THE JOURNAL OF BlOmIcAL CHesasnrr Q 1994 by The American soeiety for Biochemistry and Molecular Biology, Inc.

Val. 269, No. 9, Issue of March 4, pp. 6320-6324, 1994 Printed in U.S.A.

Leptomycin B Targets a Regulatory Cascade of crml, a Fission Yeast Nuclear Protein, Involved in Control of Higher Order Chromosome Structure and Gene Expression*

(Received for publication, September 13, 1993, and in revised form, October 29, 1993)

Kazunori NishiSI, Minoru YoshidaSn, Daisuke FujiwaraS, Mitsuo NishikawaSII, Sueharu HorinouchiS, and Teruhiko BeppuS** From the $Department of Agricultural Chemistry Faculty of Agriculture and **Biotechnology Research Center, The University of 'Ibkyo, Yayoi 1-1-1, Bunkyo-ku, lbkyo 113, Japan

The molecular action of leptomycin B (LMB), an agent inducing arrest of the eukaryotic cell cycle at G1 and 6 2 phases, was investigated by analyzing an LMB resist- ance gene of Schizosaccharomyces pombe. A genomic li- brary of an LMB-resistant mutant was screened for LMB resistance, and a DNA fragment containing an open reading frame ( O W ) of 1078 amino acids was cloned on a multicopy vector. The plasmid was found to confer drug resistance specifically to LMB. Nucleotide se- quencing revealed that the O W was a mutant gene for the essential nuclear protein crml, which had been re- ported to complement a cold-sensitive mutation causing deformed nuclear morphology. The gene product named crml-N1 had two amino acid replacements (Gly-603 to Asp and Met-646 to ne). Two allelic mutants of cnnl (crmI-809 and cnnI-lI9) were found to be hypersensi- tive and resistant, respectively, to LMB. Nuclear mor- phology of the cold-sensitive crmI-809 mutant at the restrictive temperature was almost the same as that of the wild-type cells treated with LMB. Furthermore, a low concentration of LMB induced the intracellular ac- cumulation of a 26-kDa protein in the wild-type cells, which was immunologically identical to the protein ac- cumulating in the cnI-809 mutant cells. These results strongly suggest that LMB primarily inhibits the func- tion of the crml gene which is required for maintaining higher order chromosome structures, correct gene ex- pression, and cell growth in the fission yeast.

A eukaryotic chromosome consists of not only DNA associ- ated with histones but also various classes of nuclear proteins to form higher order chromosome structure, which shows dy- namic changes during the cell cycle and probably plays crucial roles in chromosome function and nuclear environment (Gasser and Laemmli, 1987; Reeves, 1984). However, little has been revealed about the regulatory functions of these nuclear pro- teins and many other components may still remain unidenti-

from the Ministry of Education, Science and Culture, Japan. The costs * This work was supported by a grant-in-aid for Cancer Research

of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "uduertise- ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Present address: Discovery Research Laboratories 11, Discovery Re- search Division, Takeda Chemical Industries, Ltd., 2-17-85 Jusohonma- chi, Yodogawa-ku, Osaka 532, Japan. 1 To whom correspondence should be addressed: Dept. of Agricultural

Chemistry, Faculty ofAgriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3812-2111 (ext. 5124); Fax:

1) Present address: Pharmaceutical Research Laboratory, Kirin Brew- 81-3-3812-0544.

ery Co., Ltd., 1-2-2 Souja-machi, Maebashi-shi, Gunma 371, Japan.

fied. One of such putative nuclear components, crml (chromo- some region maintenance), was recently reported by Ada& and YinagidaTl9891, who isolated cold-sensitive (cs) crml mu- tants of the fission yeast Schizosaccharomyces pombe. The crml mutants carried mutations in the crml+ gene resulting in deformed nuclear domains at the restrictive temperature. The crml+ gene was cloned, and its nucleotide sequencing predicted a 1078-amino acid protein. Immunoblots and immunofluores- cence microscopy demonstrated wide distribution of a homolo- gous protein among distant eukaryotes as well as preferential localization in the nucleus and its periphery. Most recent ob- servations using fluorescence in situ hybridization suggested that the crml+ gene product was required for the interphase arrangements of centromeres and telomeres (Funabiki et al., 1993). Although the crml+ gene product is one of the proteins involved in maintaining the proper higher order chromosome structure, its molecular function has remained almost obscure.

