Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.     

Diversity of Mating-Type Chromosome Structures in the Yeast Zygosaccharomyces rouxii Caused by Ectopic Exchanges between MAT-Like Loci

We investigated sex chromosome diversity in Zygosaccharomyces rouxii (Z. rouxii). In the current study, we show that the organization of the mating-type (MAT) locus is highly variable in the Z. rouxii population, indicating the MAT, HML, and HMR loci are translocation hotspots. Although NBRC1130 and CBS732 were originally two stocks of the type strain of the species, only NBRC1130 retains the original karyotype. A reciprocal translocation between the MAT and HMR loci appears to have occurred during the early passage culture of CBS732, which was used for genome sequencing. In NBRC1733, NBRC0686, NBRC0740 and NBRC1053, the terminal region of the chromosome containing the HMR locus was replaced with the chromosomal region to the left of the MAT or HML loci. The translocation events found in NBRC1733, NBRC0686, NBRC0740, and NBRC1053 were reconstructed under our experimental conditions using the DA2 background, and the reconstruction suggests that the frequency of this type of translocation is approximately 10⁻⁷. These results suggest that the MAT and MAT-like loci were the susceptible regions in the genome, and the diversity of mating-type chromosome structures in Z. rouxii was caused by ectopic exchanges between MAT-like loci.     

Precise Mapping of the Homothallism Genes HML and HMR in SACCHAROMYCES CEREVISIAE

The HML and HMR loci carry unexpressed copies of MATa and MATα information, and a replica of that information is transposed to MAT during mating-type interchange in Saccharomyces yeasts. A negative control mechanism keeps silent the information located at the HML and HMR loci. We mapped these loci by constructing strains in which these loci are expressed. In these strains, the mating type of the segregants is dependent upon the allele at HML and HMR. This novel approach is independent of their switching function. HML is located on the left arm of chromosome III distal to his4 by about 26.8 centimorgans (cM). HMR maps on the right arm of the same chromosome distal to thr4 by about 39.8 cM and proximal to MAL2 by about 1.0 cM. The results allow the exact placement of these loci and are in accord with the observations made by Harashima and Oshima (1976).     

Directionality of yeast mating-type interconversion

The mating-type a and α alleles of the yeast Saccharomyces cerevisiae interconvert by a transposition-substitution reaction where replicas of the silent mating loci, at HML and HMR, are transmitted to the expressed mating-type locus (MAT). HML is on the left arm and HMR on the right arm, while MAT is in the middle of chromosome III. Cells with the genotype HMLα HMRa switch mating type efficiently at a frequency of about 86%. Since well over 50% of the cells switch, it is thought that switches do not occur randomly, but are directed to occur to the opposite mating-type allele. In contrast, we report that strains possessing the reverse HMLa HMRα arrangement switch (phenotype) inefficiently at a maximum of about 6%. The basis for this apparent reduced frequency of switching is that these strains preferentially yield futile homologous MAT locus switches—that is, MATa to MATa and MATα to MATα—and consequently, most of these events are undetected. We used genetically marked HM loci to demonstrate that a cells preferentially choose HMR as donor and a cells preferentially choose HML as donor, irrespective of the genetic content of the silent loci. Because of this feature, HMLα HMRa strains generate predominantly heterologous while HMLa HMRα strains produce predominantly homologous MAT switches. The control for directionality of switching therefore is not at the level of transposing heterologous mating-type information, but only at the level of choosing HML versus HMR as the donor. In strains where the preferred donor locus is deleted, the Inefficient donor becomes capable of donating efficiently. Thus the preference seems to be mediated by competition between the HM loci for donating information to MAT.     

Illegal transposition of mating-type genes in yeast

Mating-type switching in the yeast Saccharomyces cerevisiae involves the transposition of a copy of a or α information from unexpressed “library” genes, HML or HMR, to replace the sequence at the mating type locus, MAT. In normal homothallic strains, where conversions of MAT may occur as often as every cell division, the switching of MAT alleles does not alter the alleles at HML or HMR. We have discovered that several mutations within or adjacent to MAT that impair the excision of the MAT allele permit conversions of the alleles at HML or HMR in more than 1% of the cells analyzed. The two mutations within the MAT locus (MATa-inc and MATα-inc) can transpose to HML or HMR without being lost at MAT. Thus a MATα-inc HMLα HMRa HO strain can switch to MATα-inc HMLα HMRα-inc HO. Even though the α-inc and a-inc alleles prevent their own replacement at MAT, these sequences are efficiently transposed back from HMLα-inc or HMLa-inc to replace normal MAT alleles. When these alleles reappear at MAT, they are again blocked in excision. Thus the sequences used to remove an allele from MAT must differ from those used to replicate and transpose it. Two cis-acting stk mutations adjacent to MAT that block switching of MATa to MATα also induce the conversion of HMLα to HMLa. However, we have previously shown that these events do not occur in strains carrying a recessive “switch” mutant (swi1) or in strains carrying a defective allele of the HO gene. In stk1 MATa HO strains, HMLα was converted to HMLa in approximately 4% of the subclones examined. In contrast, the HMLα-inc sequence was not converted in similar stk1 MATa HO strains. Thus the excision of the α-inc sequence seems to be prevented at both MAT and HML. These results suggest that the illegal conversions of HML and HMR occur by a mechanism similar to that used for normal conversions of MAT.     

