Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1.

Moretti, Paolo; Freeman, Katie B.; Coodly, Lavanya R.; Shore, David

doi:10.1101/gad.8.19.2257

Cited by 516 publications

(581 citation statements)

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“…While Rap1p is a major transcriptional activator, it is also a major transcriptional silencer, at the silent mating type loci and at telomeric regions (reviewed in reference 49). In both cases, Rap1p recruits Sir3p and Sir4p to form a complex that inhibits the transcription of the adjacent genes (38). Since our results implicate Rap1p in mediating the repression of RP gene transcription, we asked if Sir3p and Sir4p are involved in the repression.…”

Section: Resultsmentioning

confidence: 99%

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Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis

Nierras

Warner

1999

Molecular and Cellular Biology

103

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The ribosomal proteins (RPs) of Saccharomyces cerevisiae are encoded by 137 genes that are among the most transcriptionally active in the genome. These genes are coordinately regulated: a shift up in temperature leads to a rapid, but temporary, decline in RP mRNA levels. A defect in any part of the secretory pathway leads to greatly reduced ribosome synthesis, including the rapid loss of RP mRNA. Here we demonstrate that the loss of RP mRNA is due to the rapid transcriptional silencing of the RP genes, coupled to the naturally short lifetime of their transcripts. The data suggest further that a global inhibition of polymerase II transcription leads to overestimates of the stability of individual mRNAs. The transcription of most RP genes is activated by two Rap1p binding sites, 250 to 400 bp upstream from the initiation of transcription. Rap1p is both an activator and a silencer of transcription. The swapping of promoters between RPL30 and ACT1 or GAL1 demonstrated that the Rap1p binding sites of RPL30 are sufficient to silence the transcription of ACT1 in response to a defect in the secretory pathway. Sir3p and Sir4p, implicated in the Rap1p-mediated repression of silent mating type genes and of telomere-proximal genes, do not influence such silencing of RP genes. Sir2p, implicated in the silencing both of the silent mating type genes and of genes within the ribosomal DNA locus, does not influence the repression of either RP or rRNA genes. Surprisingly, the 180-bp sequence of RPL30 that lies between the Rap1p sites and the transcription initiation site is also sufficient to silence the Gal4p-driven transcription in response to a defect in the secretory pathway, by a mechanism that requires the silencing region of Rap1p. We conclude that for Rap1p to activate the transcription of an RP gene it must bind to upstream sequences; yet for Rap1p to repress the transcription of an RP gene it need not bind to the gene directly. Thus, the cell has evolved a two-pronged approach to effect the rapid extinction of RP synthesis in response to the stress imposed by a heat shock or by a failure of the secretory pathway. Calculations based on recent transcriptome data and on the half-life of the RP mRNAs suggest that in a rapidly growing cell the transcription of RP mRNAs accounts for nearly 50% of the total transcriptional events initiated by RNA polymerase II. Thus, the sudden silencing of the RP genes must have a dramatic effect on the overall transcriptional economy of the cell.The Saccharomyces cerevisiae cell invests enormous resources in the synthesis of ribosomes. In a rapidly growing cell, the 100 or more rRNA genes are responsible for at least 60% of total transcription (59). The 137 ribosomal protein (RP) genes are among the most active in the genome (57). As a result, the cell has evolved mechanisms that tightly control the synthesis of the components of the ribosome. The 78 RPs are synthesized in nearly equimolar amounts that are matched to the synthesis of rRNA. Furthermore, the synthesis of these components is coo...

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Section: Resultsmentioning

confidence: 99%

“…At the telomeres, Rap1p binds to the [C 1-3A] n repeats (63), leading to the silencing of genes adjacent to telomeres. In both cases Rap1p recruits multiple copies of Sir3p and Sir4p that participate in the silencing, perhaps by interacting with the tails of histones H3 and H4 (38). A mutant allele, rap1 s , relieves the repression at silent mating type loci.…”

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confidence: 99%

Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis

Nierras

Warner

1999

Molecular and Cellular Biology

103

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“…Growth and manipulation of yeast were carried out according to standard procedures (Adams et al 1997). ␤-Galactosidase expression in the two-hybrid strain CTY10-5D (Bartel and Fields 1995) was measured both by liquid assay (Moretti et al 1994) and by nitrocellulose filter assay (Breeden and Nasmyth 1987). Plasmids carrying LexA-Pol12 (full-length) fusion hybrids were obtained by cloning XmaI-XhoI DNA fragments containing either POL12 or pol12-216 genes in the XmaI-SalI-digested pBTM116 vector (Bartel and Fields 1995; pGS86 and pGS96, respectively).…”

