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/MATalpha-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.
This report describes the identification, cloning, and molecular analysis of UME6 (CAR80/CARGRI), a key transcriptional regulator of early meiotic gene expression. Loss of UME6 function results in the accumulation of fully derepressed levels (70-to 100-fold increase above basal level) of early meiotic transcripts during vegetative growth. In contrast, mutations in five previously identified UME loci (UME1 to UME5), result in low to moderate derepression (2-to 10-fold increase) of early meiotic genes. The behavior of insertion and deletion alleles indicates that UME6 is dispensable for mitotic division but is required for meiosis and spore germination. Despite the high level of meiotic gene expression during vegetative growth, the generation times of ume6 mutant haploid and diploid cells are only slightly reduced. However, both ascus formation and spore viability are affected more severely. The UME6 gene encodes a 91-kD protein that contains a C6 zinc cluster motif similar to the DNA-binding domain of GAL4. The integrity of this domain is required for UME6 function. It has been reported recently that a mutation in CAR80 fails to complement an insertion allele of UME6. CAR80 is a gene required for nitrogen repression of the arginine catabolic enzymes. Here, through sequence analysis, we demonstrate that UME6 and CAR80 are identical. Analyses of UME6 mRNA during both nitrogen starvation and meiotic development indicate that its transcription is constitutive, suggesting that regulation of UME6 activity occurs at a post-transcriptional level.
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 SIRResponsive 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.
Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.
Mutations in Saccharomyces cerevisiae have been identified that derepress early meiotic genes functioning in separable pathways required for normal meiotic development. The phenotypes of these ume (unscheduled meiotic gene expression) mutations suggest that their wild-type alleles encode negative regulators acting downstream of both the celltype and nutritional controls of meiosis. These newly defined loci do not affect either general transcription or transcription of meiotic genes expressed later in meiosis and spore formation.Initiation of meiosis in Saccharomyces cerevisiae is under the control of two independent, convergent regulatory pathways, one responding to cell type and the other sensing nutritional status (1-3). The cell-type pathway operates through a transcriptional regulatory cascade in which the products of the MATa and MATa loci combine to form a negative regulator (4, 5) that inhibits the expression of RMEI (6, 7), which encodes a repressor of meiosis. RMEJ, in turn, negatively regulates IMEI, an inducer of meiosis (8), which positively regulates IME2 (9). Overexpression of either IMEI or IME2 allows meiotic functions to be expressed during mitosis (8, 9).The nutritional pathway senses glucose and nitrogen deprivation and involves a number of well-characterized genes, e.g., ARDI (10), BCYJ, CYR2, and CYR3 (11), and RAS2 (12,13). Evidence that the nutritional and cell-type pathways are initially independent is based on the observation that rmel mutants still require starvation conditions to enter meiosis (6) and, conversely, mutants that interfere with nutritional control, allowing meiosis in rich media, still require both MATa and MATa expression (14). IMEJ is regulated by both cell type and nutritional conditions and represents the first known point at which these pathways converge.The process of meiosis and gamete formation in yeast includes DNA replication, recombination, chromosome segregation at meioses I and II, and spore formation. A number of genes required for these events have been cloned and found to be developmentally regulated; i.e., they exhibit elevated message levels only during sporulation (15)(16)(17). Among these are SP013, a gene required for chromosome segregation at meiosis I (18), SPOIl, a gene involved in recombination (19), and SP016, a gene that affects the efficiency of early prophase events (R. T. Elder and R.E.E., unpublished results). The purpose of this study was to identify trans-acting regulators that directly control the expression of these genes. Our approach was to use a fusion reporter gene to recover regulatory mutations that derepress the mitotic expression of these meiosis-specific genes. Here we report the successful application of this method to meiotic control and the identification of five such trans-acting genes. MATERIALS AND METHODSStrains and Plasmids. Mutants were isolated in RSY10 (S. Frackman, University of Wisconsin-Milwaukee), an ade6 derivative of W303-1A (R. Rothstein, Columbia University College of Physicians and Surgeons): MATa ade...
We show that the extent of transcriptional regulation of many, apparently unrelated, genes in Saccharomyces cerevisiae is dependent on RPD1 (and RPD3 [M. Vidal and R. F. Gaber, Mol. Cell. Biol. 11:6317-6327, 1991]). Genes regulated by stimuli as diverse as external signals (PHOS), cell differentiation processes (SPOil and SPO13), cel type (RMEJ, FUS), HO, TY2, STE6, STE3, and BAR)), and genes whose regulatory signals remain unknown (TRK2) depend on RPD1 to achieve maximal states of transcriptional regulation. RPD1 enhances both positive and negative regulation of these genes: in rpdlA mutants, higher levels of expression are observed under repression conditions and lower levels are observed under activation conditions. We show that several independent genetic screens, designed to identify yeast transcriptional regulators, have detected the RPDJ locus (also known as SIN3, SD)), and UME4). The inferred RPD1 protein contains four regions predicted to take on helix-loop-helix-like secondary structures and three regions (acidic, glutamine rich, and proline rich) reminiscent of the activating domains of transcriptional activators.Until recently, transcriptional regulation studies have focused primarily on components directly involved in activation of gene expression (20,33,42 (57), the gene that encodes the low-affinity K+ transporter (24,25). Through derepression of TRK2, rpd mutations confer reduced potassium dependency, allowing growth on low-potassium medium. UME4 was identified as a mitotic repressor of a class of meiotic genes (SPOIl, SPO13, and SPO16) coexpressed early in meiosis (52). Mutations in UME4 allow unscheduled meiotic gene expression through increased transcription of these genes during vegetative growth. Mutations in SIN3 (SDIJ) were identified in a screen for variants that allow expression of HO in the absence of SWI5 and are thus SW15 independent. SIN3 is proposed to be required for repression of HO in daughter cells (37,50 Media. The genetic crosses and standard media used were previously described (46). Synthetic low-salt and low-phosphate media were prepared essentially as previously de-6306 MOLECULAR AND CELLULAR BIOLOGY, Dec. 1991, p. 6306-6316
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