Cell, Vol. 61, 437-446,

May 4, 1990, Copyright

0

1990 by Cell Press

Yeast Centromere Binding Protein CEIFI, of the Helix-Loop-Helix Protein Family, Is Requked for Chromosome Stability and Methionine m Mingjie Cai and Ronald W. Davis Department of Biochemistry Stanford University School of Medicine Stanford. California 94305-5307

Summary The centromere and its binding proteins constitute the kinetochore structure of metaphase chromosomes, which is crucial for the high accuracy of the chromosome segregation process. Isolation and analysis of the gene encoding a centromere binding protein from the yeast S. cerevisiae, CBF7, are described in this paper. DNA sequence analysis of the CBF7 gene reveals homology with the transforming protein myc and a family of regulatory proteins known as the helix-loop-helix (HLH) proteins. Disruption of the CBF7 gene caused a decrease in the growth rate, an increase in the rate of chromosome loss/nondisjunction, and hypersensitivity to the antimitotic drug thiabendazole. Unexpectedly, the cbf7 null mutation concomitantly resulted in a methionine auxotrophic phenotype, which suggests that CBFl, like other HLH proteins in higher eukaryotic cells, participates in the regulation of gene expression.

Chromosome segregation during cell division is a highly accurate process. The frequency of spontaneous loss or nondisjunction for a single chromosome in the yeast Saccharomyces cerevisiae is about 10m5 per cell division (Hartwell et al., 1982). Although the direct measurement for chromosome stability is not yet available for mammalian cells, indirect evidence suggests that the nondisjunction/loss rate of chromosomes in T lymphocytes will not be higher than 3 x 1O-5 (Janatipour et al., 1988). This high accuracy of chromosome segregation in eukaryotic cells is necessary to ensure the correct transmission of genetic information from mother to daughter cells. A particular chromosomal locus, termed the centromere, plays a central role in the process of chromosome segregation. It is on this locus that a multilayer, proteinaceous complex known as the kinetochore is formed during cell division, which, morphologically, serves as an anchor for microtubule attachment (for a review see Mitchison, 1988). Although very little is known about the nature and function of kinetochore proteins, studies on chromosomes of CHO cells have suggested that the kinetochore may provide the driving force for chromosome movement along the microtubule fibers (Koshland et al., 1988). Centromeric DNA has been identified and extensively characterized from the yeast S. cerevisiae (FitzgeraldHayes et al., 1982; Hieter et al., 1985b; Mann and Davis, 1986; Panzeri and Philippsen, 1982). The relationship be-

tween the DNA sequence and centromere function has been firmly established (Cumberledge and Carbon, 1987; Gaudet and Fitzgerald-Hayes, 1987; Hegemann et al., 1988; McGrew et al., 1986; Ng and Carbon, 1987; Panzeri et al., 1985). The minimal functional centromere of S. cerevisiae is contained within a 125 bp sequence, which can be divided into three elements, CDEI, CDEII, and CDEIII (Cottarel et al., 1989). CDEI is an 8 bp sequence, PuTCACPuTG (Pu, purine), which is completely conserved in all known yeast centromeres. CDEII is ~80 bp in length with an unusually high A+T content (>90%), but lacks recognizable conservation of primary sequence. CDEIII contains a 25 bp conserved sequence with partial dyad symmetry. While mutations in each of the three elements could impair centromere function, the most profound effect results from changes in the center of the partial dyad symmetry in CDEIII (Hegemann et al., 1988; McGrew et al., 1986; Ng and Carbon, 1987). Several groups have recently reported purification of proteins from S. cerevisiae that exhibit specific binding activities to the CDEI element of yeast centromere (Baker et al., 1989; 5ram and Kornberg, 1987; Cai and Davis, 1989; Jiang and Philippsen, 1989). The protein that we purified, previously named CBP-I, is now called CBFl (centromere binding factor), because the former name has been adopted for cytochrome b messenger RNA processing genes (Mayer and Dieckmann, 1989). Because CBFl is able to distinguish single base pair mutations in the CDEI region and the sequence specificity of its binding in vitro correlates well with the known sequence requirements determined for CDEI function in vivo, it is likely to be an authentic component of the kinetochore apparatus (Cai and Davis, 1989). In this paper we describe the isolation and analysis of the gene encoding CBFl. DNA sequence analysis of CBF7 revealed that it is related to a family of regulatory proteins in higher eukaryotes, the newly identified helixloop-helix (HLH) proteins (Murre et al., 1989a, 19896). The phenotypic analysis of cbfl null mutants demonstrates that CBFl is involved in the process of chromosome segregation. Furthermore, evidence is presented suggesting that CBFl may also act as a transcriptional activator. Results Cloning of the CBF7 Gene The 16 kd yeast centromere binding protein has been purified 7000-fold to homogeneity (Cai and Davis, 1989). The purified protein was subjected to sequence analysis, and the sequence of 17 of its N-terminal amino acids was obtained (see Experimental Procedures). This information was used to synthesize two nonoverlapping pools of completely degenerate oligonucleotides, which were subsequently used to screen a yeast cDNA library by the tetramethylammonium chloride washing method (Wood et al., 1985). From 100,000 clones screened, 3 were found to hybridize to both oligonucleotide probes. The homology to