Section: Yeast Strains Media and Plasmidsmentioning

confidence: 99%

“…Maintenance of telomere length about a fixed average value is achieved through a mechanism that appears to measure not the TG-repeat tract length per se, but, rather, the number of Rap1 proteins bound to it (Marcand et al 1997;Ray and Runge 1999a). The C terminus of Rap1 negatively regulates telomere elongation in cis (Kyrion et al 1992;Marcand et al 1996), a function that requires two additional proteins, Rif1 and Rif2, both of which interact physically with this domain of Rap1 (Hardy et al 1992;Moretti et al 1994;Wotton and Shore 1997). The inhibition of telomerase addition by Rap1-Rif1/2 complexes increases linearly as a function of telomere tract length (and presumably the number of Rap1-Rif1/2 complexes bound) through a mechanism that is at present unknown.…”

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confidence: 99%

Pol12, the B subunit of DNA polymerase α, functions in both telomere capping and length regulation

Grossi¹,

Puglisi²,

Dmitriev³

et al. 2004

Genes Dev.

129

173

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The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood. Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase ␣ (Pol1)-primase complex, as a new factor involved in this process. Unlike many POL1 and POL12 mutations, which also cause telomere elongation, the pol12-216 mutation described here does not lead to either reduced Pol1 function, increased telomeric single-stranded DNA, or a reduction in telomeric gene silencing. Instead, and again unlike mutations affecting POL1, pol12-216 is lethal in combination with a mutation in the telomere end-binding and capping protein Stn1. Significantly, Pol12 and Stn1 interact in both two-hybrid and biochemical assays, and their synthetic-lethal interaction appears to be caused, at least in part, by a loss of telomere capping. These data reveal a novel function for Pol12 and a new connection between DNA polymerase ␣ and Stn1. We propose that Pol12, together with Stn1, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.

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“…This complex is recruited to DNA by the telomere-binding protein Rap1 (13). A series of physical and genetic arguments imply that this SIR complex may nucleate from the telomere and spread along the chromosome to coat more centromeric regions and thereby silence intervening genes (14,15).…”

mentioning

confidence: 99%

Chromatin Domain Boundaries Delimited by a Histone-binding Protein in Yeast

Ferrari

Simmen

Dusserre

et al. 2004

Journal of Biological Chemistry

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When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.The expression state of a eukaryotic gene depends in part on its location in the chromosome. This position effect results from the organization of eukaryotic genomes into discrete functional domains, defined by local differences in chromatin structure. The expression of genes within each domain appears to be defined and maintained by the concerted action of regulatory elements such as promoters, enhancers, silencers, and locus control regions. Individual domains may be bordered by boundary elements that separate regions of permissive and silent chromatin. Experimentally, boundary elements have been defined functionally by their ability to protect against position effect when flanking the assayed gene. Examples of boundary elements in metazoans include the subtelomeric anti-silencing regions at yeast telomeres, the scs and scsЈ elements flanking the 87A1 hsp70 locus in Drosophila, and the chicken ␤-globin boundary elements (1-4). Several proteins have been associated with barrier activities in yeast, Drosophila, and mammalian cells (5).Transcriptional repression of the yeast Saccharomyces cerevisiae subtelomeric regions is one of the best-studied examples of position-dependent gene expression. This phenomenon is referred to as telomere position effect and is similar to the position effect variegation (PEV) initially described in Drosophila (6). Transcriptional silencing at yeast telomeres is associated with a heterochromatin-like structure (7,8). DNA wrapped in transcriptionally silent chromatin replicates late in S phase and is refractory to some modifying agents. In addition, nucleosomes have reduced acetylation compared with nucleosomes from active region of the genome (9, 10).Transcriptional silencing at subtelomeric regions is mediated by a multiprotein nucleosome binding complex called the SIR complex, composed of the Sir2, Sir3, and...

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Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1.

Cited by 516 publications

References 69 publications

Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis

Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis

Pol12, the B subunit of DNA polymerase α, functions in both telomere capping and length regulation

Chromatin Domain Boundaries Delimited by a Histone-binding Protein in Yeast

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