Phosphorylation of nonhistone proteins during premature chromosome condensation in a temperature-sensitive mutant, tsBN2

In tsBN2 cells, a temperature-sensitive (ts) mutant of the BHK21 cell line, with a ts-defect in its regulatory system for chromosome condensation, antigens that react with mitotic specific mouse monoclonal antibody MPM-2 were produced when premature chromosome condensation (PCC) was induced by a temperature shift. The polypeptides of antigens recognized by MPM-2 in tsBN2 cells with PCC were identical to those of antigens in mitotic cells. These antigens appeared concomitantly with chromosome condensation, which suggests that these mitotic-specific antigens may be related to chromosome condensation. As the production of mitotic-specific antigens was inhibited by W-7, a specific and potent antagonist of calmodulin, calmodulin may function in the mitotic phosphorylation of nonhistone protein.     

Chromosomes of G2 arrested cells are easily analyzed by use of the 'tsBN2' mutation

The tsBN2 cell line, a temperature sensitive (ts) mutant of the BHK21/13 cell line (Nishimoto, T. et al. Somatic Cell Genet. 4, 323-340, 1978) has a ts defect in its regulatory mechanism for the initiation of chromosome condensation, the so-called, premature chromosome condensation (PCC) being induced at a nonpermissive temperature (Nishimoto, T. et al. Cell 15, 475-483, 1978, Nishimoto, T. et al. J. Cell Physiol. 109, 299-308, 1981). Using the 'tsBN2' mutation, we analyzed chromosomes of tsBN2 cells arrested in the G2 phase with neocarzinostatin (NCS) and found considerable aberrations, such as gaps, breaks and double minutes-like fragmentation, in addition to a typical G2-chromosome (a long filamental chromosome made up of two chromatids). Our results provide evidences that the tsBN2 cell line can be used for examinations of the chromosomes of cells arrested in the G2 phase.     

Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene.

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.     

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

We cloned the hamster cdc25C cDNA by using the human cdc25C cDNA as a probe and prepared an antibody to Escherichia coli-produced hamster cdc25C protein that is specific to the human cdc25C protein. The microinjected antibody inhibited a chromosome condensation induced by tsBN2 mutation, indicating that the cdc25C protein is required for an activation of p34cdc2 kinase caused by loss of RCC1 function. The hamster cdc25C protein located in the cytoplasm, prominently in a periphery of the nuclei of cells arrested with hydroxyurea, and seemed to move into the nuclei by loss of RCC1 function. Also, we found a molecular shift of the cdc25C protein in cells showing premature chromosome condensation (PCC), in addition to normal mitotic cells. This molecular-shift appeared depending on an activation of p34cdc2 kinase.     

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.     

Chromosome condensation may enhance X-ray-related cell lethality in a temperature-sensitive mutant (tsBN2) of baby hamster kidney cells (BHK21)

In the tsBN2 cell line, which has a temperature-sensitive defect in the regulatory mechanism for chromosome condensation, the lethal effect of X rays was enhanced by incubating the cells at a nonpermissive temperature (40 degrees C) following X irradiation. This enhancement was suppressed in the presence of cycloheximide, which inhibits induction of premature chromosome condensation. The findings obtained in the case of delayed incubation at 40 degrees C and in synchronized cells indicate that X-ray-related potentially lethal damage, which can be expressed by chromosome condensation, is produced in the cells at any stage of the cell cycle, but it is repairable for all cells except those at around the late G2-M phase, where chromosome condensation occurs at a permissive temperature (33.5 degrees C). These observations suggest that the high sensitivity of late G2-M cells to X rays is caused by the events associated with chromosome condensation.     

Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation.

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.     

The induction of chromosome condensation in tsBN2, a temperature-sensitive mutant of BHK21, inhibited by the calmodulin antagonist, W-7

The induction of premature chromosome condensation (PCC) in tsBN2 cells, a temperature-sensitive (ts) mutant of BHK21/13 which shows PCC at the non-permissive temperature, was almost completely inhibited by 40 microM W-7, an antagonist of calmodulin. The mitotic phosphorylation of histone H1 and H3 was also inhibited by W-7. W-5, a chlorine-deficient analogue of W-7 and which interacts weakly with calmodulin, did not inhibit the induction of PCC, even at a dose of 80 microM. The content of calmodulin in tsBN2 cells was increased by a temperature shift to 40.5 degrees C. All these results suggested that calmodulin is required for the chromosome condensation.     

