directionality of fission yeast mating-type interconversion …...mat2 or mat3, the event being...

Copyright 0 1993 by the Genetics Society of America

Directionality of Fission Yeast Mating-Type Interconversion Is Controlled by the Location of the Donor Loci

Genevieve Thon and Amar J. S. Klar NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Laboratory of Eukaryotic Gene Expression,

Frederick, Maryland 21702-1201 Manuscript received January 26, 1993

Accepted for publication April 22, 1993

ABSTRACT Cells of homothallic strains of Schizosaccharomyces pombe efficiently switch between two mating types

called P and M. The phenotypic switches are due to conversion of the expressed mating-type locus (matl) by two closely linked silent loci, mat2-P and mat3-M, that contain unexpressed information for the P and M mating types, respectively. In this process, switching-competent cells switch to the opposite mating type in 72-90% of the cell divisions. Hence, mat2-P is a preferred donor of information to matl in M cells, whereas mat3-M is a preferred donor in P cells. We investigated the reason for the donor preference by constructing a strain in which the genetic contents of the donor loci were swapped. We found that switching to the opposite mating type was very inefficient in that strain. This shows that the location of the silent cassettes in the chromosome, rather than their content, is the deciding factor for recognition of the donor for each cell type. We propose a model in which switching is achieved by regulating accessibility of the donor loci, perhaps by changing the chromatin structure in the mating-type region, thus promoting an intrachromosomal folding of mat2 or mat3 onto matl in a cell type-specific fashion. We also present evidence for the involvement of the Swi6 and Swi6- mod trans-acting factors in the donor-choice mechanism. We suggest that these factors participate in forming the proposed folded structure.

D IRECTED recombination events are a key fea- ture of certain cell differentiation programs. One model system for the study of such events is mating-type switching in the fission yeast Schirosac- charomyces pombe (for reviews, see EGEL 1989; KLAR 1989; GUTZ and SCHMIDT 1990). S. pombe cells exist in two mating types, P and M , that are respectively determined by the matl-P and matl-M alleles of the mating-type locus (LEUPOLD 1958). In addition to the expressed matl locus, the mating-type region contains two storage cassettes, mat2-P and mat3-M, that are not transcribed (EGEL and GUTZ 1981; BEACH 1983; BEACH and KLAR 1984; EGEL 1984; KELLY et al. 1988; Figure 1 A). The P information contained at mat2 and the M information contained at mat3 are used for gene conversions of matl that result in mating-type switching. This arrangement of the mating-type region found in homothallic (i.e., switchable) strains is designated h9'. The conversions are thought to be initiated by a site-specific double-stranded break (DSB) of the DNA found at matl (BEACH 1983; BEACH and KLAR 1984; NIELSEN and EGEL 1989; SINGH and KLAR 1993).

In switching-competent h9' cells, switches to the opposite mating type occur in 72-90% of the cell divisions (MIYATA and MIYATA 1981; EGEL and EIE 1987; KLAR 1990; Figure 1 B). This high frequency of switching to the opposite mating type shows that Genetics 134 1045-1054 (August, 1993)

the donor for the matl conversion is not chosen ran- domly. Rather, the mat2-P cassette is preferably (or only) selected in M cells and, conversely, the mat3-M cassette in P cells. We have used a genetic approach to investigate the reason for this cell type-specific preference for one donor vs. the other.

Three models may be envisioned to explain the preferential switching to the opposite mating type. First, all information required for choosing the proper donor could be contained within the donor cassette (model 1). Second, the cells may choose a particular donor because of its chromosomal location (model 2). A third possibility is that donor choice may be random. By this mechanism, multiple switches could occur in each cell cycle, the process stopping only after a switch to the opposite allele has been executed (model 3). To distinguish among these models, we constructed a strain in which mat2 contains the M information and mat3 the P information. We refer to this arrangement of the mating-type region as ho9, that is, the reverse of h9'. If cassette content is sufficient for directing the switch (models 1 and 3), ho9 cells should switch to the opposite mating type with an efficiency similar to that of h9' cells. If location of the donor locus is the deciding factor (model 2), then ho9 cells should not switch to the opposite mating type efficiently.