On the other hand, leptomycin B (LMB)l (Fig. l), which was originally isolated by our group as an antifungal antibiotic from a Streptomyces strain (Hamamoto et al., 1983a, 1983b), causes characteristic inhibition of the eukaryotic cell proliferation be- ing accompanied by abnormal nuclear structures (Hamamoto et al, 1985; Komiyama et al., 1985~). Since our discovery of lep- tomycins, five other related compounds with the same skeletal structure have been isolated from various species of Strepto- myces, all of which showed similar strong cytostatic effects on both mammalian cells and the fission yeast S. pombe (Komi- yama et al., 1985b; Hayakawa et al., 1987; Abe et al., 1993a, 199313). Some of these leptomycin-related compounds showed i n vivo antitumor activity against experimental tumors and are actually considered as antitumor agents (Komiyama et al., 1985a, 1985c; Hayakawa et al., 1987). We found that LMB caused a specific arrest of the cell cycle at both G1 and G2 phases in cultured mammalian cells. Upon removal of the agent, a large fraction of the tetraploid cells appeared, since the G2-arrested cells resumed the cell cycle without passage through M phase (Yoshida et al., 1990b). Although trichostatin A, a potent inhibitor of histone deacetylase (Yoshida et al., 1990a), gave a similar effect (Yoshida and Beppu, 1988), vari- ous biochemical analyses have indicated that the molecular target of LMB is completely different.

In order to identify the target of LMB, we started to clone LMB resistance genes from LMB-resistant mutants of s. pombe. One of the cloned genes was identified to be a homolog of the mammalian multidrug resistance gene and named pmdl+ (pombe Gr i - l ike gene). When the gene cloned on a multicopy vector was introduced into wild-type S. pombe, the

The abbreviations used are: LMB, leptomycin B; DAPI, 4',6-diami- dino-2-phenylindole; kb, kilobase pairb); ORF, open reading frame.

6320

Inhibition of crml Function by Leptomycin B 632 1

0 HO

FIG. 1. Chemical structure of LMB. transformant showed elevated resistance to not only LMB but also several structurally unrelated antifungal agents (Nishi et al., 1992). Since the pmdl' gene seemed not to be a gene di- rectly involved in the molecular mechanism of the LMl3 inhi- bition, we triedko clone another LMB resistance gene. The gene thus obtained coincided with the crml gene described above. This paper describes cloning of the LMB resistance gene which was identified to be a mutant of the crml' gene. Analyses of the mutant as well as the crmlCs mutant strongly suggest that the crml protein is the molecular target of LMB. The role of the crml protein is also discussed.

FXPERIMENTAL PROCEDURES Chemicals-LMB was purified from a culture broth of Streptomyces

sp. strain ATS1287 as reported previously (Hamamoto et al., 1983a). Purified LMB was dissolved in ethanol as stock solutions and stored at -20 "C. Cycloheximide and valinomycin were purchased from Sigma. Staurosporine was generously provided by Kyowa Hakko Kogyo Co. Ltd. (Tokyo). AU the other chemicals used in this study were of reagent grade.

Strains, Plasmids, and Media-The S. pombe strains used were the wild-type L972 (h-), JY266 fh+ leu1-32), AC1 (h- Zeu1-32 crrnl-SOS), AD45 (h- ku1-32 crml-119), KN4 (h- ade6-M210 pmdl::ura4 ura4- Dl8 ku1-32), and LM102 (h- leu132 crml-Nl). The cold-sensitive crml mutant strains, AC1 and AD45, were generously supplied by M. Yanagida (Kyoto University). The strain LM102 is one of the LMB- resistant mutants, which was reported previously (Nishi et al., 1992). E. coli TGl (A(1ac-pro), supE, thi, hsdD5, F' traD36, proAB, l a d , lacZAM15), and JM105 ( A ( l a ~ - p ~ ) , thi, strA, endA, obcB15, hsdR4, F' tmD36,proAB, lacIq, lacZAM16) were used for cloning and sequencing. Plasmid pLM27 containing the crml-Nl gene was isolated from the genomic DNA library of the LM102 strain. Plasmid pCRM1-284 con- taining the crml' gene was generously provided by M. Yanagida (Kyoto University). S. pombe strains were grown in either SD medium con- taining 0.67% yeast nitrogen base without amino acid (Difco) and 2% glucose or YF'D medium containing 1% yeast extract, 2% polypepton, and 2% glucose. Solid media contained 1.5% agar.