Transformation of temperature-sensitive growth mutant of BHK21 cell line to wild-type phenotype with hamster and mouse DNA

A temperature-sensitive (ts) mutant, tsBN2, which was derived from BHK21 and is defective in the regulatory mechanism for chromosome condensation, was transformed to the temperature-resistant (ts+) phenotype by means of DNA-mediated gene transfer with hamster and mouse DNA. Treatment of mouse DNA with the restriction enzymes EcoRI, HindIII, PstI and SalI, but not with XhoI, almost completely abolished the transforming activity. A fluctuation test, originally devised by Luria and Delbrück, was used to estimate the reversion and transformation frequencies of tsBN2 cultures.     

X-ray-related potentially lethal damage expressed by chromosome condensation and the influence of caffeine

Caffeine has been reported to induce premature chromosome condensation (PCC) in S-phase cells in the presence of an inhibitor of DNA synthesis. We found that when S-phase cells are treated with caffeine and hydroxyurea after X irradiation, substantially more potentially lethal damage (PLD) is expressed, but the addition of cycloheximide, which inhibits PCC induction in S-phase cells, in the presence of caffeine and hydroxyurea reduces the expression of PLD to the same level as seen with caffeine alone. This can be interpreted to mean that the expression of PLD seen with caffeine in the absence of an inhibitor of DNA synthesis is not associated with chromosome condensation. Evidence that PCC induction in S-phase cells and the influence of caffeine on PLD expression were suppressed by incubation at 40 degrees C of tsBN75 cells with a ts defect in ubiquitin-activating enzyme indicates the involvement of ubiquitin in these two processes. These observations as well as previous findings on ubiquitin suggest to us that caffeine induces changes in DNA-chromatin conformation, which are caused by induction of PCC or ubiquitination of chromosomal protein. Such changes occurring postirradiation would favor expression of PLD.     

Specific site of histone H3 phosphorylation related to the maintenance of premature chromosome condensation. Evidence for catalytically induced interchange of the subunits

Previously we have found that histone H1 and H3 of tsBN2 cells showing premature chromosome condensation (PCC) at nonpermissive temperature (40.5 degrees C) were phosphorylated extensively as in mitotic cells (Ajiro, K., Nishimoto, T., and Takahashi, T. (1983) J. Biol. Chem. 258, 4534-4538). Under the influence of various chemicals, both the prevention of the PCC induction and the suppression of H3 phosphorylation occurred simultaneously, whereas H1 phosphorylation did not. At the minimum concentration for the inhibition of PCC induction, H1 phosphorylation remained at the control level, but H3 phosphorylation was completely suppressed. Tryptic peptide analysis revealed that the H3 phosphopeptide in PCC was single, and it was observed in the same position as in mitosis. The results suggest that specific site(s) of H3 phosphorylation related to the maintenance of a condensed state of chromatin.     

Non-apoptotic chromosome condensation induced by stress: delineation of interchromosomal spaces

Chromosomes are known to occupy distinct territories, suggesting the existence of definite borders. Visualization of these borders requires chromatin condensation like that seen in prophase cells. We developed a novel method to induce chromosome condensation in all cells regardless of cell cycle stage using a complex set of stresses. The cells were not apoptotic, as indicated by the absence of DNA damage, maintenance of the intact lamina and scaffold attachment factor A, and by the continuation of metabolic processes as well as proliferative capacity. That the appearance of chromosome condensation did not represent a premature mitotic event was shown by the absence of fibrillarin and Ki67 envelopment of chromosomes, continued protein synthesis and the reversibility of chromosome condensation. That chromosome condensation was achieved was demonstrated by the removal of chromatin from the nuclear envelope and chromosome painting. Specific genetic sites known to be at the surface of chromosomes retained their positions as shown by in situ hybridization. Stress-induced chromosome condensation was used to prove that specific nuclear domains such as ND10 are interchromosomally located and that green fluorescent protein-tagged ND10-associated proteins are useful markers for chromosomal boundaries after adenovirus 5 track formation in vivo. From these observations we conclude that chromosomal territories appear to have boundaries that exclude developing macromolecular aggregates. Received: 26 November 1999 / Accepted: 29 February 2000     

Molecular cloning of a human cDNA encoding a novel protein, DAD1, whose defect causes apoptotic cell death in hamster BHK21 cells.