Interconversion of Yeast Cell Types by Transposable Genes

The a and α cell types of budding yeast Saccharomyces cerevisiae are controlled by alternate alleles of the mating-type locus (MAT), MATa and MATα. The cell types can be interconverted by switching alleles of MAT. The loci HMRa and HMLα, which are loosely linked to MAT, are involved in mating-type switching. Experimental evidence for their role in MAT interconversion is presented. As a result of switching, the homothallic and heterothallic strains containing the amber and ochre mutations within the HMRa locus yield corresponding amber and ochre mutant mata loci. Similarly, the hmlα mutant strain generates matα mutant alleles. That is, specific mutations from HMRa and HMLα are transmitted to MAT. A replica of the mating-type coding information originating from these loci is transposed to MAT, where it replaces the existing information. Furthermore, "Hawthorne deletions" in strains containing hmra-amber/ochre result in production of mata-amber/ochre alleles. Therefore, genetic information for MATa resides at HMRa. The switches occur in a defined set of clonally related cells. Thus, the efficient interconversion of yeast cell types is mediated by an unidirectional transfer of genetic information between nonallelic sites in a nonrandom and programmed fashion. The results are inconsistent with the "flip-flop" models, but satisfy a key prediction of the general controlling element and the specific cassette models proposed for mating-type interchange.     

Long-range heterochromatin association is mediated by silencing and double-strand DNA break repair proteins

The eukaryotic genome is highly organized in the nucleus, and this organization affects various nuclear processes. However, the molecular details of higher-order organization of chromatin remain obscure. In the present study, we show that the Saccharomyces cerevisiae silenced loci HML and HMR cluster in three-dimensional space throughout the cell cycle and independently of the telomeres. Long-range HML–HMR interactions require the homologous recombination (HR) repair pathway and phosphorylated H2A (γ-H2A). γ-H2A is constitutively present at silenced loci in unperturbed cells, its localization requires heterochromatin, and it is restricted to the silenced domain by the transfer DNA boundary element. SMC proteins and Scc2 localize to the silenced domain, and Scc2 binding requires the presence of γ-H2A. These findings illustrate a novel pathway for heterochromatin organization and suggest a role for HR repair proteins in genomic organization.     

Nonsex Genes in the Mating Type Locus of Candida albicans Play Roles in a/α Biofilm Formation,Including Impermeability and Fluconazole Resistance

The mating type locus (MTL) of Candida albicans contains the mating type genes and has, therefore, been assumed to play an exclusive role in the mating process. In mating-incompetent a/α cells, two of the mating type genes, MTL a1 and MTLα2, encode components of the a1-α2 corepressor that suppresses mating and switching. But the MTL locus of C. albicans also contains three apparently unrelated “nonsex” genes (NSGs), PIK, PAP and OBP, the first two essential for growth. Since it had been previously demonstrated that deleting either the a/α copy of the entire MTL locus, or either MTLa1 or MTLα2, affected virulence, we hypothesized that the NSGs in the MTL locus may also play a role in pathogenesis. Here by mutational analysis, it is demonstrated that both the mating type and nonsex genes in the MTL locus play roles in a/α biofilm formation, and that OBP is essential for impermeability and fluconazole resistance.     

Candida albicans Forms a Specialized “Sexual” as Well as “Pathogenic” Biofilm

Candida albicans forms two types of biofilm in RPMI 1640 medium, depending upon the configuration of the mating type locus. In the prevalent a/α configuration, cells form a biofilm that is impermeable, impenetrable by leukocytes, and fluconazole resistant. It is regulated by the Ras1/cyclic AMP (cAMP) pathway. In the a/a or α/α configuration, white cells form a biofilm that is architecturally similar to an a/α biofilm but, in contrast, is permeable, penetrable, and fluconazole susceptible. It is regulated by the mitogen-activated protein (MAP) kinase pathway. The MTL-homozygous biofilm has been shown to facilitate chemotropism, a step in the mating process. This has led to the hypothesis that specialized MTL-homozygous biofilms facilitate mating. If true, then MTL-homozygous biofilms should have an advantage over MTL-heterozygous biofilms in supporting mating. We have tested this prediction using a complementation strategy and show that minority opaque a/a and α/α cells seeded in MTL-homozygous biofilms mate at frequencies 1 to 2 orders of magnitude higher than in MTL-heterozygous biofilms. No difference in mating frequencies was observed between seeded patches of MTL-heterozygous and MTL-homozygous cells grown on agar at 28°C in air or 20% CO₂ and at 37°C. Mating frequencies are negligible in seeded patches of both a/α and a/a cells, in contrast to seeded biofilms. Together, these results support the hypothesis that MTL-homozygous (a/a or α/α) white cells form a specialized “sexual biofilm.”     