Pedigree analysis of switching of ho9 cells demon-

1046 G. Thon and A. J. S. Klar

B M M /"\

/ \ / \ /\ P M P M

M P M P M P M M FIGURE I.-S. pombe h90 mating-type region and the rules of

switching. (A) Mating-type region. matl, mat2 and mat3 are evenly spaced in the right arm of chromosome I I (EGEL and GUTZ 1981; BEACH 1983; BEACH and KLAR 1984). The three cassettes are bordered by identical DNA sequences: H1 (59 bp) on the side distal to the centromere (CENII) and H2 (135 bp) on the centromere- proximal side. An additional region of homology, H3 (57 bp), is shared only by mat2 and mat3. matl is transcribed and contains P or M sequences; substitution of one sequence for the other leads to mating-type switching. mat2 (1 1 13 bp) and mat3 (1 127 bp) encode functions required for the expression of the P and M cell types, respectively, but are transcriptionally silent (KELLY et al. 1988). A DSB, found at the junction of the allele-specific sequence and the H1 region at matl (BEACH 1983; BEACH and KLAR 1984; NIELSEN and EGEL 1989), is thought to initiate gene conversion of matl by mat2 or mat3, the event being resolved within H2. The "cold spot" indicates a total lack of meiotic recombination in the mat2-mat3 interval (EGEL 1984). The matl, mat2 and mat3 cassettes are found on 10.4, 6.3 and 4.2 HindIII restriction fragments, respectively (BEACH 1983). Lollipops represent the HindIII restriction sites flanking the cassettes. The figure is not drawn to scale. (B) Rules of switching. hPo cells switch to the opposite mating type following a regular pattern: (1) Only one in four "granddaughters" of a cell ever switches mating type (MIYATA and MIYATA 1981). (2) The "sister" of a newly switched cell is switching-competent as it usually produces one switched daughter. The other daughter is unswitched but switching-competent. This generates lineages of switching-competent cells whose probability of switching to the opposite mating type is 72-90% (EGEL and EIE 1987; KLAR 1990). These rules of switching apply to cells of either mating type. That the observed probability of heterologous switching is higher than 50% demon- strates that the donor choice for the matl conversion is nonrandom.

strates that the content of the cassettes is not chosen per se, ruling out models 1 and 3, but rather, that the location of the silent information is important for the directionality of mating-type switching. Based on this result, we propose a model in which the availability of the mat2 and mat3 loci for converting matl varies with the cell type. This model postulates the existence of trans-acting factors involved in folding the mating- type region to preferentially bring one or the other donor locus close to matl. We present evidence that swi6, a gene required for efficient mating-type switching in h90 cells (EGEL, BEACH and KLAR 1984) and also known t o function to inhibit meiotic crossing over between mat2 and mat3 (KLAR and BONADUCE 1991;

LORENZ, HEIM and SCHMIDT 1992), plays a role in the directionality of switching. Supporting this evidence, we find that a locus that suppresses the swi6- mutation, swib-mod, also affects the donor choice.

MATERIALS AND METHODS

Strains, transformation and culture conditions: All strains were constructed in this laboratory using standard genetic techniques. DNA-mediated transformation was performed using the lithium acetate protocol developed by ITO et al. (1 983) for Saccharomyces cerevisiae transformation and used for S. pombe by HEYER, SIPICZKI and KOHLI (1986). The genotypes of the strains used are reported in Table 1. The media (complete medium, medium lacking an amino acid or nitrogen-free medium to induce sporulation) were as in MORENO, KLAR and NURSE ( 1 991).

Random spores analysis: Spores were obtained by treat- ing sporulated cultures with snail enzyme (NEE-154 Glusu- lase, f rom Du Pont) to selectively eliminate the vegetative cells (MUNZ and LEUPOLD 1979). Lysis of the vegetative cells was verified by microscopic examination before plating ap- propriate dilutions of the spore material.

Assay of the efficiency of switching by iodine staining: Estimates of the efficiency of switching were obtained by staining individual colonies grown on sporulation medium with iodine vapors (BRESH, MULLER and ECEL 1968). A starch-like compound produced by sporulating cells results in black staining of the colonies after exposure to iodine vapors. Intensity of the black stain is an indication of the efficiency of switching to the opposite mating type at the colony level. Cells that switch their mating type efficiently both from P t o M and from M t o P form colonies in which mating and sporulation are extremely efficient due to the homogeneous distribution of the two cell types. Such colonies are stained very darkly by iodine vapors. Cells that switch their mating type with a lower efficiency, at least in one direction, form lighter staining colonies because mating and sporulation within the colony are not as efficient. When the rates of switching are low in both directions, two types of colonies are formed. One type, initiated by P cells, is predominantly composed of P cells while the other type, initiated by M cells, is predominantly composed of M cells. When spores of these cells are plated at densities such that some of the resulting colonies are in contact with each other, iodine staining is observed at the junctions of approximately 50% of pairs of colonies, showing that these colonies contain cells of mostly opposite mating type. In contrast, strains that switch efficiently in one direction and inefficiently in the other direction form colonies of a single predominant mating type, that is the type whose switching is inefficient. The regions of contact between colonies from such strains are not stained by iodine. The predominant mating type in such light-staining individual colonies can be assessed by mixing the cells in patches with stable P (matl-P, mat2,3A::LEU2) and stable M (matl-M, mat2,3A::LEU2) tester strains described earlier (KLAR and MICLIO 1986). Iodine staining of the patch identifies the predominant mating type of the cells in question. In all such experiments, random spores derived from sporulated cultures were plated. This regime assures that we tested clones originating from both mating types. Staining of over 50 colonies was tested for each genotype.

Construction of mat2-M and mat3-P cassettes in vitro: A cloned 6.3-kilobase pairs (kb) Hind111 genomic fragment containing mat,?-P and a cloned 4.2-kb Hind111 genomic fragment containing mat3-M (BEACH 1983; Figure 1) were used to construct a mat2-M and a mat?-P cassette (Figure 2).

