A mutation that permits the expression of normally silent copies of mating-type information in Saccharomyces cerevisiae

Studies of heterothallic and homothallic strains of Saccharomyces cerevisiae have led to the suggestion that mating-type information is located at three distinct sites on chromosome 3, although only information at the mating-type (MAT) locus is expressed (Hicks, Strathern and Herskowitz, 1977). We have found that the recessive mutation cmt permits expression of the normally silent copies of mating-type information at the HMa and HM alpha loci. In haploid strains carrying HMa and HM alpha, the cmt mutation allows the simultaneous expression of both a and alpha information, leading to a nonmating ("MATa/MAT alpha") phenotype. The effects of cmt can be masked by changing the mating-type information at HMa or HM alpha. For example, a cell of genotype MATa hma HM alpha cmt has an a mating type, while a MAT alpha hma HM alpha cmt strain is nonmating. Expression of mating-type information at the HM loci can correct the mating and sporulation defects of the mata* and mat alpha 10 alleles. Meiotic segregants recovered from cmt/cmt diploids carrying the mat mutations demonstrate that these mutants are not "healed" to normal MAT alleles, as is the case in parallel studies using the homothallism gene HO.--All of the results are consistent with the notion that the HMa and hm alpha alleles both code for alpha information, while HM alpha and hma both code for a information. The cmt mutation demonstrates that these normally silent copies of mating-type and sporulation information can be expressed and that the information at these loci is functionally equivalent to that found at MAT. The cmt mutation does not cause interconversions of mating-type alleles at MAT, and it is not genetically linked to MAT, HMa, HM alpha or HO. In cmt heterozygotes, cmt becomes homozygous at a frequency greater than 1% when the genotype at the MAT locus is mata*/MAT alpha or mat alpha 10/MATa.     

Heterochromatin regulates cell type-specific long-range chromatin interactions essential for directed recombination

Mating-type switching in Schizosaccharomyces pombe involves replacing genetic information at the expressed mat1 locus with sequences copied from one of two silent donor loci, mat2-P or mat3-M, located within a 20-kb heterochromatic domain. Donor selection is dictated by cell type: mat2 is the preferred donor in M cells, and mat3 is the preferred donor in P cells. Here we show that a recombination-promoting complex (RPC) containing Swi2 and Swi5 proteins exhibits cell type-specific localization pattern at the silent mating-type region and this differential localization modulates donor preference during mating-type switching. In P cells, RPC localization is restricted to a recombination enhancer located adjacent to mat3, but in M cells, RPC spreads in cis across the entire silent mating-type interval in a heterochromatin-dependent manner. Our analyses implicate heterochromatin in long-range regulatory interactions and suggest that heterochromatin imposes at the mating-type region structural organization that is important for the donor-choice mechanism.     

Swi6, a Gene Required for Mating-Type Switching, Prohibits Meiotic Recombination in the Mat2-Mat3 ``cold Spot'''' of Fission Yeast

Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 "hot spot" for transposition should be contrasted with the "cold spot" of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination.     

Schizosaccharomyces pombe switches mating type by the synthesis-dependent strand-annealing mechanism

Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event. A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism.     

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

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 (mat1) 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 mat1 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 mat1 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.     

The fission yeast homologue of CENP-B, Abp1, regulates directionality of mating-type switching

In fission yeast, mating-type switching involves replacing genetic information contained at the expressed mat1 locus by that of either the mat2P or mat3M donor loci. Donor selection is nonrandom, as mat1P cells preferentially use mat3M for switching, whereas mat1M cells use mat2P. Switching directionality is determined by the cell-type-specific distribution of the Swi2-Swi5 complex that, in mat1P cells, localises to mat3M and, only in mat1M cells, spreads to mat2P in a heterochromatin-dependent manner. Mechanisms regulating spreading of Swi2-Swi5 across heterochromatin are not fully understood. Here, we show that the fission yeast homologue of CENP-B, Abp1, binds to the silent domain of the mating-type locus and regulates directionality of switching. Deletion of abp1 prevents utilisation of mat2P, as when heterochromatin is disrupted and spreading of Swi2-Swi5 is impaired. Our results show that, indeed, deletion of abp1 abolishes spreading of Swi2-Swi5 to mat2P. However, in abp1Delta cells, heterochromatin organisation at the mating-type locus is preserved, indicating that Abp1 is actually required for efficient spreading of Swi2-Swi5 through heterochromatin. Cbh1 and Cbh2, which are also homologous to CENP-B, have only a minor contribution to the regulation of directionality of switching, which is in contrast with the strong effects observed for Abp1.     

Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast.

Position-effect control at the silent mat2-mat3 interval and at centromeres and telomeres in fission yeast is suggested to be mediated through the assembly of heterochromatin-like structures. Therefore, trans-acting genes that affect silencing may encode either chromatin proteins, factors that modify them, or factors that affect chromatin assembly. Here, we report the identification of an essential gene, clr6 (cryptic loci regulator), which encodes a putative histone deacetylase that when mutated affects epigenetically maintained repression at the mat2-mat3 region and at centromeres and reduces the fidelity of chromosome segregation. Furthermore, we show that the Clr3 protein, when mutated, alleviates recombination block at mat region as well as silencing at donor loci and at centromeres and telomeres, also shares strong homology to known histone deacetylases. Genetic analyses indicate that silencing might be regulated by at least two overlapping histone deacetylase activities. We also found that transient inhibition of histone deacetylase activity by trichostatin A results in the increased missegregation of chromosomes in subsequent generations and, remarkably, alters the imprint at the mat locus, causing the heritable conversion of the repressed epigenetic state to the expressed state. This work supports the model that the level of histone deacetylation has a role in the assembly of repressive heterochromatin and provides insight into the mechanism of epigenetic inheritance.     

Two Portable Recombination Enhancers Direct Donor Choice in Fission Yeast Heterochromatin

Mating-type switching in fission yeast results from gene conversions of the active mat1 locus by heterochromatic donors. mat1 is preferentially converted by mat2-P in M cells and by mat3-M in P cells. Here, we report that donor choice is governed by two portable recombination enhancers capable of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the mat2-mat3 heterochromatic domain. Cells whose silent cassettes are swapped to mat2-M mat3-P switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were transposed together with the cassette contents switched like wild type. Trans-acting mutations that impair directionality affected the wild-type and swapped cassettes in identical ways when the recombination enhancers were transposed together with their cognate cassette, showing essential regulatory steps occur through the recombination enhancers. Our observations lead to a model where heterochromatin biases competitions between the two recombination enhancers to achieve directionality.     

Mating-Type Mutations in SCHIZOSACCHAROMYCES POMBE: Isolation of Mutants and Analysis of Strains with an h or h Phenotype

Mutants defective in various steps of the sexual cycle have been isolated from homothallic strains of Schizosaccharomyces pombe by Bresch, Müller and Egel (1968). These mutants include heterothallic h(+) and h(-) strains. We have isolated additional h(+) and h(- ) mutants from homothallic strains. Those mutants which are due to mutations in the mating-type region were analyzed in detail. Our results show that the mating-type gene mat2 not only has a function in copulation and meiosis, but that it also regulates the formation of the map1 gene product (map1 is a mating-type auxiliary gene). Some of the h( -) mutants have lost only one of the three functions while others are defective in at least two, and perhaps all three, functions. Further, we show that the mat1(-) allele of h(90) strains can mutate to mat1(+) but that mutations in mat2 appear to affect the mutational behavior of mat1. Finally, we describe a new inactive mating-type allele, mat2*, which is different from mat2(0) in that it can mutate to mat2(+).     

The Mutator Gene Swi8 Effects Specific Mutations in the Mating-Type Region of Schizosaccharomyces Pombe

The swi8(+) gene of Schizosaccharomyces pombe appears to be involved in the termination step of copy synthesis during mating-type (MT) switching. Mutations in swi8 confer a general mutator phenotype and, in particular, generate specific mutations in the MT region. Sequencing of the MT cassettes of the h(90) swi8-137 mutant revealed three altered sites. One is situated at the switching (smt) signal adjacent to the H1 homology box of the expression locus mat1:1. It reduces the rate of MT switching. The alteration at the smt signal arose frequently in other h(90) swi8 strains and is probably caused by gene conversion in which the sequence adjacent to the H1 box of mat2:2 is used as template. This change might be generated during the process of MT switching when hybrid DNA formation is anomalously extended into the more heterologous region flanking the H1 homology box. In addition to the gene conversion at mat1:1, two mutations were found in the H3 homology boxes of the silent cassettes mat2:2 and mat3:3.     

The fission yeast ubiquitin-conjugating enzymes UbcP3, Ubc15, and Rhp6 affect transcriptional silencing of the mating-type region

Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.