Evolution and Functional Trajectory of Sir1 in Gene Silencing

We used the budding yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii to examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles of SIR and RNA interference (RNAi) genes in T. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins in T. delbrueckii revealed a different topography of chromatin at the HML and HMR loci than was observed in S. cerevisiae. S. cerevisiae Sir1, enriched at the silencers of HMLα and HMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast to S. cerevisiae SIR1''s partially dispensable role in silencing, the T. delbrueckii SIR1 paralog KOS3 was essential for silencing. KOS3 was also found at telomeres with T. delbrueckii Sir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping in T. delbrueckii revealed single silencers at HML and HMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. The KOS3 gene mapped near HMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing, T. delbrueckii RNAi genes AGO1 and DCR1 did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.     

MFalpha1, the gene encoding the alpha mating pheromone of Candida albicans

Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the α mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the α pheromone gene. Here we report the isolation and characterization of the gene MFα1, which codes for the precursor of the α mating pheromone in C. albicans. Two active α-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfα1 null mutant of C. albicans. The mfα1 null mutant fails to mate as MTLα, while MTLa mfα1 cells are still mating competent. Experiments performed with the synthetic α-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLα kex2 mutant strain when added exogenously, thereby confirming their roles as α mating pheromones.     

Dpb11, the budding yeast homolog of TopBP1, functions with the checkpoint clamp in recombination repair

Dpb11 is required for the loading of DNA polymerases α and on to DNA in chromosomal DNA replication and interacts with the DNA damage checkpoint protein Ddc1 in Saccharomyces cerevisiae. The interaction between the homologs of Dpb11 and Ddc1 in human cells and fission yeast is thought to reflect their involvement in the checkpoint response. Here we show that dpb11-1 cells, carrying a mutated Dpb11 that cannot interact with Ddc1, are defective in the repair of methyl methanesulfonate (MMS)-induced DNA damage but not in the DNA damage checkpoint at the permissive temperature. Epistatic analyses suggested that Dpb11 is involved in the Rad51/Rad52-dependent recombination pathway. Ddc1 as well as Dpb11 were required for homologous recombination induced by MMS. Moreover, we found the in vivo association of Dpb11 and Ddc1 with not only the HO-induced double-strand break (DSB) site at MAT locus but also the donor sequence HML during homologous recombination between MAT and HML. Rad51 was required for their association with the HML donor locus, but not with DSB site at the MAT locus. In addition, the association of Dpb11 with the MAT and HML locus after induction of HO-induced DSB was dependent on Ddc1. These results indicate that, besides the involvement in the replication and checkpoint, Dpb11 functions with Ddc1 in the recombination repair process itself.     

Efficient Mating-Type Switching in Candida glabrata Induces Cell Death

Candida glabrata is an apparently asexual haploid yeast that is phylogenetically closer to Saccharomyces cerevisiae than to Candida albicans. Its genome contains three MAT-like cassettes, MAT, which encodes either MATa or MATalpha information in different strains, and the additional loci, HML and HMR. The genome also contains an HO gene homolog, but this yeast has never been shown to switch mating-types spontaneously, as S. cerevisiae does. We have recently sequenced the genomes of the five species that, together with C. glabrata, make up the Nakaseomyces clade. All contain MAT-like cassettes and an HO gene homolog. In this work, we express the HO gene of all Nakaseomyces and of S. cerevisiae in C. glabrata. All can induce mating-type switching, but, despite the larger phylogenetic distance, the most efficient endonuclease is the one from S. cerevisiae. Efficient mating-type switching in C. glabrata is accompanied by a high cell mortality, and sometimes results in conversion of the additional cassette HML. Mortality probably results from the cutting of the HO recognition sites that are present, in HML and possibly HMR, contrary to what happens naturally in S. cerevisiae. This has implications in the life-cycle of C. glabrata, as we show that efficient MAT switching is lethal for most cells, induces chromosomal rearrangements in survivors, and that the endogenous HO is probably rarely active indeed.     

A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MATα cells bud in an axial pattern, and MATa/α cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MATα cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/α cells were defective in the bipolar budding pattern, but MATa and MATα cells still exhibit a normal axial budding pattern. We also observed that MATa/α actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/α cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/α cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.     

The trans action of HMRa in mating type interconversion

Summary HML and HMR are the sites of cryptic mating type genes in the yeast Saccharomyces cerevisiae. In the presence of the HO gene, the information from HML or HMR (an a or cassette) is transferred to the mating type locus (MAT). HML, HMR, and MAT are located on chromosome III, yet are widely separeted. Similarly, in other yeasts, at least some of the genes involved in mating type interconversion are linked to the mating type locus. We demonstrate here that a cassette donor (HMR) and the cassette target (MAT) need not be physically linked for successful mating type interconversion. In particular, we show that HMR a on one chromosome can donate an a cassette to the mating type locus on a homologous chromosome III.