Genetic Methodsatandard genetic methods were carried out ac- cording to Gutz et al. (1974). S. pombe was transformed according to the lithium acetate protocol of It0 et al. (1983). Standard molecular cloning techniques were followed as described by Maniatis et al. (1982). Chro- mosomal and plasmid DNAs were isolated by using E. coli as described by Beach et al. (1982).

DNA Sequence Analysis-The nucleotide sequence of the crml-N1 gene was determined by the dideoxy method using single-stranded M13 phages (Sanger et al., 1977). Sequenase kit (U. S. Biochemical Co.) was used for sequencing reaction.

Assay of Drug Sensitivity-Drug sensitivity of cells was assayed by measuring the minimal inhibitory concentration as follows. YPD plates containing various concentrations of each drug were prepared just be- fore we. The cells carrying the plasmids grown on SD plates without leucine at 27 "C for 2 days were streaked with sterile toothpicks onto YPD plates containing drugs and incubated at 27 "C for 2-3 days. Colony formation on each plate was observed.

Assay of p25 Production-The cell extract o f S. pombe was prepared as described by Moreno et al. (1991). The cytoplasmic proteins in the extracts were separated by slab gel electrophoresis using SDS-poly- acrylamide gels (Laemmli, 1970), followed by staining with Coomassie Brilliant Blue R-260 (Nacalai tesque). For Western blot analysis, the proteins were transferred to polyvinylidene difluoride membrane (Mil- lipore) from the SDS-polyacrylamide gel. The blots were blocked with 3% gelatin and incubated for 1 h at room temperature with rabbit p25 antibody. For detection, the blots were incubated with horseradish per- oxidase-conjugated goat anti-rabbit IgG (Amersham Corp.), and the

TAEILE I Drug resistance conferred by pLM27 (crml-Nl) and pmdl'

vector pDB248', pLM27 (containing the crml-Nl gene on pDB248'), or Wild-type S. pombe strain (JY266; h+ leul) was transformed with the

pLM24 (containing thepmdl' gene on pDB248'). The drug sensitivities of these transformants were determined by measuring the minimal inhibitory concentrations (MIC) on YPD plates.

MIC pDB248' pLM27 pLM24

Leptomycin B, ng/ml 20 80 100 Cycloheximide, pg/ml 30 30 100 Valinomycin, p g / d 5 5 100 Staurosporine, d m 1 1 1 5

antigen-antibody complexes were visualized by incubation with 4-chloro-1-naphthol (Bio-Rad) and hydrogen peroxide.

DAPI Staining-Nuclei of S. pombe were stained with DAF'I (Sigma) as described by Toda et al. (1981).

RESULTS Cloning of an LMB Resistance Gene"T0 identify the target

molecule of LMB, we conducted cloning experiments of the LMB resistance gene from a LMB-resistant mutant of S. pombe LM102. A genomic gene library of LM102 was constructed by using a multicopy shuttle vector pDB248' (Beach et al., 1982) in Escherichia coli TG1. The library DNAs were introduced into strain JY266 (h' Zeul) and Leu' transformants were selected. We next screened the transformants for colonies capable of growing on YPD plates containing 50 ng/ml LMB by replica plating. A plasmid pLM27, which contained a 16-kb DNA on pDB248', was thus cloned from the genomic library of LM102 strain as a DNA that dominantly conferred LMB resistance. When pLM27 was introduced into the wild-type S. pombe strain, the transformant showed increased resistance to LMB (80 ng/ml) but no cross-resistance to the other agents examined (Table I ) . In contrast, the transformant carrying the pmdl' gene on the same vector plasmid showed the resistance to not only LMB (100 ng/ml) but also cycloheximide, valinomycin, and staurosporine. These results suggest that pLM27 contains a gene able to confer resistance specific to LMB when it is intro- duced on a multicopy vector.