Directionality of Switching

TABLE 1

S. pombe strains

1047

Strain ~ ~~~~

mat regiona mi6 mi6 -mod

SP62 h 90 + SP887 hW + + SP926 h9' swi6-115 SP952 h" swi6-115 + SP982 h" + PC8 matP-M + + PC1 1 mat3-P RV::ura4 + + PC17 mat3-P RV::ura4 swi6-115 + PC1 9 ho9 + + PC25 matP-M swi6-115 + PC27 ho9 + PC68 ho9 swi6-115 + PC74 ho9 swi6-115 -

-

-

-

-

Auxotrophic markers

leul-32, ura4 leul-32, ura4-Dl8, ade6-MZ10 h i d , ade6-MZ16 his.2, ade6-M210 h i d , ade6-M216 leul-32, ura4-DI8, ade6-MZ10 leul-32, ura4-DI8, ade6-M216 leul-32, ura4-Dl8, ade6-MZ16 leul-32, ura4-Dl8, ade6-MZ10 leul-32, ura4-DI8, ade6-MZ10 ade6-M210 ura4-Dl8, ade6-MZ16 leul-32, ade6-MZ10

PC251 ho9 - + + - leul-32 ade6-M210 matI-PAI7::LEU2,mat2,3A::leu2 + + + ade6-MZ16

PC595 h90 swi6-I15 - leul-32, ura4, ade6-MZ10 h9' is a wild-type mat2-P, mat3-M arrangement while ho9 contains the swapped matP-M, mat3-P cassettes.

To make such constructions possible, an NcoI restriction site was introduced in vitro at the distal boundary of the mat3- M cassette by oligonucleotide-directed mutagenesis (oligonucleotide used: 5'CTCTCGTTCGTTTCCATGGTATC- CAAATATGTTTGTTTGGC3') with an in vitro mutagenesis system version 2 from Amersham. Parental and mutated plasmids differ only in the first base pair located centromere- distal to the homology box H 1. That difference is indicated by an asterisk above the sequences of mat3-M (wild-type) and mat3-M' (mutated) in Figure 2A. The plasmids carrying mat3-M' and mat2-P were then both cleaved by the endonucleases BglI and NcoI. The BglI-NcoI fragments containing the silent information were swapped between them, to pro- duce a mat2-M (plasmid pGT65) and a mat3-P cassette (plasmid pGT66). In a subsequent construction, a 1.8-kb HindIII restriction fragment containing the S. pombe ura4 gene (BACH 1987) was filled in at the ends and inserted into an EcoRV site located 150 base pairs (bp) distal to mat3 in the mat3-P construct, creating the mat3-P RV::ura4 construct (plasmid pGT67). All cloning steps were performed using standard techniques (SAMBROOK, FRITSCH and MANIATIS 1989).

Construction of ho9 strains: The mat2-M and mat3-P cassettes were introduced in a h9' strain by DNA-mediated transformation in two sequential steps. The first step consisted in replacing the mat2-P cassette with the mat2-M cassette constructed as described above. T o this end, SP887 cells were cotransformed with the replication-competent LEU2-containing plasmid pIRT2 (HINDLEY et al. 1987) and the 6.3-kb Hind111 fragment containing mat2-M. Substitu- tion of mat2-P with mat2-M should generate transformants lacking the P information and therefore incapable of sporulating. The Spo- transformants were identified as they do not stain with iodine vapors. Replacement of the mate-P cassette by a mat2-M cassette was verified by Southern blot analysis (Figure 2B, lane labeled 2-M, and strain PG8 in Table 1). The second step of the ho9 strain construction consisted in replacing the mat3-M cassette with a mat3-P cassette in mat2-M (PC8) cells. This was accomplished by cotransforming PC8 cells with the plasmid pIRT2 and the 4.2 kb HindIII fragment containing the mat3-P cassette constructed as described above and screening the resulting Leu+ colonies with iodine vapors. Southern blot analysis of the stable Spa+, Leu+ transformants revealed that mad-M

had been replaced by mat3-P in these transformants (Figure 2B, strain PC19 in Table 1). In addition to the stable Spo+ transformants, a large number of unstable Spo+ transformants were found in which haploid cells were undergoing aberrant meiosis. These transformants still contained a chromosomal mat3-M cassette, and in addition contained a mat3- P cassette on an unstable episome. Presumably, coexpression of both mating types in these transformants triggers "haploid meiosis," as observed previously (KELLY et al. 1988; THON and KLAR 1992). Such transformants were not char- acterized further.