Cell 436

Figure 1. Sequence ing the CEF7 Gene

of the Bamtil-Hpal

Genomic

Fragment

Contain.

The sequence is 1672 bp in length. The numbers on the left side indicate the DNA sequence. The numbers on the right side indicate the ammo acid sequence. The oligopeptide sequence obtained from sequence analysis of the N-terminus of purified 16 kd CBFl protein is underlined (amino acids 214-231).

the oligonucleotide probes was further localized to a 268 bp Ball-Pstl fragment, which when sequenced correctly matched the oligopeptide sequence from which the probes were derived. This 268 bp cDNA insert was then used as a probe to obtain the genomic coding sequence from a yeast genomic library. The CM7 gene was finally

localized to a 3.0 kb genomic Baml-fl fragment (pNN450), whose complete sequence was then determined. By means of blotting and hybridization of yeast chromosomes separated on CHEF gels (Chu et al., 1986) the CBFI gene was found to reside on chromosome X (data not shown).

The Yeast 439

Centromere

CBFI N-myc

107 264

CBFl N-myc

135 292

CEFI N-myc

163 316

CBFI N-myc

IS0 346

CBFl N-myc

215 373

CBFI N-myc

242 401

CBFI N-myc

266 429

Figure 2 Homologous Human N-myc Protein Identities changes

Binding

Protein

The deduced amino acid sequence 01 CBFl was used to search the NBRF/PIR protein data base via the BIONET service of Intelligenetics, Inc., using the FASTA program of Pearson and Lipman (1988). The most significant homology detected was with the transforming protein N-myc (Kohl et al., 1986) which displays 22.9% identity with CBFl over a 175 residue overlap (Figure 2). This homology led to the identification of the HLH motif in CBFl, which was recently brought to light in several higher eukaryotic regulatory proteins (Murre et al., 1989a). A comparison of the HLH motif of CBFl with other known HLH proteins is illustrated in Figure 3.

Regions

in Predicted

CBFl

Protein

and the

are displayed in open boxes, and conservative amino acid are shown rn stippled boxes. The overall identity is 22.9%.

CBFl Is Homologous to myc and Other HLH Proteins DNA sequence analysis of the 3.0 kb BamHl fragment revealed an uninterrupted open reading frame of 1053 nucleotides capable of encoding a 351 amino acid polypeptide with a calculated molecular size of 39,427 daltons. The DNA sequence of the 1872 bp BamHI-Hpal fragment that comprises the CBF7 gene is shown in Figure 1. The 17 amino acids of the N-terminus of the previously purified 16 kd CBFl (underlined in Figure 1) match the gene sequence perfectly, starting from amino acid 214, and predict a protein fragment of 16,203 daltons in size. This suggests that the 16 kd CBFl protein is either a degradation or processing product.

HELIX

CBFl N-myc L-myc C-myc h da twi MyoD CMDl Myf-5 Myogenin El2 E47 AS-C/T4 AS-C/T5 Figure

3. Sequence

AIN SFL RFL SFF CLN ALK AFK AFE AFE AFE AFE AFK AFR SFA GFS Comparison

Disruption of the CBFl Gene In agreement with the suggestion that the HLH motif is involved in DNA binding (see Discussion), this motif is contained within the 16 kd CBFl fragment we previously purified that binds DNA in a highly specific manner. To generate a null mutation in the CBF7 gene, the 268 bp Ball-Pstl fragment of the cloned gene, which includes the HLH motif, was deleted and substituted by the TRP7 gene. This deletion/insertion construct, when linearized by BamHI-Hpal digestion, was used to transform a diploid yeast strain YNN413, and Trp+ transformants were selected. The expected replacement of one copy of the wildtype CBFl gene by the deletion/insertion construct was confirmed by Southern analysis (data not shown). One of these CBF7 heterozygotes, YNN414, was randomly chosen for further investigation. Sporulation of YNN414 gave rise to four viable spores with 2:2 segregation for normal and small colony size (Figure 4). All small colonies were found to be Trp+, while normal-sized colonies were Trp-. The CBF7 gene in spore clones of both phenotypes was examined by Southern analysis. As expected, only small colonies had the disrupted CBF7 gene (data not shown), indicating that the

