No abstract

Meiotic development in yeast is characterized by the sequential induction of temporally distinct classes of genes. Genes that are induced at the middle stages of the pathway share a promoter element, termed the middle sporulation element (MSE), which interacts with the Ndt80 transcriptional activator. We have found that a subclass of MSEs are strong repressor sites during mitosis. SUM1 and HST1, genes previously associated with transcriptional silencing, are required for MSE-mediated repression. Sum1 binds specifically in vitro to MSEs that function as strong repressor sites in vivo. Repression by Sum1 is gene specific and does not extend to neighboring genes. These results suggest that mechanisms used to silence large regions of chromatin may also be used to regulate the expression of specific genes during development. NDT80 is regulated during mitosis by both the Sum1 and Ume6 repressors. These results suggest that progression through sporulation may be controlled by the regulated competition between the Sum1 repressor and Ndt80 activator at key MSEs.

A key transition in meiosis is the exit from prophase and entry into the nuclear divisions, which in the yeast Saccharomyces cerevisiae depends upon induction of the middle sporulation genes. Ndt80 is the primary transcriptional activator of the middle sporulation genes and binds to a DNA sequence element termed the middle sporulation element (MSE). Sum1 is a transcriptional repressor that binds to MSEs and represses middle sporulation genes during mitosis and early sporulation. We demonstrate that Sum1 and Ndt80 have overlapping yet distinct sequence requirements for binding to and acting at variant MSEs. Whole-genome expression analysis identified a subset of middle sporulation genes that was derepressed in a sum1 mutant. A comparison of the MSEs in the Sum1-repressible promoters and MSEs from other middle sporulation genes revealed that there are distinct classes of MSEs. We show that Sum1 and Ndt80 compete for binding to MSEs and that small changes in the sequence of an MSE can yield large differences in which protein is bound. Our results provide a mechanism for differentially regulating the expression of middle sporulation genes through the competition between the Sum1 repressor and the Ndt80 activator.

No abstract

Sfp1, an unusual zinc finger protein, was previously identified as a gene that, when overexpressed, imparted a nuclear localization defect. sfp1 cells have a reduced size and a slow growth phenotype. In this study we show that SFP1 plays a role in ribosome biogenesis. An sfp1 strain is hypersensitive to drugs that inhibit translational machinery. sfp1 strains also have defects in global translation as well as defects in rRNA processing and 60S ribosomal subunit export. Microarray analysis has previously shown that ectopically expressed SFP1 induces the transcription of a large subset of genes involved in ribosome biogenesis. Many of these induced genes contain conserved promoter elements (RRPE and PAC). Our results show that activation of transcription from a reporter construct containing two RRPE sites flanking a single PAC element is SFP1 dependent. However, we have been unable to detect direct binding of the protein to these elements. This suggests that regulation of genes containing RRPEs is dependent upon Sfp1 but that Sfp1 may not directly bind to these conserved promoter elements; rather, activation may occur through an indirect mechanism.

Transcriptional repression is often correlated with the alteration of chromatin structure through modifications of the nucleosomes in the promoter region, such as by deacetylation of the N-terminal histone tails. This is presumed to make the promoter region inaccessible to other regulatory factors and the general transcription machinery. To accomplish this, histone deacetylases are recruited to specific promoters via DNA-binding proteins and tethering factors. We have previously reported the requirement for the NAD ؉ -dependent histone deacetylase Hst1 and the DNA-binding protein Sum1 for vegetative repression of many middle sporulation genes in Saccharomyces cerevisiae. Here we report the identification of a novel tethering factor, Rfm1, that is required for Hst1-mediated repression. Rfm1 interacts with both Sum1 and Hst1 and is required for the Sum1-Hst1 interaction. DNA microarray and Northern blot analyses showed that Rfm1 is required for repression of the same subset of Sum1-repressed genes that require Hst1. These results suggest that Rfm1 is a specificity factor that targets the Hst1 deacetylase to a subset of Sum1-regulated genes.

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-␣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.One of the main paradigms for the control of gene expression is that regulatory proteins bind to the promoter regions of genes to activate or repress transcription. However, it is now clear that noncoding RNAs (ncRNAs) also play important roles in gene regulation. For example, RNA interference (RNAi)-mediated regulation controls gene expression in Caenorhabditis elegans, Arabidopsis thaliana, humans, and many other organisms (16,24). However, a large number of ncRNAs do not appear to be involved in RNAimediated regulation. For example, more than 900 ncRNAs are expressed in the yeast Saccharomyces cerevisiae (11,15,33,40,45). Several of these ncRNAs act to regulate gene expression in yeast (2,5,18,26). However, S. cerevisiae lacks the enzymes Dicer and Argonaute, which are required for RNAi, and therefore, it must utilize different mechanisms for ncRNA-mediated regulation (12). In this paper we investigate how two antisense ncRNAs regulate the expression of genes required for meiosis in yeast.Under starvation conditions, diploid yeast undergoes meiosis and sporulation to form four haploid spores. This process involves the expression of more than 500 genes that are highly regulated in a coordinated manner (7,28). Entry into the meiotic pathway is controlled by the expression of IME1, the master initiator of meiosis (20, 29). There are two signals that regulate IME1 expression (Fig. 1A). One signal relates to the nutritional status of the cell, activating IME1 expression when the cell is starved of both nitrogen and a fermentable carbon source (14, 37). The second signal operates through cell-typespecific regulation, which allows the expression of meiotic genes only in a/␣ diploid cells. Cell-type-specific regulation is controlled by the a1-␣2 repressor complex, which reg...

We have isolated 64 different missense mutations at 36 out of 53 residue positions in the Arc repressor of bacteriophage P22. Many of the mutant proteins with substitutions in the C-terminal 40 residues of Arc have reduced intracellular levels and probably have altered structures or stabilities. Mutations in the N-terminal ten residues of Arc cause large decreases in operator DNA binding affinity without affecting the ability of Arc to fold into a stable three-dimensional structure. We argue that these N-terminal residues are important for operator recognition but that they are not part of a conventional helix-turn-helix DNA binding structure. These results suggest that Arc may use a new mechanism for sequence specific DNA binding.