We also constructed an ho9 strain in another way, by obtaining crossing over events between mat2 and mat3. We felt that this was necessary because the second step of the construction described above was biased in that only Spo+ colonies were examined. It is possible that cells with swapped donor cassettes never switch to the opposite mating type and therefore should never stain with iodine vapors. We cotransformed SP887 cells with pIRT2 and the 6.0-kb HindIII fragment containing the mat3-P RV::ura4 construct described above. Leu+ transformants were selected. Ho- mologous recombinants at mat3 were identified among the Leu+ Spo- transformants by Southern blot hybridization (strain PG11, Table 1). [We found that the ura4 gene inserted near mat3 in the chromosome could not be used as a selectable marker as its expression is presumably repressed by the same mechanism that silences mat3 (THON and KLAR 1992).] The swi6- mutation (mi6-115 allele) was crossed into the strains PG8 and PG11 to construct the strains PG25 and PC1 7, respectively. Diploids were produced by the mating of PG25 and PG17 cells, allowed to sporulate and subjected to random spore analysis. swi6- mutants undergo meiotic recombination in the mat2-mat3 interval (KLAR and BONADUCE 1991 ; LORENZ, HEIM and SCHMIDT 1992), which is prohibited in swi6+ cells (EGEL 1984). As expected, approximately 1 % of the colonies grown from the spores of the PG25 X PC17 diploid were stained lightly by iodine vapors, a phenotype identical to that of h9", swi6- colonies (EGEL, BEACH and KLAR 1984). In addition, about 1% of the colonies exhibited a novel darker iodine-staining phenotype. Southern blot hybridization established that the cells forming the light-staining colonies indeed had h90 chromo- somes, whereas the cells forming darker-staining colonies had mat2-M, mat3-P RV::ura4 mating-type regions. A sub-


A NWl I 711 Ncol

h09 - H 3 H 2 O H 1 H 3 H 2 B H l - I mat2-M mat3-P

t

mar3-M -GGCAC;CCTCGTAGGCTT-- M -CGllTCCATGhATC--

mar3-M -GGCPGCCTCGTAGGCTT-- M -CGl"fCCAT&TATC--

mar2-P -GGCAGCCTCGTAGGCTT-- p -CGTlTCCATGGTTTG-- Bgl I Ncol

4.2 kb- - - mat3

Probe P Probe M

FIGURE 2.-Construction of the ho9 strain. (A) Construction of mat2-M and mat3-P cassettes. The mat2-P and mat3-M DNA fragments located between the conserved elements H1 and H3 were swapped in vitro between the two cassettes and the rearranged cassettes introduced into the chromosome in place of mat2-P and maf3-M (see MATERIALS AND METHODS). 'BglI" and 'NcoI" indicate restriction sites for the BglI and NcoI endonucleases that were used for constructing the swapped cassettes. The nucleotide replaced by oligonucleotidedirected mutagenesis to create an NcoI site in mat3- M (see MATERIALS AND METHODS) is indicated by an asterisk. DNA sequences are from KELLY et al. (1 988). (B) Southern blot analysis of Hind111 restriction fragments of DNA derived from hW strain SP887. from the intermediate PC8 strain with two silent M cassettes (2") and hm strain PG19, probed with the 10.4-kb Hind111 restriction fragment containing matl-P (probe P, left) or matl-M (probe M, right). Different levels of hybridization of various fragments are explained by the different extent of shared sequences with the probes employed. The 5.4-kb matl-proximal (P) and 5.0-kb matl- distal ( D ) fragments result from site-specific double-stranded break of the 10.4-kb matlcontaining fragment in vivo (BEACH 1983).

sequent genetic cross removed the mi6- mutation from the ho9 strain. The mat2-M, mat3-P RV::ural, mi6+ cells mated and sporulated inefficiently. Their colonies displayed a light iodine-staining phenotype identical to the phenotype of ho9 strains constructed by the first procedure. In summary, the two construction strategies generated ho9 strains with the same sporulation phenotype, which also behaved identically

in subsequent crosses. Pedigree analysis of ha loid cells: Efficiency of mating-

type interconversion in h9'and ho9 haploid cells was meas- ured as described previously (MIYATA and MIYATA 1981). The procedure consists of microscopically observing zygote formation in the undisturbed progeny of single cells grown to the four cell stage on solid mating-inducing medium. Only one zygote is ever formed among four related cells indicating a single switch at the four cell stage.

Pedigree analysis of diploid cells: The above-mentioned procedure cannot be used to determine the efficiency of switching in a particular direction. Determination of that requires pedigree analysis, testing the competence of diploid cells to undergo meiosis and sporulation according to the procedure of ECEL and EIE (1 987) and KLAR (1 990). The efficiency of P-to" switching in the ho9 chromosome was assayed by pedigree analysis of diploid cells of strain PG25 1 (ho9/matl-PA1 7::LEU2 mat2,3k:leu2). The mat2,3k:LEU2 allele is an 18-kb deletion of the donor loci that are replaced by the S. cereuisiae LEU2 gene (KLAR and MIGLIO 1986). The matl-PAI7::LEU2 allele carries a small deletion at matl that prevents formation of the DSB and, consequently, matl switching (ARCANCIOLI and KLAR 1991). Therefore only switching of the ho9 chromosome is possible in PC25 1 cells. A matl-P/matl-PAI7::LEU2 cell does not sporulate as it lacks an expressed matl-M allele. Such cells divide on solid sporulation medium with a generation time of 6-8 hr. As soon as the switching process converts the matl-P allele to the matl-M allele in the ho9 chromosome, the switched cell stops growing and sporulates. Thus, assay of sporulation of individual PC25 1 cells in cell pedigrees tests the pattern and the efficiency of switching from matl-P to matl-M in the ho9 chromosome.