I

HELIX

-----"R----------E ---EL"K---------NE ---TLAS---------CS ---ELEN---------NE TKKDPAR---------HS LKSDKP--------------TL----------PSDK p---------------NQRL p---------------NQRL p---------------NQRL p---------------NQRL LNSEKP-------------Q LKSDKJ,-------------Q IITDLTKG--G-GRGPHKKI VIADLSNGRRGIGPGANKKL

of the HLH Motif between

CBFl

and Other

Known

II

ETDE AEEH GAEK AEEQ RQQA QQVR RMLS ALLR ALLR ELLR ALLS QQVR QQVR DLVD KVLH HLH Proteins

Homologous residues are boxed. Two bars on the top indicate the amphipathic helices. Asterisks mark hydrophobic residues that position on one face of the helix (Murre et al., 1989a). Amino acid sequences are taken from CBFl (222-275) human N-myc (393-437; Kohl et al., 1986) human L-myc (289-338; DePinho et al., 1987) human c-rnyc (348-401; Battey et al., 1983) hairy (31-93; Rushlow et al.. 198Q), daughterless (554-813; Caudy et al., 1988). hvist(357-407; Thisse et al., 1988) MyoD (108-164; Davis et al., 1987) CMDl (100-156; Lin et al., 1989) myf-5 (83-139; Braun et al., 1989). myogenin (81-136; Wright et al., 1989). El2 (336-393: Murre et al., 1989a). E47 (336-393; Murre et al., 1989a), T4 (102-167; Villares and Cabrera, 1987). and T5 (27-95; Villares and Cabrera, 1987).

Cell 440

Table

1. Yeast

Strain

Genotype

YNN413

a/a ade2/ade2 his3/his3 leu2/leu2 trpl/trpl ura3/ura3 ala cbfl::TRPl/+ ade2/ade2 his3/his3 leuZNeu2 trp Vtrp 1 ura3/ura3 (I cbfl::TRPl ade2 his3 leu2 trpl ura3 a ade2 his3 leu2 trp7 ura3 a cbfl::TRPl ade2 his3 leu2 trp7 ura3 a ade2 his3 feu2 trpl ura3 a ade2 his3 trp7 ura3 a ade2 leu2 trp7 ura3 a cbfl::TRP7 ade2 his3 lys2 trpl ura3 a cbfl::TRPl ade2 leu2 trpl ura3 a leu2Al lys5

YNN414

Figure

4. The Slow Growth

Phenotype

of the cbfl::TRP7

Mutant

A diploid strain (YNN414) heterologous for the CBF7 gene made by one-step gene disruption (Rothstein, 1983) was sporulated and tetrads dissected. Each small colony contained the cbfl::TRPi allele.

CM7 gene is not essential but is important for cell growth. A more quantitative measurement of the effect of CM7 deletion upon growth rate was obtained by measuring the growth rate of CM7 wild-type cells and cbfl::Tf?P7 mutants in YPD medium. The doubling time of the wild-type cells was 1.8 hr, whereas that of the cbfl::TRP7 mutants was 2.35 hr. To determine whether disruption of the CBF7 gene eliminated the CDEI binding activity, nuclear extracts were prepared from the cbfl::TRP7 mutants as well as from its isogenie wild-type cells. Electrophoretic mobility retardation assays were performed according to the procedure previously reported (Cai and Davis, 1989) and are shown in Figure 5. The double retarded bands produced by wild-type nuclear extracts have been shown to be specific for the CDEI sequence (Cai and Davis, 1989). The cbfl:.TRP7 mutant does not exhibit the centromere binding activity that is present in wild-type cells. This absence of the CDEI binding activity from the cbfl::TRP7 mutant cell was not due to improper preparation of the nuclear extracts, be-

SK1 NUCICU ex*racts @I)

0

Figure 5. The cbfl Binding Activity

124

Disruption

Mutant

CBFl

cbfl::TRPl

124

124

No Longer

Contains

the CDEI

Nuclear extracts were made from a cbfl::TRP7 mutant (YNN415), its isogenic CBFl wild type (YNN416). and a wild-type diploid (SKl) from which the 16 kd CBFl was originally purified (Cai and Davis, 1989). In vitro binding reactions and electrophoretic mobility retardation assays were performed as described (Cai and Davis, 1989). The protein concentrations of the nuclear extracts are 1900 uglml, 750 ug/ml, and 800 pglml for SKI, YNN418 (CBFI), and YNN415 (cbfl::TRPl), respectively.