The results of the subcloning analysis of the insert on pLM27 are shown in Fig. 2. Plasmids pLM27-D1, pLM27-D2, and pLM27-D3 were constructed from pLM27 by BamHI digestion, BgZII-XbaI double digestion and BamHI-XbaI double digestion, respectively, each of which was followed by self-ligation. Plas- mid pLM27-D4 was constructed by the insertion of the 5.5-kb BgZII fragment of the pLM27 into the vector pDB248' at the BamHI site. Among these plasmids, only pLM27-Dl could con- fer the LMB resistance in wild-type s. pombe. Failure to confer the resistance by pLM27-D2, pLM27-D3, and pLM27-D4 sug- gested the presence of the resistance gene in the middle of the pLM27 insert. Therefore, we next constructed a plasmid con- taining a 4.0-kb SphI-XbaI fragment (pLM27-D5), which suc- cessfully conferred the LMB resistance. Nucleotide sequencing of the SphI-XbaI fragment revealed the presence of an open reading frame (ORF) of 1078 amino acids.

Identity of the LMB Resistance Gene to the crml Gene-A homology search of the sequence of the ORF revealed extremely high similarity to the crml' gene, which was cloned from wild- type s. pombe as a gene complementing abnormal nuclear mor- phology and the cold sensitivity in the crml mutant at the restrictive temperature (Adachi and Yanagida, 1989). The de- duced amino acid sequence of the ORF for LMB resistance showed two amino acid replacements ofAsp and Ile for Gly-503 and Met-546, respectively (Table 11). Except for the 2 base exchanges from G to A causing these two amino acid replace- ments, the whole nucleotide sequence of the ORF together with

6322 Inhibition of crml Function by Leptomycin B

Plasmid LMB- c"----l

P O c o x s G G x 5 t t, ' resistance pLM27 I" I

ORF _II) 1 +

c "4

-0 *"C

"o cc

pLM27-Dl - + pLM27-D2 - - pLM27-D3 - - pLM27-D4 pLM27-DS + pCRM1-284 +

-

FIG. 2. Restriction map and subcloning analysis of the l&kb insert in plasmid pLM27. A closed box represents the sequenced

arrow indicates the location of an open reading frame and its direction. region, and the sequence strategy is shown by thin arrows. A thick

b c a t e d fragments subcloned on pDB248' are shown by thin lines. Plasmids pLM27-D1, pLM27-D2, and pLM27-D3 were constructed by deleting 1.6-kb EamHI, 8.0-kb EglII, and 8.9-kb EamHI-EglII frag- ments of the insert, respectively, from pLM27. Plasmid pLM27-D4 was

at the BamHI site. Plasmid pLM27-D5 consisted of the 4.4-kb SphI- constructed by cloning the 5.2-kb EglII fragment on the vector pDB248'

XbaI fragment derived from the insert and pDB248'. Plasmid pCRM1- 284 was supplied from M. Yanagida (Kyoto University). The 4.3-kb SphI-BglII fragment in pCRM1-284 contains the coding region of crml'. The plasmids thus obtained were examined for the ability to confer resistance to LMB in the wild-type strain JY266. Abbreviations for the restriction sites are: E , EamHI; G, EglII; P, PstI; S, SphI; and X, XbaI, respectively.

TABLE I1 Location of point mutations in crml-Nl gene

The sequence of the 4413 nucleotides of the SphI-XbaI fragment (see Fig. 2) were determined and compared with that of crml' which had recently been corrected by lbda et al. (1992). Two base exchanges caus- ing amino acid substitutions were found in crml-N1, which are indi- cated by bold letters. The sequence data are available from the EMBIJ GenBanWDDBJ under accession number D16355.