RESULTS AND DISCUSSION

The efficiency of mating-type interconversion is reduced when the content of the donor loci is swapped: We constructed a strain that contains a matZ-M cassette in place of the mat2-P cassette, and a mat3-P cassette in place of the mat3-M cassette (ho9 arrangement of the mating-type region, shown in Figure 2). Like the mat2-P and mat3-M cassettes of h9' cells, the mat2-M and mat3-P cassettes of ho9 cells were transcriptionally silent. This was shown by the fact that haploid ho9 cells placed on sporulation medium did not undergo "haploid meiosis," a phenotype expected from cells that express both P and M information (KELLY et al. 1988). The level of DSB in ho9 cells was also similar to that of h9' cells (Figure 2B). We assayed the efficiency of switching to the opposite mating type in ho9 cells by pedigree analysis (Figure 3), as well as by iodine staining at the colony level (Figure 4). We found that switching to the opposite mating type was much reduced in ho9 cells as compared with h9' cells (Figures 3A and 4, left two colonies). This result rules out models 1 and 3 (see the Intro- duction) and is consistent with the model of donor- selection by position rather than donor genetic content.

It is clear that ho9 cells switch inefficiently to the opposite matl allele. Either fewer switches are performed, or predominantly futile homologoustassette

Directionality of Switching 1049

A / p \ p P

M

M ' ' M , ,

Frequency of 1 switched cell among 4 "cousins":

h90 78% (86/110)

ha9 18% (23/126)

2"

predominant predominant interactions interactions

in Pcells in M cells

FIGURE 3.-(A) Efficiency of switching in ho9 and hW strains of S. pombe. Efficiency of switching was determined for haploid cells of strains PC27 (hop and SP982 (hy according to MIYATA and MI- YATA (1981). The numbers in parenthesis indicate actual switches of one in four cousin cells. (B) Model for the directionality of mating-type interconversion. Donor choice is accomplished by cell type-specific intrachromosomal folding of the donor loci onto m a t l . In matl-P cells, mat3 is a more accessible donor than mat2, which favors heterologous switching in the hW chromosome and homologous (or inefficient) switching in the hW chromosome. Conversely, in M cells, ma12 is more accessible than mat3.

switches occur, in which mat2-M donates information to mall-M and mat3-P to matl-P. These possibilities cannot be distinguished at present as there is no assay

swi6-mod -

s wi6 + s wi6 -

h09

h90

available for quantitating homologous mating-type switching in S. pombe. In principle, pedigree analysis of a strain with a novel genotype, matl-P, mat2-M, mat3-M, should diferentiate between the two possibilities. Unfortunately, this experiment is impossible to carry out since the matl-P allele will be quickly changed to mall", prohibiting construction of such a strain. Although we were unable to perform a defin- itive test, we note three observations that are compatible with homologous mating-type switches occurring efficiently in ho9 cells. First, the level of DSB at matl was similar in ho9 and h9' cells (Figure 2B). Therefore, recombination events at matl may occur equally efficiently in both genotypes. In this context though, it is important to realize that S. pombe cells can form and maintain a DSB at matl without switching, since cells deleted for both donor loci are viable and contain wild-type levels of DSB (KLAR and MIGLIO 1986). Thus, ho9 cells do not need to switch to form and heal their DSB. Second, in the mi@, mi6-mod+ genetic background, ho9 cells switched from matl-P to matl- M at a rate of 46%, suggesting that ho9 cells are not defective in switching per se (see below). Third, the observed rates of heterologous switching of 78% in h9' and 18% in ho9 (Figure 3A) are compatible with the notion that nearly 100% of the cells, both ho9 and h9', generate one switched cell in four granddaughters. Approximatively 80% of the h9' cells would switch to the opposite mating type, 20% switching homologously because of some leakiness in the directionality control. Conversely, the directionality control would cause around 80% of the ho9 cells to switch homologously, the leakiness of the control allowing the remaining 20% to switch heterologously.