Strains

YNN415 YNN416 YNN417 YNN418 YNN419 YNN420 YNN421 YNN422 YNN423

Plasmid

pGARS1 pGARS1

cause factors binding to the regulatory region of the RNRP (small subunit of ribonucleotide reductase) gene (Elledge and Davis, 1989) were readily detected in the cbfl::TffP7 nuclear extracts (data not shown). CM7 Is Involved in Chromosome Segregation One possible cause for slow growth of the haploid cbfl::TRP7 mutant is the cell’s inability to maintain and transmit its chromosomes as properly as wild-type cells, owing to the loss of CBFl from the kinetochore apparatus. The effects of the cbfl::TRP7 mutation on chromosome stability were first examined using the colony color sectoring assay (Hieter et al., 1985a). Yeast cells with a nonsense mutation in the AMP gene will accumulate a red pigment and thus turn red upon growth on solid medium. This red color will disappear when the cell contains a tRNA suppressor that can suppress the ade2 nonsense mutation. This phenomenon has been utilized to measure minichromosome stability (Hieter et al., 1985a, 1985b). A 10 kb circular plasmid (pGARS1, Table 1) bearing CEN4, ARS7, URA3, and SUP77 markers was used to transform the cbfl::TRP7 mutant as well as its isogenic wild-type strain. After growing the transformed cells to exponential phase in selective medium, cells were plated onto a synthetic medium supplemented with a limited amount of adenine (see Experimental Procedures). The sectoring pattern is shown in Figure 6. It is obvious that the cbfl::TRP7 mutant has lost the plasmid at a higher frequency than the wild-type cell, since mutant colonies were almost all red, whereas wild-type colonies were predominantly white. Quantitative measurement of the rate of plasmid loss is given in Table 2. The effect of the cbfl::TRP7 mutation on stability of native chromosomes was also examined. A convenient and widely used method is the quantitative mass mating assay (Clarke and Carbon, 1983). A diploid yeast strain can become mating competent when one of its mating loci (MAT) is lost, owing to either chromosome loss or mitotic recombination on chromosome 3 (Figure 7). To distinguish chromosome loss from recombination events, the diploid strain to be examined is made heterozygous for the LEUP gene, which is located on the opposite arm of chromosome 3 from the MATlocus. By measuring the mating type

The Yeast 441

Centromere

Binding

Protein

CBFI

cbfl::TRPl

Figure 6. The cbfI::TRfI Mutant Loses a Minichromosome Faster Than the CBFI Wild-Type Ceil YNN417 (cbf7::TRPl) and YNN418 (CBFI) were grown in uracil-free medium to select the minichromosome. Late log-phase cells were diluted with sterile water and plated on sectoring plates (see Experimental~ Procedures). Cells were allowed lo grow at 3ooC for at least 48 hr and placed at 4% for 24 hr to help color development

than in the wild-type cell. Similar results were also obtained from two different strains (data not shown). Figure 8 shows that the cbfl::TRP7 mutant was more sensitive than the wild type to the antimitotic drug thiabendazole (Matsuzaki et al., 1988), therefore providing additional evidence supporting the involvement of CBf7 in the chromosome segregation process.

Table 2. The Quantitative Measurement of the Minichromosome Stability in CBFI (YNN418) and cbfl::TRP 1 (YNN417) Cellsa Percent Generations

in YPD

0 6 20

Ura+ Cells

YNN417

YNN418

80 30 5

100 95 65

The cbfl::TRPT Disruption Causes Methionine Auxotrophy When the cbfl::TRP7 mutant (YNN415) was inoculated into defined medium supplemented with all of its known auxotrophic requirements (adenine, histidine, leucine, and uracil), it failed to grow. This cbfl::TRP7 strain was then tested for its ability to grow on defined media that lacked single nutritional supplements, and it was found that the strain had a novel requirement for methionine. This methionine auxotrophic phenotype was inseparable genetically from the cbfl::TffP7 genotype. As shown in 6igure 9, all Trp+ segregants from sporulation of the heterozygote YNN414 were Met-. Further analysis showed that the cbf7::TRPI

a Cells were grown in a selective medium (uracil-free) to the late logphase and diluted 1 W-fold into YPD medium lo allow the minichromosome lo escape. At indicated points, cells were taken and diluted with sterile water and plated onto YPD as well as uracil-free plates. Duplicate samples were plated. Growing cells were diluted with YPD medium again when they reached the late log-phase lo ensure their continuous dividing.

