Transcriptome profiling studies have recently uncovered a large number of noncoding RNA transcripts (ncRNAs) in eukaryotic organisms, and there is growing interest in their role in the cell. For example, in haploid Saccharomyces cerevisiae cells, the expression of an overlapping antisense ncRNA, referred to here as RME2 (Regulator of Meiosis 2), prevents IME4 expression. In diploid cells, the a1-Œ±2 complex represses the transcription of RME2, allowing IME4 to be induced during meiosis. In this study we show that antisense transcription across the IME4 promoter region does not block transcription factors from binding and is not required for repression. Mutational analyses found that sequences within the IME4 open reading frame (ORF) are required for the repression mediated by RME2 transcription. These results support a model where transcription of RME2 blocks the elongation of the full-length IME4 transcript but not its initiation. We have found that another antisense transcript, called RME3, represses ZIP2 in a cell-type-specific manner. These results suggest that regulated antisense transcription may be a widespread mechanism for the control of gene expression and may account for the roles of some of the previously uncharacterized ncRNAs in yeast.
Sir2 and Hst1 are NAD(+)-dependent histone deacetylases of budding yeast that are related by strong sequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repression. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2 interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomere chromosomal regions. To identify the determinants of gene-specific versus regional repression, we created a series of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform both regional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereas gene-specific repression required Rfm1 and Sum1. Our findings demonstrate that the nonconserved N-terminal region and two amino acids within the enzymatic core domain account for cofactor specificity and proper targeting of these proteins. These results suggest that the differences in the silencing and repression functions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather may be the result of distinct cofactor specificities.
The yeast Mcm1 protein is a member of the MADS box family of transcription factors that interacts with several cofactors to differentially regulate genes involved in cell-type determination, mating, cell cycle control and arginine metabolism. Residues 18 to 96 of the protein, which form the core DNA-binding domain of Mcm1, are sufficient to carry out many Mcm1-dependent functions. However, deletion of residues 2 to 17, which form the nonessential N-terminal (NT) arm, confers a salt-sensitive phenotype, suggesting that the NT arm is required for the activation of salt response genes. We used a strategy that combined information from the mutational analysis of the Mcm1-binding site with microarray expression data under salt stress conditions to identify a new subset of Mcm1-regulated genes. Northern blot analysis showed that the transcript levels of several genes encoding associated with the cell wall, especially YGP1, decrease significantly upon deletion of the Mcm1 NT arm. Deletion of the Mcm1 NT arm results in a calcofluor white-sensitive phenotype, which is often associated with defects in transcription of cell wall genes. In addition, the deletion makes cells sensitive to CaCl2 and alkaline pH. We found that the defect caused by removal of the NT arm is not due to changes in Mcm1 protein level, stability, DNA-binding affinity, or DNA bending. This suggests that residues 2 to 17 of Mcm1 may be involved in recruiting a cofactor to the promoters of these genes to activate transcription.