Two previous observations suggest that the normal arrangement of the silent information with respect to

swi6-mod +

s wi6 + s wi6 -

FIGURE 4.-1odine-staining phenotypes of hW and hW colonies and the effects of the swi6-I15 allele and its modifier. Wild-type hW cells switch, mate to form zygotes, sporulate efficiently and consequently pro- duce dark-staining colonies. Pro- nounced imbalances in the ratio of P- t o " cells lead to the extremely light- staining phenotypes of the hW, sw'6-, swi6-mod- and ho9, swi6+, swi6-mod+ strains. The colonies displayed are of the following strains: ho9, JWW, swi6- mod-: PC27; hW, swi6-, swi6-mod-: PG74; ho9, swi6+. m'6-mod+: PG19; hW, swi6-, swi6-mod+: PG68; hW, swi6+, swi6-mod-: SP982; h", S W K , swi6-mod-: SP926; hW, swi6+, m'6- mod+: SP887; hW, swi6-, swi6-mod+: SP952. White areas, particularly in the hW, swi6+ colonies, are artifacts resulting from reflections of the light used to take the photograph.


matl is essential for efficient switching. First, a sub- stantial reduction of switching occurs when the donor information is derived from the homolog (KLAR and BONADUCE 1991). Second, although the physical dis- tance between mat2 and mat3 is approximately 15 kb, no meiotic crossing over events were found in the interval at a resolution of 0.001 cM (EGEL 1984). According to the overall correspondance between genetic and physical distances in the S . pombe genome, 15 kb should place mat2 and mat3 about 3 cM apart. Reduced recombination is also found in the matl-mat2 interval (LEUPOLD 1958; KLAR and MICLIO 1986; KLAR and BONADUCE 1991). The existence of this “cold spot” for meiotic recombination between and around mat2 and mat3 indicates that the chromosome attains a novel structure in the mating-type region, prohibiting homologous chromosomal interaction in this region. We propose that such a structural arrangement promotes utilization of a specific donor regard- less of its genetic content (Figure 3 B ) .

Trans-acting factors involved in the directionality of mating-type switching: We searched for trans- acting factors involved in forming the folded structure proposed in our model (Figure 3 B ) . Our model pre- dicts that mutations in genes encoding such factors might increase heterologous switching in ho9 and decrease heterologous switching in h9’ by allowing the cassette at the “wrong” location, that is, the one that contains the same information as matl in hyo, and the opposite information in hoy, to act as a donor. Muta- tions that diminish or prohibit usage of a specific donor should affect the ratios of P to M cells in an opposite fashion in populations of hyo and hoy cells. In fact, the existence and behavior of such mutants can test our model.

Swi6 is needed more for mat2 utilization than for mat3 utilization: Among the 11 groups of mutations known to affect switching in h90, the mutation in sw26 (swi6-1 I5 allele) reduces switching without affecting the amount of DSB at matl (EGEL, BEACH and KLAR 1984). Also, this mutation increases meiotic crossing over in the “cold spot” between mat2 and mat3 by more than lOOO-fold, to a frequency similar to the recombination frequency in other parts of the genome (KLAR and BONADUCE 1991 ; LORENZ, HEIM and SCHMIDT 1992). This last observation points to a possible role of SwiG in promoting intrachromosomal interaction between the silent loci and mat l , perhaps by folding the donor loci onto mat l . Such an arrangement would sequester this region, possibly prohibiting crossing over in the mat2-mat3 interval (KLAR et al. 1991). It was, therefore, of interest to determine the phenotype of the swi6-115 mutation in a strain bearing the ho9 gene arrangement.

We used the iodine staining procedure to compare the efficiency of heterologous switching in hy” and hoy

cells in presence or absence of the swi6-115 mutation (Figure 4). More than 50 sporulated colonies of each strain were examined microscopically after exposure to iodine vapors. No noticeable staining variation was observed between colonies of a given strain. The same phenotypes were observed when spores were used to initiate the colonies, in which case half the colonies were derived from P spores and half from M spores. The predominant mating type adopted by inefficiently switching cells was determined with the mating procedures described in MATERIALS AND METHODS. Here also colonies founded by spores, that is by both P and M cells, were examined. In addition, we assessed the ratio of P to M cells in cultures of all the strains displayed in Figure 4 by Southern blot analysis of the matl DNA (Figure 5). Southern blot analysis provides an independent method for determining the predominant mating type of each strain, including the ones that form dark-staining colonies for which the P-to-M ratio cannot be determined using the iodine staining technique. A summary of the iodine staining phenotypes and predominant mating types of the eight strains examined is given in Table 2.

As shown previously (EGEL, BEACH and KLAR 1984), the swi6-I 15 allele reduced mating-type switching in h9’ cells, leading to the formation of colonies stained lightly by iodine vapors (Figure 4, compare h9’, swi6- 115 in column 2 with h9*, swi6+ in column 1). Individ- ual colonies containing the swi6-115 allele displayed very similar phenotypes and staining was not observed at the edge of adjoining colonies, indicating that they all predominantly contained cells of the same mating type. Mating and sporulation were observed when h9”, swi6-I15 colonies were placed in contact with colonies of P tester-cells, but not when they were placed in contact with colonies of M tester-cells, indicating that hyo, swi6-115 colonies contained predominantly M cells. That most hPO, swi6-115 cells were of the M mating type was confirmed by examining the content of mat1 by Southern blot analysis (Figure 5B, lane 3). We conclude that the swi6-115 mutation affects switches from M to P more adversely than switches in the converse direction in h9’ cells. This result suggests that the requirement for SwiG is more stringent for utilizing mat2 than for utilizing mat3 as a donor.