as well as the LEU2 marker, chromosome loss and recombination events are readily distinguished from each other (Figure 7). The results of this experiment, as shown in Table 3, demonstrate that chromosome 3 in the cbf7::TRPI diploid mutant is lost at a frequency about lo-fold higher

Icu 2

-

LEU 2 Mitotic

leu 2 LEU2

recombinsrion/

~

MATa

-

hlATa

Figure 7. The Scheme Chromosome 3 Loss

MATa MATa

*

~mosorne

loss

leu 2

-

or

mra

or

leu 2

-

MATa

LEU2

-

MATa

LEU2

-

MATa

Designed

10 Measure

Diploid cells will become mating competent when one of their MAT loci is lost, as a result of either chromosome loss or mitottc recomb!nation. Only those cells whose mating competency is accompanied by a Leu- phenotype are derived from a chromosome loss event.

Cell 442

Table

3. Quantitative

Mass

Mating

Assays

to Measure

the Rate of Chromosome

3 Loss=

Number of Diploid Cells on Each Plate

Number of Colonies on Leu-Free Plates

Number of Colonies on Leu-Containing Plates

Number of MATa Leu-

cbfl::TRPl cbfl::TRPI

1.9 x 10s

120 (*lo)

850 ( + 50)

730

3.8 x 10m4

E CBFl

3.5 x IO”

40 (*lo)

150 (k20)

110

3.1 x 10-S

Cells

Frequency of Chromosome 3 Loss

a The quantitative mass mating assay was used to measure the rate of chromosome 3 loss. A cbfl::TRP7 homozygote was made by a cross between YNN421 and YNN422 (Table 1). An isogenic wild-type diploid strain was made from a cross between YNN419 and YNN420 (Table 1). Each of the diploid strains was mixed with 4.0 x lo* of tester strain (YNN423, Table 1) at the concentrations of 1.0 x l@ (mutant) and 1.8 x IO* (wild type). Cells were concentrated on nitrocellulose filters and placed on a YPD plate for 4.5 hr at 30°C and then suspended into 5 ml of water. Triplicate samples (0.1 ml each) were plated onto minimal plates with or without leucine. The rate of chromosome 3 loss is represented as the ratio of the number of MATa Leu- colonies and the number of diploid cells participating in the mating assay (see Figure 7).

allele did not create a requirement for adenine, leucine, or uracil (data not shown).

histidine,

Discussion CDEI-Specific Centromere Binding Proteins of the Yeast S. cerevisiae Studies on centromere binding proteins are important for understanding the mechanism of chromosome segregation. In recent years, several groups have published their work on identification and characterization of yeast centromere binding proteins, which, interestingly, were all found to be specific to the CDEI sequence. Bram and Kornberg (1987) first identified a CDEI binding activity, CPl, in crude yeast extracts. Although they did not purify the activity, they were able to show the protein size to be about 57-64 kd. Very recently, three independent reports regarding purification of CDEI binding proteins were published. The protein we purified, CBFl (formerly CBP-I), was 16 kd in size (Cai and Davis, 1989). Two other reports provided CDEI binding proteins similar in size to that estimated by Bram and Kornberg (1987), 58 kd (Baker et al., 1989) and 64 kd (Jiang and Philippsen, 1989). Both proteins have been named CPl. The cloning and sequencing of the gene encoding CBFl is now reported. The 16 kd CBFl protein we previously purified is a fragment of a 39 kd protein with DNA binding activity. The smaller size is possibly due to the use of a wild type instead of a protease-deficient yeast strain as a source for protein purification. However, this result is still very different from that obtained by the other groups.

CBFl

cbfl::TRPl

CBFl

cbfl::TRPl

CBFl

Two distinct possibilities are readily apparent. First, there are at least two different CDEI binding proteins in yeast, 60 kd CPl and 39 kd CBFl. They both recognize the CDEI sequence but may play specific roles in vivo. In light of this, the 39 kd CBFl may be the same as the 37 kd CDEI binding protein reported by Jiang and Philippsen (1989) which the authors proposed to be a degradation product of CPl. The second possibility is that there is only one CDEI binding protein, CBFl, and it migrates as 60 kd because of some unusual modifications and/or sequence effects. These two possibilities will become clear when the sequence of CPl is available for comparison. Conservation of the HLH Motif from Human to Yeast Murre et al. (1989a) recently isolated two human genes K chain enhancer binding that encode immunoglobulin proteins, El2 and E47. Both proteins were found to be related to a group of regulatory proteins previously known as the myc gene family (Davis et al., 1987). A structural feature common to all these proteins is a 60 or so amino acid stretch designated by Murre et al. as the helix-loophelix (HLH) motif (Murre et al., 1989a), because two amphipathic helices from this region could be hypothesized. This HLH motif was demonstrated to play an important role in both protein dimerization and DNA binding (Murre et al., 1989a). Various members of the HLH family were shown to be able to form heterodimers and bind to a common DNA sequence (Murre et al., 1989b). In addition to a list of human and Drosophila HLH proteins, the yeast centromere binding protein CBFl is now

cbfl::TFU’l

Figure 8. The cbfl::TRPI Mutant Is More Sensitive to the Antimitotic Drug Thiabendazole I I