No. of amino crml' m l - N l acid residue A m i n o acid Codon Amino acid Codon

503 GlY GGC ASP GAC 546 Met ATG Ile ATA

the long noncoding upstream and downstream regions was identical to that of crml' (data not shown). Since the crml+ gene was reported to be unique in the genome, we named this cloned gene c m l - N l as a mutant of the crml+. To examine whether these replacements in crml-Nl are re-

sponsible for LMB resistance, we introduced the plasmid pCRM1-284 that contained crml+ gene (supplied by M. Yanagida, Kyoto University) into the wild-type S. pombe JY266 strain and compared its LMB resistance with that of the strain carrying pLM27. Unexpectedly, the strain showed almost the same level of resistance to LMB as that conferred by pLM27 (Fig. 2). The result might be explained by assuming the el- evated intracellular concentration of the molecular target of LMB, crml+ gene product, which was amplified by the multi- copy vector. Since these experiments using extrachromosomal crml genes gave no direct evidence that the amino acid replace- ments in crml-N1 caused LMB resistance in the resistant mu- tant LM102, we next examined a possible correlation between the phenotype of LMB resistance and the presence of the chro- mosomal crml-N1 gene. We integrated a genetic marker LEU2 into the chromosomal region adjacent to the crml-N1 gene by using homologous recombination with the noncoding region of the cloned fragment. The 1.6-kb BamHI fragment of pLM27 insert (Fig. 2) was ligated to an integration vector, YIp33, con- taining LEU2 from s. cerevisiae, which could complement the S. pombe leul. The composite plasmid was linearized with BgZII (YIp33 has no BglII site, whereas the 1.6-kb fragment has one BglII site) and then integrated into the chromosome of

TABLE I11 Drug Sensitivities of the allelic crml mutants

Drug sensitivities of wild-type S. pombe strain (JY266; h+ leul) and

inhibitory concentrations (MIC) on YPD plates. three allelic crml mutants were determined by measuring minimal

Drug MIC

Wild-type= crml-Nlb crml-809' crml-119d

Leptomycin B, n g / d 20 100 2.5 80 Cycloheximide, pg/ml 30 30 30 30 Staurosporine, pg/ml 1 1 2 2 a Wild-type, JY266 (h+ leul ).

crml-N1, LM102 (h- leul crml-Nl). cml-809, AC1 (h- leul crml-809). crml-119, AD45 (h- leu1 crml-119).

LM102 (Leu-) to obtain mitotically stable Leu' integrants. Southern hybridization of their genomic DNA with the BamHI fragment as a probe confirmed the integration by homologous recombination. The analysis of the tetrads from the heterozy- gous diploids between the integrant (Leu') and JY266 (Leu-) showed a 2:2 segregation pattern for Leu+:Leu-. All the Leu' segregants showed the same extent of LMB resistance as that of LM102 (data not shown). Cosegregation between the LMB resistance and the marker introduced to the vicinity of the mutated crml gene (crml-NI ) confirmed that the mutation in the chromosomal gene with a single copy is sufficient to cause LMB resistance.

We further tested whether other allelic mutations in the chromosomal crml gene caused the altered LMB resistance. LMB sensitivities of the cold-sensitive crml mutants, crml- 809 and crml-119 (Adachi and Yanagida, 1989), were compared with that of the wild-type strain. As shown in Table 111, the crml-809 mutant was found to be highly sensitive to LMB and could not grow at 2.5 ng/ml of LMB, which was far lower than the minimal inhibitory concentration for the wild-type strain (20 nglml). On the other hand, another cold-sensitive crml mutant crml-119 showed increased resistance to LMB (80 ng/ ml). In contrast to the dramatic changes in the sensitivity to LMB, these mutants showed no significant decrease or increase in the sensitivities to other drugs.

Comparison of the Phenotypes between the Wild-type Cells Treated with LMB and the Cold-sensitive crml-809 Mutant Cells-The crml mutants of S. pombe were originally isolated according to their cold-sensitive phenotype accompanying the deformed nuclear chromosome structure at the restrictive tem- perature. On the other hand, LMB was discovered as an anti- fungal antibiotic inducing cell elongation in s. pombe and de- formed chromosomes in both s. pombe and HeLa cell nuclei. We compared the abnormal nuclear morphology in the crml-809 mutant at the restrictive temperature and that induced by LMB in the wild-type S. pombe strain by using the fluorescence microscopy with DAPI staining (Fig. 3). crml-809 showed de- formed nuclear chromosomes consisting of fragmented and con- densed segments at the restrictive temperature (Fig. 30). Closely similar morphology was observed in the wild-type cells treated with 50 ndml of LMB (Fig. 3B).