In contrast to its effect in hyo cells, the swi6-115 mutation did not decrease switching to the opposite allele in ho9 cells. In fact, the mutation slightly increased the iodine staining of hoy colonies (Figure 4, compare hoy, swi6-115 in column 2 with ho9, swi6+ in column 1). Our interpretation for the swi6 mutation not decreasing heterologous switching in ho9 cells is that in swi6-115 cells the mating-type region is more “unfolded” than in swi6+ cells. The unfolding would diminish interaction with a preferred donor. This would allow random interaction with either donor,


A TABLE 2 DdeI NsiI DdeI

1 I

I I matl-M I1 -1.2 kb-

Genotype Intensity of

mal region swi6 mi6-mod iodine staining M:P

hY0 hYl1 h 911 - - hVll -

+ - +++++ M = P + + +++++ M = P

+ M >> P + +++ M > P

++ huy

M = P + + + M >> P

DdeI NsiI DdeI

+0.6 kb+ hov + - - Probe

B hW hm

I I I I swi6: + + - - + + - -

swi6-mod: - + - + - + - +

1.2kb- - - (matl-M)

0.6 kb + (matl-P)

1 2 3 4 5 6 7 8 FIGURE 5.-Effect of the m . 6 - I 1 5 allele and its modifier on the

predominant mating type adopted by hPO and ho9 cells. (A) Size of the restriction fragments generated by DdeI cleavage at the centromere-proximal border of the mall" and mal l -P cassettes, as predicted from sequence analysis (KELLY et al. 1988). The Ddel- NsiI restriction fragment indicated by a solid bar was used as probe in panel B. (B) Southern blot analysis of the mat l content in h9' and ho9 cells in the presence and absence of swi6-115 and its modifier. Total genomic DNA was digested with the restriction enzyme DdeI, size fractionated and hybridized to the 0.4-kb Ddel-NsiI restriction fragment of mat l indicated in A. "mat l -M" indicates the 1.2-kb Ddel restriction fragment originating from mall -M. 'mal l -P" indicates the 0.6-kb DdeI restriction fragment originating from mal l -P . The relevant genotype of the strains analyzed is indicated at the top. The following strains were used: 1, SP982; 2, SP887; 3, SP926; 4. SP952; 5, PG27; 6. PG19; 7, PG74; 8, PG68.

resulting in increased heterologous switching in the ho9 chromosome. Furthermore, hoy, swi6-I 15 colonies contained more P than M cells, which was assessed both by mating tests and by estimating the matl-"to- matl-P DNA ratio by Southern blot analysis (Figure 5B, lane 7). This observation reinforces the conclusion that Swi6 functions more for utilizing mat2 than for utilizing mat3 as a donor for the matl conversion.

M to P ratio was determined by mating tests and confirmed by Southern blot analysis shown in Figure 5.

The swiCmodifier affects directionality: In addition to the effect of the swi6-115 mutation, we investigated the effect of an as yet uncharacterized "modifier/suppressor" of swi6-115, designated swi6-mod, that is present in some of our strains (swib-rnod+ strains). swi6-mod segregated in crosses as a single locus unlinked to m i 6 or to the mating-type region (data not shown). A summary of swi6-mod+ effect on staining and predominant mating type of the cultures examined is presented in Table 2. swi6-mod+ partially sup- pressed the swi6-I 15 phenotype in h90 cells by increasing the proportion of P cells in the colonies (compare hyo, swib-115, swi6-mod-, Figure 4 column 2 and Fig- ure 5B lane 3 to hyo, swi6-115, swib-mod+, Figure 4 column 4 and Figure 5B lane 4; the latter contains more P cells). In ho9 cells, swi6-mod+ affected the swi6- I15 phenotype by increasing the proportion of M cells in the colonies (compare ho9, swi6-115, swi6-mod-, Figure 4, column 2, and Figure 5B, lane 7, to ho9, swi6-115, swib-mod+, Figure 4, column 4, and Figure 5B, lane 8; the latter contains more M cells). As mat2 contains the P information in h9' and the M information in ho9, both effects are consistent with swi6-mod+ either increasing mat2 to matl interactions or decreasing mat3 to matl interactions.

The swi6 modifier also affected directionality in ho9, swi6+ cells. Each ho9, swi6+, swib-mod+ colony, whether initiated by a P or by a M cell, was predominantly composed of M cells, and consequently exhibited much reduced iodine staining (compare ho9, swi6+, m i d m o d - , Figure 4, column 1, and Figure 5B, lane 5, to ho9, swi6+, swi6-mod+, Figure 4, column 3, and Figure 5B, lane 6; the latter contains more M cells). This result is consistent with an increased use of mat2 as a donor rather than a simple decreased use of mat3 in swi6-mod+ cells. This conclusion was established more directly by pedigree analysis. Switches from P to M were remarkably frequent in ho9, swi6+, swi6- mod+ cells. They occurred at a rate of 46% (7 1 among 154 PG251 diploid cells switched), confirming that swib-mod+ increases the use of mat2.


