Rochelle Easton Esposito

A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA

The yeast SIR2 gene is involved in regulating nucleosome phasing and transcription in the mating type system. We have found that SIR2 also plays another important role in the cell. Specifically, in wild-type SIR2 strains recombination between the tandemly repeated ribosomal RNA genes is depressed. In sir2 mutants, both mitotic and meiotic intrachromosomal recombination increase 10- to 15-fold. In striking contrast to its effect on rDNA, the SIR2 gene does not affect intrachromosomal recombination between non-rDNA gene duplications. Furthermore, in the absence of the SIR2 gene product, rDNA acquires a partial dependency on recombination gene functions (RAD50 and RAD52) that are normally dispensable for exchange in the rDNA array. Thus, SIR2 may function in excluding the rDNA region from the general recombination system. Here we demonstrate that SIR2s effect is not restricted to controlling mating type expression, but rather that SIR2 functions in a more general way in the genome.

The core meiotic transcriptome in budding yeasts

We used high-density oligonucleotide microarrays to analyse the genomes and meiotic expression patterns of two yeast strains, SK1 and W303, that display distinct kinetics and efficiencies of sporulation. Hybridization of genomic DNA to arrays revealed numerous gene deletions and polymorphisms in both backgrounds. The expression analysis yielded approximately 1,600 meiotically regulated genes in each strain, with a core set of approximately 60% displaying similar patterns in both strains. Most of these (95%) are MATa/MATα-dependent and are not similarly expressed in near-isogenic meiosis-deficient controls. The transcript profiles correlate with the distribution of defined meiotic promoter elements and with the time of known gene function.

Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA.

The yeast SIR2 gene maintains inactive chromatin domains required for transcriptional repression at the silent mating‐type loci and telomeres. We previously demonstrated that SIR2 also acts to repress mitotic and meiotic recombination between the tandem ribosomal RNA gene array (rDNA). Here we address whether rDNA chromatin structure is altered by loss of SIR2 function by in vitro and in vivo assays of sensitivity to micrococcal nuclease and dam methyltransferase, respectively, and present the first chromatin study that maps sites of SIR2 action within the rDNA locus. Control studies at the MATα locus also revealed a previously undetected MNase‐sensitive site at the a1‐α2 divergent promoter which is protected in sir2 mutant cells by the derepressed a1‐α2 regulator. In rDNA, SIR2 is required for a more closed chromatin structure in two regions: SRR1, the major SIR‐Responsive Region in the non‐transcribed spacer, and SRR2, in the 18S rRNA coding region. None of the changes in rDNA detected in sir2 mutants are due to the presence of the a1‐α2 repressor. Reduced recombination in the rDNA correlates with a small, reproducible transcriptional silencing position effect. Deletion and overexpression studies demonstrate that SIR2, but not SIR1, SIR3 or SIR4, is required for this rDNA position effect. Significantly, rDNA transcriptional silencing and rDNA chromatin accessibility respond to SIR2 dosage, indicating that SIR2 is a limiting component required for chromatin modeling in rDNA.

A screen for genes required for meiosis and spore formation based on whole-genome expression

BACKGROUND Meiosis is the process by which gametes are generated with half the ploidy of somatic cells. This reduction is achieved by three major differences in chromosome behavior during meiosis as compared to mitosis: the production of chiasmata by recombination, the protection of centromere-proximal sister chromatid cohesion, and the monoorientation of sister kinetochores during meiosis I. Mistakes in any of these processes lead to chromosome missegregation. RESULTS To identify genes involved in meiotic chromosome behavior in Saccharomyces cerevisiae, we deleted 301 open reading frames (ORFs) which are preferentially expressed in meiotic cells according to microarray gene expression data. To facilitate the detection of chromosome missegregation mutants, chromosome V of the parental strain was marked by GFP. Thirty-three ORFs were required for the formation of wild-type asci, eight of which were needed for proper chromosome segregation. One of these (MAM1) is essential for the monoorientation of sister kinetochores during meiosis I. Two genes (MND1 and MND2) are implicated in the recombination process and another two (SMA1 and SMA2) in prospore membrane formation. CONCLUSIONS Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.