The tsBN7 cell line, one of the mutant lines temperature sensitive for growth which have been isolated from the BHK21 cell line, was found to die by apoptosis following a shift to the nonpermissive temperature. The induced apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, but not by the bcl-2-encoded protein. By DNA-mediated gene transfer, we cloned a cDNA that complements the tsBN7 mutation. It encodes a novel hydrophobic protein, designated DAD1, which is well conserved (100% identical amino acids between humans and hamsters). By comparing the base sequences of the parental BHK21 and tsBN7 DAD1 cDNAs, we found that the DAD1-encoding gene is mutated in tsBN7 cells. The DAD1 protein disappeared in tsBN7 cells following a shift to the nonpermissive temperature, suggesting that loss of the DAD1 protein triggers apoptosis.     

Histone H1 and H3 phosphorylation during premature chromosome condensation in a temperature-sensitive mutant (tsBN2) of baby hamster kidney cells

The histone phosphorylations of temperature-sensitive mutant cells (tsBN2) were investigated during the induction of premature chromosome condensation (PCC). At the permissive temperature (33.5 degrees C), the histones of the cells were phosphorylated typically as in any other mammalian cell. However, at the nonpermissive temperature (40.5 degrees C), both histone H1 and H3 were phosphorylated extensively as in mitotic cells, and the increase in these phosphorylations throughout S to G2 phase was closely correlated to the frequency of cells showing PCC. The pattern of H1 subtype phosphorylations 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 G1 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.     

Nucleocytoplasmic Recycling of the Nuclear Localization Signal Receptor α Subunit In Vivo Is Dependent on a Nuclear Export Signal, Energy, and RCC1

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The α subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the β subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207–217 or a heterologous nuclear export signal, but not a mutant form of residues 207–217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.     

Relationship between histone phosphorylation and premature chromosome condensation

The relationship between histone phosphorylation and chromosome condensation was investigated by determining changes in phosphorylation levels of histones H1 and H3 following fusion between mitotic and interphase cells and subsequent premature chromosome condensation. We detected significant increases in the levels of phosphorylation of H1 and H3 from interphase chromatin in which a majority of nuclei had undergone premature chromosome condensation. In addition, we noted significant decreases in the phosphate content of the highly phosphorylated mitotic H1 and H3, presumably resulting from phosphatase activity contributed by the interphase component of mitotic/interphase fused cells. These observations further strengthen the correlation between histone phosphorylation and the changes in chromosome condensation associated with the initiation of mitosis. This study also suggests that maintenance of the mitotic chromosomes in a highly condensed state does not require the continued presence of histones in a highly phosphorylated form.     

Meiotic maturation of mouse oocytes in vitro: association of newly synthesized proteins with condensing chromosomes.

The nature, intracellular distribution, and role of proteins synthesized during meiotic maturation of mouse oocytes in vitro have been examined. Proteins synthesized during the initial stages of maturation are concentrated within the nucleus (germinal vesicle) and become intimately associated with the condensing chromosomes. Inhibition of protein synthesis during this period does not prevent germinal vesicle dissolution or chromosome condensation, but meiotic progression is blocked reversibly at the circular bivalent stage. A protein is synthesized during meiotic maturation of the mouse oocyte which exhibits several of the characteristics of the very lysine-rich histone, FI; this and other histones are phosphorylated during the initial stages of maturation. These results are discussed in relation to studies of meiotic maturation of oocytes from non-mammalian species and chromosome condensation in both oocytes and mitotic cells.     

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.