TABLE 3

Effect of d 6 on targeted recombination at the ura4 locus

Transformed No. Leu‘ No. stable Ura+ No. stable strain transformants transformants Ura+/No. Leu‘

SP62 (swi6+) 6800 155 2.3% PC595 (swi6-) 5880 170 2.9%

In summary, the swi6-115 mutation reduced the ability of mat2 to act as the donor in both h9’ and ho9 arrangement; mi6-mod+ increased that ability. We can propose two ways of action for swi6-mod+. A first mode of action would be a direct increase of mat2 utilization. Alternatively, swi6-mod+ may act indirectly by inhib- iting mat3 utilization, if by a competition effect a decreased use of mat3 increased the use of mat2. Our results do not distinguish between these possibilities. We imagine that mi6 mediates donor choice by con- ferring a specific chromatin structure to the mating- type region; such a structure may promote folding of the donor loci onto m a t l . Consistent with this sugges- tion, a low level of expression of the donor loci has been shown to occur in swi6- strains (LORENZ, HEIM and SCHMIDT 1992).

Swi6 prohibits targeted homologous recombination at the mad locus: During the construction of the ho9 strain by targeted recombination, we found that integration of a homologous DNA fragment at the mat3 locus occurred at a much lower frequency than homologous integration at other S . pombe loci. We obtained the relative numbers of homologous integration events at mat3 and, for comparison, at the ura4 locus from a single transformation experiment. In that experiment, a single preparation of competent h9’, ura4- (SP62) cells was split and half was cotransformed with 0.2 pg of pIRT2 DNA and 5 pg of the 1.8-kb Hind111 fragment containing the ura4 gene (BACH 1987). Leu+ transformants in which the ura4+ allele had substituted for the ura4- allele were counted (Table 3). Integration at ura4 occurred at a relatively high frequency, similar to what is commonly observed for homologous integration at other S . pombe loci (MORENO, KLAR and NURSE 199 1). The other half of the competent cells was cotransformed with 0.2 pg of the LEU2 carrying plasmid pIRT2 and 10 pg of the 6-kb Hind111 fragment containing the mat3-P RV;:ura4 construct. Leu+ transformants were selected and, among them, the ones in which mat3-P RV::ura4 had substituted for mat3-M were counted (Table 4). In that particular experiment, no recombinant at mat3 was found (Table 4). This observation strongly suggests that the recombination block in the mating-type region, originally discovered in cells undergoing meiosis (EGEL 1984), is also present in mitotically dividing cells.

As the mi6-115 mutation allows crossing over in the mat2-mat3 region, it was of interest to investigate

TABLE 4

Effect of mi6 on targeted recombination at the mat3 locus

Transformed No. Leu’ RV::ura4 No. mat3-P RV::ura4/ No. mat3-P

strain transformants transformants No. Leu+

SP62 (swi6+) 7440 0


BROACH 1992). In that yeast, the donor loci are located near telomeres, more than 200 kb to the left and nearly 100 kb to the right of the active MAT locus. The pattern of switching in a cell lineage, as well as the functions required for switching, differ greatly between the two organisms [see KLAR (1989) for a comparative review]. Whether similar mecha- nisms operate to dictate donor preference in both organisms remains to be determined.

In S. pombe, the observed reduced switching and iodine staining of ho9 colonies provide us with a simple tool to isolate additional directionality mutants. Mu- tations that randomize switching should increase staining of ho9 colonies. It should be noted that mutations that randomize the choice of donor loci should only reduce the rate of heterologous switching in h90 to 50%, possibly in only one direction. Such mutations will not significantly reduce the level of iodine staining of h90 colonies and, consequently, would have been easily missed by previously used screens (GUTZ, MEADE and WALKER 1975; GUTZ and SCHMIDT 1985). That all genes required for efficient mating-type switching were not identified by these screens is illus- trated by the accidental discovery of the rikl mutation by EGEL, WILLER and NIELSEN (1 989). The effect of the rzkl mutation would be worth assaying in ho9 cells as its pleiotropic phenotype in h9’ cells includes reduced mating-type switching and release of the cold spot for meiotic recombination between mat2 and mat3 (EGEL, WILLER and NIELSEN 1989). Further characterization of m i 6 , swi6-mod and other potential directionality mutations should shed light not only on the mechanism of selection of one specific location (positive vs. negative regulation), but possibly also on how the unusual long-range interaction between the recipient and the donors is physically mediated.

We thank JOAN CURCIO, STANLEY BROWN, GIL SHARON and ANNE ARTHUR for their comments on the manuscript. We are also grateful to our colleagues of the Laboratory of Eukaryotic Gene Expression for helpful discussions. This work was supported by the National Cancer Institute, Department of Health and Human Serv- ices, under contract N01-CO-74101 with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply en- dorsement by the U.S. Government.

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Communicating editor: E. W. JONES

directionality of fission yeast mating-type interconversion …...mat2 or mat3, the event being...

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