Meiosis and Ascospore Development

INTRODUCTION Sporulation in yeast includes meiosis and ascospore development. It has been the focus of numerous studies for two primary reasons. First, it provides a relatively simple model system for the investigation of eukaryotic differentiation and the manner in which a complex series of biochemical, morphological, and genetic events are coordinated into a successful developmental pathway. Second, two events of major genetic consequence occur during meiosis: genetic recombination and chromosome segregation. Both of these events play a profound role in the generation of new genotypes and euploid genomes during sexual reproduction. Despite the central importance of the meiotic process, specific knowledge of the genetic and biochemical control of gametogenesis in eukaryotic organisms is very limited. Utility of Yeast For Studies of Sporulation As an experimental system, yeast presents the opportunity to study meiosis and gamete development in an organism that possesses all of the technical advantages of microbial systems, while exhibiting chromosome behavior typical of higher eukaryotic cells. It has a number of attractive features that make it particularly well-suited for an analysis of meiotic cell differentiation: (1) Yeast has well-developed genetics and is readily manipulated biochemically. (2) Large numbers of single cells can be stimulated to undergo meiosis in a defined medium. (3) Meiosis can be interrupted and viable cells recovered at various stages of development. (4) All of the meiotic products of a given meiosis can be recovered in association with one another, permitting precise reconstruction of exchange and segregation events. (5) Aspects of the life cycle and...

The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast.

The Ume6 transcription factor in yeast is known to both repress and activate expression of diverse genes during growth and meiotic development. To obtain a more complete profile of the functions regulated by this protein, microarray analysis was used to examine transcription in wild-type and ume6Δ diploids during vegetative growth in glucose and acetate. Two different genetic backgrounds (W303 and SK1) were examined to identify a core set of strain-independent Ume6-regulated genes. Among genes whose expression is controlled by Ume6 in both backgrounds, 82 contain homologies to the Ume6-binding site (URS1) and are expected to be directly regulated by Ume6. The vast majority of those whose functions are known participate in carbon/nitrogen metabolism and/or meiosis. Approximately half of the Ume6 direct targets are induced during meiosis, with most falling into the early meiotic expression class (cluster 4), and a smaller subset in the middle and later classes (clusters 5–7). Based on these data, we propose that Ume6 serves a unique role in diploid cells, coupling metabolic responses to nutritional cues with the initiation and progression of meiosis. Finally, expression patterns in the two genetic backgrounds suggest that SK1 is better adapted to respiration and W303 to fermentation, which may in part account for the more efficient and synchronous sporulation of SK1.

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.

Identification of the Sin3-Binding Site in Ume6 Defines a Two-Step Process for Conversion of Ume6 from a Transcriptional Repressor to an Activator in Yeast

ABSTRACT The DNA-binding protein Ume6 is required for both repression and activation of meiosis-specific genes, through interaction with the Sin3 corepressor and Rpd3 histone deacetylase and the meiotic activator Ime1. Here we show that fusion of a heterologous activation domain to Ume6 is unable to convert it into a constitutive activator of early meiotic gene transcription, indicating that an additional function is needed to overcome repression at these promoters. Mutations in UME6 allowing the fusion to activate lie in a predicted amphipathic alpha helix and specifically disrupt interaction with Sin3 but not with Teal, an activator of Ty transcription also found to interact with Ume6 in a two-hybrid screen. The mutations cause a loss of repression by Ume6 and precisely identify the Ume6 Sin3-binding domain, which we show interacts with the paired amphipathic helix 2 region of Sin3. Analysis of these mutants indicates that conversion of Ume6 to an activator involves two genetically distinct steps that act to relieve Sin3-mediated repression and provide an activation domain to Ume6. The mutants further demonstrate that premature expression and lack of subsequent rerepression of Ume6-Sin3-regulated genes are not deleterious to meiotic progression and suggest that the essential role of Sin3 in meiosis is independent of Ume6. The model for Ume6 function arising from these studies indicates that Ume6 is similar in many respects to metazoan regulators that utilize Sin3, such as the Myc-Mad-Max system and nuclear hormone receptors, and provides new insights into the control of transcriptional repression and activation by the Ume6-URS1 regulatory complex in yeast.

The genetic control of sporulation inSaccharomyces

SummaryHeat-sensitive sporulation-deficient (spo) mutants ofS. cerevisiae may be either dominant or recessive. The number of loci which can mutate to thespo phenotype has been estimated to be 48±27 from complementation studies. Comparison of the wild type and mutants by light microscopy after exposure to sporulation medium at the restrictive temperature and Giemsa staining has shown that mutant populations can not complete the meiotic nuclear divisions.

Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae.

We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.