Abstract. We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the threedimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere-associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten loci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3-strain, and similarly, Sir3p staining is no longer punctate in a sir4-strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.S EVERAL lines of evidence, including in situ hybridization with whole chromosome probes, suggest that the organization of chromosomes within the interphase nucleus is not random (for review see Cremer et al., 1993). Indeed, it is generally assumed that three-dimensional nuclear organization is likely to facilitate essential nuclear functions, such as transcription, the processing and transport of mRNA, replication, and recombination. Evidence for the organization of chromosomes in prophase nuclei was provided over a hundred years ago by Rabl's observation that salamander chromosomes are positioned in nuclei with centromeres clustered at one pole and the telomeres at the opposite pole (RAN, 1885). Work by Sedat and his colleagues later lent support to this notion through the study of the polytene salivary gland chromosomes of Drosophila melanogaster. They found that centromeres, fused into the chromocenter, abut the nuclear envelope within a restricted area while telomeres tended to cluster at the opposite pole (Mathog et al., 1984;Hochstrasser et al., 1986). A peripheral localization of telomeres has been also reported in Trypanosoma (Chung et

Transcriptional repression at the silent mating-type loci in yeast requires the targeting of silent information regulator (Sir) proteins through specific interactions formed at cis-acting silencer elements. We show here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere. Repression is restored by creation of a new telomere 13 kb from the integrated reporter or by elevated expression of SIRl, SIRS, and/or SIR4. Coupled expression represses in an additive manner, suggesting that all three factors are in limiting concentrations. When overexpressed, Sir3 and Sir4 are dispersed throughout the nucleoplasm, in contrast to wild-type cells where they are clustered in a limited number of foci together with telomeres. Efficient silencer function thus seems to require either proximity to a pool of concentrated Sir proteins, that is, proximity to telomeres, or delocalization of the silencing factors.

No abstract

The Ccr4-Not complex is a global regulator of transcription that affects genes positively and negatively and is thought to regulate transcription factor IID function. Two components of this complex, Caf1p and Ccr4p, are directly involved in mRNA deadenylation, and Caf1p is associated with Dhh1p, a putative RNA helicase thought to be a component of the decapping complex. In this work, we tried to determine whether Dhh1p might interact with the Ccr4-Not complex. We found that, first, not mutations displayed severe synthetic phenotypes when combined with a dhh1-null mutation. Second, overexpression of Not1p was toxic in dhh1-null cells. Third, a not mutant phenotype was suppressed by deletion of DHH1 and mimicked by overexpression of DHH1. Fourth, dhh1-null mutants displayed resistance to heat shock, a phenotype observed for all mutants that affect the Ccr4-Not complex. Finally, like Caf1p and Ccr4p, Dhh1p co-immunoprecipitated with the nonessential Nterminal domain of Not1p, and the levels of Caf1p and Dhh1p were dependent upon this Not1p domain. Taken together, our results suggest that the Ccr4-Not complex, via the N-terminal region of Not1p, is necessary for the maintenance of stable cellular levels of Dhh1p and Caf1p, thus contributing to regulation of mRNA decay in addition to transcription.

Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis‐acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long‐range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild‐type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir‐containing complexes bind, nucleating the silent chromatin state.