig. 5a) but no H3 phosphorylation. At 40.5 "C, however, H1 was clearly phosphorylated and the H3 phosphorylation was ob- vious (Fig. 56). The bands on x-ray frlm were scanned to make the difference more evident in the temperature at 33.5 "C and 40.5 "C.

Effect of Metabolic Inhibitor on HI a n d H3 Phosphoryla-

d. c - .. a'

H1 b-

a

e

b C

i 9

d

h

C P FIG. 3. Phosphorylation patterns of H1 subtypes from the

same tsBN2 sample shown in Fig. 2 a-h. Histone HI (3 fig) was resolved in sodium dodecyl sulfate-gel electrophoresis. The proteins were stained with 0.28 Coomassie blue. After destaining, the gels were dried and autoradiographed for 5 days. C, Coomassie blue staining, P, "YP-labeling autoradiography.

tion during PCC Induction-Previously, we reported that PCC induction was inhibited by cyclohexamide, but not by actinomycin D (13). T o c o n f m this, the H1 and H3 phospho- rylation was investigated in the presence of these metabolic inhibitors.

As the control, one culture was labeled at 33.5 "C in the presence of HU. The incorporation of "'P into histone H1 was low and no H3 phosphorylation was observed (Fig. 6a). A series of cultures was shifted up to 40.5 "C without the release from HU block. An extensive H1 and H3 phosphorylation was observed in the presence of HU (Fig. 66). A similar phospho- rylation pattern appeared in the presence of 2 pg/ml of acti- nomycin D (Fig. 6c), although the extent of phosphorylation was lower than in its absence. Treatment with 10 pg/ml of cyclohexamide, however, reduced the extent of H1 phospho- rylation considerably and no H3 phosphorylation was ob- served (Fig. 6d). These data are consistent with the previous results in which the frequency of PCC was microscopically determined (13).

DISCUSSION

The phosphorylation of histones in tsBN2 cells under the permissive temperature (33.5 "C) was consistent with those in other studies on different kinds of mammalian cells (2, 7, 8).

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Chromatin Structure 4537

w A::.

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FIG. 4. Autoradiographs of '*P-labeled tryptic phosphopeptides of H1 from PCC and mitotic cells. Cultures of tsBN2 were synchronized at the GI/S boundary as described previously (16). The cultures were then shifted to 40.5 "C in the presence of HU and labeled from the 1st to the 4th h after the temperature shift (PCC sample). Other cultures were incubated at 33.5 "C in normal medium after the release from the HU treatment and then labeled by 32P from the 7th to the 12th h (M phase sample) after the HU release. To accumulate mitotic cells, 0.3 pg/ml of colcemid was added. H1 was extracted and purified by chromatography on Amberlite CG-50 resin (5). Fifty pg of H1 from PCC (-3400 cpm) and 50 pg of HI from M phase (-3500 cpm) cells were digested with 2% trypsin, and then resolved by electrophoresis and chromatography on TLC plates (6).

Under the nonpermissive temperature (40.5 "C), however, these typical cell-cycle specific patterns of histone phospho- rylation seemed to be converted to those of the mitotic state. There was a strong correlation between the increasing H1 and H3 phosphorylation (Fig. 2) and the PCC index (Table I), the degree of PCC induced during the cell-cycle progression from S to GP phase (13), and the PCC rate in the presence of three metabolic inhibitors (HU, actinomycin D, and cyclohexamide) (13). In all observed cases of PCC and mitosis, H1 and H3 were coincidentally phosphorylated. The sites of H1 phospho- rylation in PCC and M were the same in the TLC peptide map. These results strongly suggested that the same protein kinase was active during the induction of PCC, although it could not be confirmed whether the site of H3 phosphoryla- tion is the same as that of mitosis.

The previous results indicated that RNA and protein syn- thesis were rapidly inhibited when a tsBN2 culture was shifted to a high temperature in GI phase (13). These results suggest that, even in GI cells, a low level of chromatin condensation, which could not be identified as a morphological change in nuclei, might take place at a high temperature.

We demonstrated that H1 and H3 phosphorylation clearly

occurred at the nonpermissive temperature at any given time in interphase cells. The induction of PCC occurred with no relation to the mitotic chromosome structure in the normal cell cycle. Thus, the approach of Gurley et al. (11) is not restricted only to normal mitotic condensation. However, it is not yet known whether these histone phosphorylations are a driving force or only a trigger for the PCC formation.

A new RNA synthesis may not be required for the histone phosphorylation. However, protein synthesis seemed to be required for it. The data were consistent with those of Krystal and Poccia (21), who suggested that some protein factor is needed for PCC formation of sperm pronuclei in addition to a high level of H1 phosphorylation. In the presence of cyclo- hexamide, H1 phosphorylation was only slightly inhibited, and there was no phosphorylation of histone H3. These data suggested that 1) the H3 phosphorylation could be more specifically involved in both PCC and mitotic chromosome condensations than those of HI, and 2) the chromatin con- densation in both PCC and M occurred by quite a similar mechanism due to one or another factor related directly or indirectly to protein kinase activity and to histone H1 and H3 phosphorylation.

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4538 Chromatin Structure Hl H3

I I

a

b

I I FIG. 5. Histone H1 and H3 phosphorylation from tsBN2 at

GI phase under the permissive (a) and nonpermissive (6) tem- peratures. Cultures of tsBN2 were synchronized at G I phase by the low serum and isoleucine deprivation as described previously (16). Following the addition of normal medium, a half series of dishes was shifted up to 40.5 "C and labeled by [''P]orthophosphate for the period from the 1st to the 4th h after the temperature shift. Histones were extracted and resolved in acid/urea/Triton X-100-containing gel as indicated in Fig. 1. Densitometric scan of gels and x-ray autoradi- ograph of H1 and H3 region at 565 nm. - - -, absorbance; -, autoradiography.

Q b c d

H 1

H 3

Ir L

rl; .. 0

A P FIG. 6. Effect of metabolic inhibitor on H1 and H3 phospho-

rylation. A series of cultures were labeled by ['"Plorthophosphate in the presence of HU at 33.5 "C ( a ) or at 40.5 OC ( 6 4 ) as described in the legend to Fig. 2. b, with only HU; c, with HU plus 2 pg/ml of actinomycin D, d, with HU plus 10 p g / d of cycloheximide. A, Amido black staining, P, "'P-labeled autoradiography.

Acknowledgments-We express our thanks to Y. Nishikawa and A. Hayashi for their excellent technical assistance, and to I. Inagaki who assisted in the preparation of this manuscript.

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K Ajiro, T Nishimoto and T Takahashiin a temperature-sensitive mutant (tsBN2) of baby hamster kidney cells.

Histone H1 and H3 phosphorylation during premature chromosome condensation

1983, 258:4534-4538.J. Biol. Chem. 

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