0

25

THIABENDAZQLE

50 Qqhd)

Thiabendazole (Sigma) was dissolved methylformamide and mixed with melted agar at indicated concentrations. Only thylformamide was added to the control

in diYPD dimeplate.

The Yeast 443

Centromere

Binding

Protein

Methionhe-free

Tryptophan-free Frgure 9. The Methiomne

Auxotrophic

Phenotype

of cbfl::TRP7

Mutants

Spores from the plate shown m Frgure 4 were grown up rn YPD plate and replica-plated onto tryptophan-free of cells was from one tetrad. All Trp’ cells failed to grow on the methionine-free plate

shown to be a new member of this rapidly growing family. Consistent with the results that the HLH motif in El2 and E47 is responsible for DNA binding, the truncated CBFl (the 16 kd CBFl fragment, which contains the HLH motif) is capable of highly specific binding to its recognition site (Cai and Davis, 1989). In fact, the HLH motif occupies almost the first half of the 16 kd active fragment. It is therefore reasonable to believe that the CDEI binding activity of 16 kd protein is provided by the HLH motif. It is also possible that the HLH motif in CBFl plays a role in the formation of heterodimers that bind CDEI sequence, because it has been suggested that the double retarded bands in the electrophoretic mobility retardation assay performed with yeast nuclear extracts were not generated by CBFl alone (Cai and Davis, 1989). The other putative CDEI binding protein is dependent on CBFl to bind DNA, since both retarded bands disappeared when the CBF7 gene was disrupted (Figure 5). It is interesting to note that the HLH DNA binding proteins share homologies not only in their DNA binding domain, but also in the DNA sequence this domain recognizes, as illustrated in Table 4. MyoD, E12, E47, and CBFl are the only members of the HLH protein family for which DNA binding has been demonstrated and the respective binding site identified. The MyoD binding sequences were recently elucidated by methylation interference assay (Lassar et al., 1989). The binding sites of these HLH proteins have a consensus core sequence NNCANNTG. This consensus core sequence is in fact conserved in all known immunoglobulin intron enhancers (Church et al., 1985) and yeast centromeres (Hieter et al., 1985b). Both the T and G residues in yeast CDEI have been shown to be involved in centromere function (Hegemann et al., 1988), and mutations at either position were found to cause a dramatic decrease in binding efficiency to the purified 16 kd CBFl fragment (Cai and Davis, 1989). This

and methionil ne-free

plates. Each line

raises the possibility that the NNCANNTG core sequence may be an essential structural feature common to all of the recognition sequences of various HLH DNA binding proteins. Although other HLH proteins have been suggested as DNA binding transcriptional regulators, e.g., myc (Ramsay et al., 1986) hairy(Rushlow et al., 1989) daughterless (Cline, 1989). myogenin (Wright et al., 1989) and acheetescute (Villares and Cabrera, 1987) their actual recognition sites are as yet unknown. It is tempting to speculate that they may also contain this NNCANNTG core sequence. CBFl Is Involved in Chromosome Segregation CBFl (the 16 kd active fragment) was pu,rified according to its CDEI binding activity assayed in vitro. Although the sequence specificity of its binding correlates well with the results obtained by in vivo studies, its biological function

Table 4 Conservation of the HLH Proteins

HLH

Proteins

of DNA Binding

Bindmg Assayed

Sequences In Vitro=

Sequences

Locahon

of the Bmding

MyoD

GACATGTG AACACCTG

MCK enhancer MCK enhancer

E12. E47

GGCAGGTG TGCAGGTG

h enhancer )r enhancer

CBFl

uTCACuTG

Yeast

Consensus

nnCAnnTG

Site

E2 E5

centromeres

a There are two MyoD binding snes in the muscle creatme kinase (MCK) enhancer, both of which were identified by methylation interference as well as mutagenesis studies (Lassar et al., 1989). Both El2 and E47 proteins bind the K E2 box specifically, and the bindmg could be effectively competed by the n E5 box (Murre et al.. 1989a). SpecrfIC binding of the CDEI sequence of the yeast centromere by purified CBFl has been described (Cai and Davts, 1989). The u residues rn the binding sequence represent purines.