Another important phenotype of the crml mutants is the intracellular accumulation of a specific protein termed p25 at both the permissive and restrictive temperatures. If indeed LMB inhibited the function of crml, then similar overexpres- sion of p25 would be observed. To verify this possibility, we prepared extracts from the wild-type cells treated with various concentrations of LMB as well as from crml-809 cells and analyzed the production of p25 with SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue. The amount of a protein corresponding to the relative molecular mass of 25 kDa was found to increase greatly, when cells were treated with 5 ng/ml LMB, an insufficient dosage for cell

Inhibition of crml Function by Leptomycin B 6323

cells treated with LMB and cold-sensitive cnnl-809 mutant FIG. 3. Comparison of nuclear morphology between wild-type

cells. Cells were stained with DAPI and observed under fluorescence microscopy. Cytoplasmic granular fluorescence represents mitochon- drial DNA. A, wild-type cells grown in 32 "C without LMB. B, wild-type cells treated with 50 ngml LMB for 6 h at 32 "C. C, cold-sensitive crml-809 cells grown at the permissive temperature of 32 "C. D, c m l - 809 cells grown at the restrictive temperature of 20 "C. Bur, 5 pm.

growth inhibition (Fig. 4). Increased amounts of the protein were accumulated as the LMB dosage was increased. Western blotting by using anti-p25 antibody confirmed that the accu- mulated protein was identical to p25 (data not shown). In ad- dition, we observed that the LMB challenge caused accumula- tion of several other proteins, all of which were also observed in crml-809. These results indicate that the effect of LMB is identical to the phenotypic expression by the crml-809 muta- tion.

DISCUSSION This paper describes three lines of the genetic and biological

evidence that LMB inhibits the function of the crml gene prod- uct in s. pombe. First, mutations in the genomic crml+ gene caused the increase (crml-Nl and cml-119) or the decrease (crml-809) in LMB resistance. Second, amplification of the wild-type crml+ gene as well as the crml-Nl gene could confer LMB resistance in the wild-type cells. Third, the apparent phe- notypes such as abnormal nuclear morphology and overproduc- tion of a cytoplasmic protein p25 were identical between the wild-type cells treated with LMB and the cold-sensitive crml mutants. The crml protein is reported to be localized in the nucleus and ita periphery of the fission yeast S. pombe, and the homologous protein is present in the budding yeast Sacchuro- myces cerevisiae and HeLa cell nuclei (Adachi and Yanagida, 1989). It seems reasonable that growth inhibition and abnor- mal nuclear morphology induced in mammalian cells by LMB (Komiyama et al., 1985c; Yoshida et al., 1990b) is also due to the inhibition of the hnction similar to crml. However, it is still unclear whether LMB can physically interact with the crml protein. Western blotting with anti-crml antibody showed that

kDa m-- t

20 - type S. pornbe during treatment with LMB. Cell extracts were

FIG. 4. Accumulation of a cytoplasmic protein, p25, in wild-

prepared from the wild-type cells treated with various concentrations of LMB for 6 h at 32 "C and from the crml-809 cells grown at 20 "C. Cells were broken by glass beads with shaking, and proteins in the soluble fractions of the extracts were separated with 12.5% SDS-polyacryl- amide gel electrophoresis and stained with Coomassie Brilliant Blue. An arrow indicates the position of p25. Arrowheads represent the bands that are enhanced by LMB, other than p25. Lane 1, wild-type cells without treatment; lane 2, crml-809 (strain ACI) grown at 20 "C; and lanes 3-6, wild-type cells treated with 1,2,5, and 10 ndml, respectively, of LMB.

the mobility and the cellular level of the crml protein analyzed on an SDS-polyacrylamide gel were not affected by the treat- ment with LMB.2 Binding assays using labeled LMB may be necessary for obtaining the conclusive results.

