Class struggle: expression profiling and categorizing cancer.
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Animals↗
Biomedical subjects
P A Covitz
Publications and source records attributed to P A Covitz.
Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1.
The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.
Binding Sites↗
Requirement for RGR1 and SIN4 in RME1-dependent repression in Saccharomyces cerevisiae.
RME1 is a zinc-finger protein homolog that functions as a repressor of the meiotic activator IME1. RME1 is unusual among yeast repressors in two respects: it acts over a considerable distance (2 kbp) and it can activate transcription from a binding site separated from its natural flanking region. To identify genes required for RME1 to exert repression, we have selected mutants with improved RME1-dependent activation. One rare mutant was defective in RME1-dependent repression of an artificial reporter gene as well as the native IME1 gene. The mutation permits sporulation of a/a diploids, which express RME1 from its natural promoter, and of a/alpha diploids constructed to express RME1 from the GAL1 promoter. The mutation also causes temperature-sensitive growth and a methionine or cysteine requirement. Analysis of a complementing genomic clone indicates that the mutation lies in a known essential gene, RGR1. Prior studies have indicated a functional relationship between RGR1 and SIN4 (also called TSF3); we have found that a sin4 null mutation also causes a defect in RME1-dependent repression and a methionine or cysteine requirement. The rgr1 and sin4 mutations do not cause a reduction of RME1 polypeptide levels. The defect in RME1-dependent repression may result from effects of sin4 and, presumably, rgr1 on chromatin structure.
Cloning, Molecular↗
Repression by the yeast meiotic inhibitor RME1.
The RME1 gene product, a negative regulator of meiosis with three zinc finger motifs, acts by preventing transcript accumulation from IME1, whose product is required for meiotic gene expression. We have isolated a 404-bp segment from a region 2 kb upstream of IME1 that is sufficient for RME1-dependent repression of a heterologous promoter. This DNA contains an RME1-response element (RRE) and another region called the modulation region. The modulation region is required for repression because DNA containing the RRE alone did not repress but was able to confer RME1-dependent transcriptional activation of a reporter gene. In gel mobility retardation assays, RME1 formed a specific complex with the RRE, and RRE point mutations that reduced the affinity for RME1 also blocked repression and activation. Footprinting of the RME1-RRE complex revealed a 21-bp protected region that included the positions of these RRE mutations. We conclude that RME1 binding to this RRE is required for repression. Thus, the mechanism of meiotic inhibition by RME1 is direct transcriptional repression of IME1.
Base Sequence↗
The yeast RME1 gene encodes a putative zinc finger protein that is directly repressed by a1-alpha 2.
In the yeast Saccharomyces cerevisiae, a/alpha cells can enter meiosis whereas a and alpha cells cannot. The a/alpha cell type is determined by presence of a repressor, a1-alpha 2. Previous studies indicate that a/alpha cells lack an inhibitor of meiosis, the RME1 gene product, and that a and alpha cells express RME1. We report here the sequence of RME1 and functional analysis of its regulatory and coding regions. The 5'-region of RME1 includes a sequence resembling a1-alpha 2 repression sites. Deletion of this site at RME1 relieves repression by a1-alpha 2, and insertion of the site into a heterologous regulatory region (CYC1) confers weak repression in a/alpha cells. These observations indicate that RME1 is directly repressed by a1-alpha 2. The RME1 product has three regions that resemble C2H2 zinc fingers, which are characteristic of a class of nucleic-acid-binding proteins. Substitution of serine for cysteine in each of the putative fingers abolishes RME1 function; serine substitutions in the second and third putative fingers do not affect RME1 stability. These findings indicate that at least two putative zinc fingers are critical for RME1 structure or activity. Therefore RME1, which is formally a negative regulator of the meiotic gene IME1, may act directly as a repressor.
Amino Acid Sequence↗