Cdl 444

as a genuine component of the yeast kinetochore has to be evaluated by genetic analysis. The results presented in this paper strongly suggest that CBFl is involved in the process of chromosome segregation. Genetic studies on the relationship between the centromerit DNA structure and its chromosome-stabilizing function have shown that the CDEIII subregion is the most important element for mitotic centromere function, whereas CDEI plays only a minor role (Cumberledge and Carbon, 1987; Hegemann et al., 1988; Panzeri et al., 1985). Point mutations as well as complete deletion of CDEI from the centromere decreased mitotic chromosome stability at most by one order of magnitude (Cumberledge and Carbon, 1987; Hegemann et al., 1988). In agreement with these studies, our results showed that the haploid cbfl null mutant is viable, indicating that CBFl is not essential for centromere function. However, the cbfl::TRPl mutant does exhibit increased chromosome loss as tested in both haploid and homozygous diploid mutants. Chromosome 3 is lost at a lo-fold increased rate, which is quantitatively comparable with the level obtained with the mutants whose CDEI elements on specific chromosomes are altered or deleted. The other phenotype of the cbfl::TRPl mutant, the hypersensitivity to the antimitotic drug thiabendazole, also suggests that the CBFl gene is involved in mitosis. Genetic analysis of cbfl mutants, therefore, supports the notion obtained through biochemical studies that CBFl is likely to be an authentic component of the yeast kinetochore.

above studies. Since this phenotype is inseparable from the cbfl::TRPl genotype, it is likely that the Met- phenotype is a result of the inactivation of transcription of one or more MET genes, owing to the loss of CBFl activity as a UAS binding protein. The CBFl-mediated regulation does not seem to play a major role in expression of genes encoding electron transport chain proteins, which also contain the CDEI sequence in their upstream region (Dorsman et al., 1988), since the cbfl::TRPl mutant is able to grow on nonfermentable carbon source (data not shown). These genes, unlike MET2, MET3, MET25, and SAM2, have only one copy of the CDEI sequence. It is therefore possible that the structural genes for electron transport chain proteins are regulated by a mechanism where CBFl is either not involved or plays only a minor role. The Cflfl gene may be allelic with one of the previously described methionine auxotrophic mutants. The physiological relationship, if there is any, between chromosome stability and methionine metabolism is unknown. However, it is interesting to note that many mammalian cancer cells also exhibit these two phenotypes: chromosome instability (for a review see Yunis, 1983) and methionine auxotrophy (for a review see Hoffman, 1984). It has been shown that normal rat epithelial cells become methionine dependent upon transformation by the activated H-ras-1 oncogene (Vanhamme and Szpirer, 1987). These results suggest a novel and unexpected connection between the control of methionine synthesis and the control of cell cycle.

CBFl May Act as a Transcriptional Activator The possibility that CDEI acts not only as a component of yeast centromere but also as a UAS (upstream activation sequence) element for transcription was first suggestgd by Bram and Kornberg (1987), who noticed that the CDEI sequence is present in upstream regions of a number of genes from yeast and other eukaryotes. They also speculated that the factor (CPl) bound to the centromere CDEI sequence may act as a transcription factor. Dorsman et al. (1988) later found the CDEI sequence in upstream regions of a number of nuclear genes coding for mitochondrial proteins and a factor (GFII) from cell extracts that could form specific complex with these CDEI-containing fragments in vitro. The direct evidence to support this hypothesis was provided recently by Thomas et al. (1989), who dissected the upstream region of the MET25 gene and identified two direct repeats of CDEI sequence as UAS elements. They demonstrated that the two CDEl-containing UAS sequences are both required for efficient MET25 expression. Deletion of one copy of the CDEI-containing UAS sequences reduced MET25 gene expression by 57%-740/o, while deletion of both CDEI-containing UAS sequences resulted in 90% reduction in gene expression. Duplicate CDEI sequences are also found in the upstream regions of MET2, MET3, and SAM2 (coding for S-adenosylmethionine synthetase) genes (Thomas et al., 1989). The authors suggested that these methionine metabolism genes may be regulated in a similar manner. The fact that a cbfl null mutation caused a methionine auxotrophic requirement is clearly consistent with the

Experimental

Procedures

Yeast Strains and Genetic Methods The yeast strains used in this work are listed in Table 1. The diploid strain YNN413 (obtained from R. Fuller) was made from a cross between isogenic a and a cells and used as a parental strain for CBF7 gene disruption and genetic analysis. The plasmid present in YNN417 and YNN418, pGARS1. contains ARS7, CEN4, SUP17, and UffA3 markers and has been used in a colony color assay lo measure minichromosome stability (Snyder et al., 1988). Standard yeast genetic methods were used for phenotype analysis, strain crosses, and tetrad dissection (Sherman et al., 1986). Yeast transformation was performed with the lithium method (lto et al., 1983). The CBFl gene drsruption was done according to Rothstein (1983).