"he phenotype of cold-sensitive crml mutants indicated that crml protein played an important role in the regulation of not only higher order chromosome structure but also gene expres- sion such as the p25 gene. Recently, Toda et al. (1992) indicated that crml was a negative regulator of papl, an AP-1-like tran- scription factor in s. pombe. They cloned the gene encoding p25 and found that an AP-1 site was present in the upstream non- coding region of the p25 gene. The transcription was actually dependent on papl and was repressed by crml. Disruption of the papl gene could rescue the c r m P mutation. These results suggest that the crml protein regulates the papl activity by direct or indirect modification or a conformational change. The present results that LMB induced the overproduction of p25 in the wild-type cells can also be explained by the activation of papl due to the inhibition of crml function (Fig. 5). However, the molecular function of crml protein is still unknown. In addition, it seems possible that crml interacts with other regu- latory components, since papl disruption failed to rescue the complete deletion of the crml+ gene and overexpression of papl or p25 did not cause any cold-sensitive phenotype (Toda et al., 1992). To obtain further clues to understanding the molecular function of crml, the combined use of LMB and genetic ap- proach using the fission yeast system may be promising. We isolated a mutant that showed extremely increased resistance to LMB, whose phenotype was expressed only when the muta- tion was present together with the crml-809 mutation.2 In this double mutant, the growth inhibition by LMB completely sup- pressed but the overproduction of p25 still remained. Identifi- cation of the genetic determinants in such the mutants will help elucidate the crml function as well as the molecular action of LMB.

Eukaryotic chromatin is a highly dynamic macromolecular complex that undergoes continuous structural modification during transcription, replication, and chromosome distribution in mitosis. It therefore seems probable that chemicals inhibit- ing the regulation of higher order chromosome structure cause ~~

* D. F'ujiwara, M. Yoshida, T. 'Ibda, M. Yanagida, and T. Beppu, un- published results.

6324 Inhibition of crml Function by Leptomycin B

1

I ! LMB Ipapll I ! LMB "

of p%.A, according to the previous report (lbda et aE., 1992), the fission RG. 5. Schematic representation of LMB-induced expression

yeast papl protein, an AP-1-like transcription factor, activates the ex- pression of the p25 gene by binding the AP-1 site present in the up- stream of the gene. The papl-dependent transcription of the p25 gene is negatively regulated by the crml protein, probably due to the direct or indirect inhibition of the papl function. B , LMB inhibits the function of the crml protein, which may cause activation of papl. The transcription of the p25 gene may be enhanced by the activated papl, resulting in the overproduction of p25.

the pleiotropic consequences on chromatin functions or cell cycle progression. In fact, the inhibition of mammalian histone deacetylase by trichostatin A or n-butyrate induced changes in the transcriptional activities as well as specific arrest of the cell cycle at both G1 and G2 phases (Larno et al., 1984; Yoshida and Beppu, 1988; Csordas, 1990). DNA topoisomerase I1 inhibitors have been shown to block the G2/M transition in the cell cycle (Crissman et al., 1988; Ishida et al., 1991; Lanks and Lehman, 1990). Our previous studies showed that LMB caused specific arrest of the cell cycle at both G1 and G2 phases in normal fibroblast cells (Yoshida et al., 1990b). Furthermore, we also demonstrated that LMB enhanced cell killing by x-ray in baby hamster kidney (BHK) cells, at concentrations that had no

significant cytotoxic effect by itself (Sasaki et al., 1992). The most likely reason for the fixation of x-ray-induced potential lethal damage by LMB is the inhibition of repair process by alteration of the nuclear structure. Thus, LMB will be useful as a tool for the role of higher order chromosome structure through the regulatory function of crml.

Acknowledgments-We thank M. Yanagida and T. Toda, Department of Biophysics, Faculty of Science, Kyoto University, for providing the S. pombe crml mutants, plasmid pCRM1-284, and anti-p25 antibody.

REFERENCES

Abe, K., Yoshida, M., Horinouchi, S. , and Beppu, T. (1993a) J. Antibiot. (Tokyo) 46,

Abe. K., Yoshida, M., Naoki, H., Horinouchi, S. , and Beppu, T. (1993b) J. Antibiot.

Adachi, Y., and Yanagida, M. (1989) J. Cell Biol. 108, 1195-1207 Beach, D., piper, M., and Nurse, P. (1982) Mol. & Gen. Genet. 187,326-329 Crissman, H. A., Wilder, M. E., and lbbey, R. A. (1988) Cancer Res. 48,5742-5746 Csordas, A. (1990) Biochern. J. 266.23-38 Funabiki, H., Hagan, I., Uzawa, S., and Yanagida, M. (1993) J. Cell Biol. 121,

Gasser, S . M., and Laemmli, U. K (1987) Zkmds Genet. 3, 16-22 Gutz, H., Heslot, H., Leupold, U., and Loprieno, N. (1974) Schizosaccharornyces

pornbe. Handbook ofgenetics (King, R. C., ed) pp. 395-446, Plenum Press, New

Hamamoto, T., Cunji, S., Tsuji, H., and Beppu, T. (1983a) J. Antibiotics 36,639-645 York

Hamamoto, T., Seto, H., and Beppu, T. (1983b) J. Antibwt. (Tokyo) 36,646450 Hamamoto, T., Uozumi, T., and Beppu, T. (1985) J. Antibiot. (Tbkyo) 38, 1573-1580 Hayakawa, Y., Adachi, K., and Komeshima, N. (1987) J. Antibwt. (Tbkyo) 40,

Ishida, R., Miki, T., Narita, T., Yui, R., Sato, M., Utsumi, K. R., Tanabe, K, and

Ita, H., Fukuda, Y., Murata, K, and Kimura, A. (1983) J. Bacterial. 159,163-168 Komiyama, K, Okada, K., Hirokawa, Y., Masuda, K, lbmisaka, S. , and Umezawa,

Komiyama, K, Okada, K., Oka, H., Tomisaka, S. , Miyano, T., Funayama, S. , and

Komiyama, K., Okada, K., Tomisaka, S. , Umezawa, I., Hamamoto, T., and Beppu,

LaemmIi, U. K. (1970) Nature 227,680-685 Lanks, K. W., and Lehman, J. M. (1990) Cancer Res. SO, 47764778 Larno, S. , Ronot, X., Adolphe, M., and k h a t , P. (1984) J. Cell. Physiol. 120,

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Labo- 384-390

ratory Manunl, Cold Spring Harbor Laboratory, pp. 31-38, Cold Spring Harbor, NY

728-734

(Tokyo) 46,73&740

961-976

1343-1352

Andoh, T. (1991) Cancer Res. 61.49094916

I. (1985a) J. Antibiot. (Tokyo) SS, 224-229

Umezawa, I. (1985b) J. Antibiot. (Tokyo) SS,22&223

T. (1985~) J. Antibiot. (Tokyo) 39,427-429

Moreno, S., mar, A,, and Nurse, P. (1991) Mefhods EnzyrnoZ. 194,795-823 Nishi, K, Yoshida, M., Nishimura, M., Nishikawa, M., Nishiyama, M., Horinouchi,

Reeves, R. (1984) Biochirn. Biophys. Acta 782,343-393 S. , and Beppu, T. (1992) Mol. Microbiol. 6, 761-769

Sanger, F., Nicklen, S. , and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,

Toda, T., Yamamoto, M., and Yanagida, M. (1981) J. Cell Sci. 62,271-287 Sasaki, H., Yoshida, M., and Beppu, T. (1992) Radiat. Res. 129, 163-170

lbda, T., Shimanuki, M., Saka, Y., Yamano, H., Adachi, Y., Shirakawa, M., Kyogoku, Y., and Yanagida, M. (1992) Mol. Cell. Bwl. 12,5474-5484

Yoshida, M., and Beppu, T. (1988) Exp. Cell Res. 177,122-131 Yoshida, M., Qima, M., Akita, M., and Beppu, T. (1990a) J. Biol. Chern. 286,

Yoshida, M., Nishikawa, M., Nishi, K, A b e , K., Horinouchi, S . , and Beppu, T.

5463-5467

17174-17179

(1990b) Exp. Cell Res. 187, 150-156