Cloning of the CBFI Gene Sequence analysis of the N-terminal region of purified 16 kd CBFl (Cai and Davis, 1989) was performed in the Protein Structure Laboratory (Department of Biochemistry and Biophysics, Uncversity of California, Davis, California). Seventeen amino acids were obtained (amino acid 6 was unidentified): ATTDE(

)KKQRKDSHKEVE I II

Two completely degenerate oligonucleotide pools, corresponding to the underlined fragment I and fragment II, were synthesized as follows: Probe I (17 nucleotldes)

S-AA(A,G)AA(A,G)CA(A,G)(C,A)G(A,C,G;r)AA(A.G)GA-3

Probe II (14 nucleotides)’

WA(C,T)AA(A.G)GA(A,G)GT(A,C,G,T)GA-3

Probes were end-labeled with T4 polynucleotide kinase and [u-~*P] ATP and used to screen a yeast cDNA library (obtained from S. Elledge), according lo Wood et al. (1985). From 100,000 clones screened, 3 were obtained that could hybridize to both probes. The homology to

The Yeast 445

Centromere

Binding

Protein

oligonucleotide probes was further localized to a 266 bp Ball-Pstl fragment, which was sequenced and shown lo correctly match the oligopeptide sequence from which the probes were derived. The cDNA msert was then used as a probe to screen a yeast genomic library (obtained from S. Elledge). The CBFl gene was finally cloned as a 3.0 kb BamHl fragment (pNN450) whose complete sequence was determined. DNA Sequencing and Computer Analysis The BamHl fragment of the CBFI gene (pNN450) was cloned into the Bluescript KS plasmid (Stratagene), and a deletion series was made unidirectionally according to Henikoff (1984). DNA sequencing was performed using the dideoxy chain termination method (Sanger et al., 1977) with Sequenase, a modified T7 DNA polymerase (US Biochemicals). DNA sequence data were analyzed on the BIONET computer of Intelligenetics, Inc. (Mountain View, California). Search of sequence data bases for homologous sequences was done via FASTA-MAIL program of the BIONET computer. Quantitative Mass Mating Assay Quantitative mass mating experiments were performed lo determine the frequency of chromosome 3 loss. The procedure was derived from Clarke and Carbon (1983). A homozygous cbf7t:TRPI diploid mutant was made from a cross between YNN421 and YNN422 (Table 1). A wild-type diploid lo be used as a control strain was made from a cross between YNN419 and YNN420 (Table 1). Exponentially growing diploid cells were mixed with a haploid mating type tester strain (YNN423, Table 1) and concentrated onto a 0.45 mm nitrocellulose filter (Millipore Corp.) at a density of about 1 x lo8 for each strain. The filter was transferred to a YPD plate and incubated for 4-5 hr at 30°C. The cells were then washed from the filter into sterile water and plated on selective media. MATa Leu- cells were scored. The chromosome loss frequency is expressed as a ratio of the number of MATa Leu- cells and the number of diploid cells participating in the mating process. Other Methods The colony color assay (Hieter et al., 1985a) was employed to measure mimchromosome stability in diploid cbfl::TRPI mutants. Color sectoring was allowed to occur on minimal media supplemented with all auxatrophic requirements, exceot adenine. which was added at a concentration of 6 pglml rather than 20 Fglml as described in Sherman et al. (1988). This level of adenine is optimal for both cell growth and color formation (Hieter et al., 1985a). Preparation of yeast nuclear extracts and electrophoretic mobility retardation assays for DNA-protein interaction were performed as previously described (Cai and Davis, 1989). Acknowledgments We thank S. Elledge, J. McCusker, S. Klapholz, I. Chambers, and R. Fuller for their valuable discussions and S. Elledge for providing yeast libraries. We also thank Intelligenetics, Inc. for assistance with computer analysis. This work was supported by National Institutes of Health grant 5R37 GM21891 lo R. W. D.; M. Cai is a predoctoral fellow of the Markey Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. SectIon 1734 solely lo indicate this fact. Received

January

25, 1990; revised

March

7, 1990.

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Methods

in Yeast

GenBank

Accession

The accession M33620.

number

D. A., and Lin, V. K. (1989). Myogenin, has a domain homologous to MyoD. chromosomal

basis

of human

Base chlogene

a factor Cell 56,

neoplasia.

Number for the sequence

reported

in this paper

is