Edgar Trelles-Sticken

Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast.

Recent experiments have shown that gene repression can be correlated with relocation of genes to heterochromatin-rich silent domains. Here, we investigate whether nuclear architecture and spatial positioning can contribute directly to the transcriptional activity of a genetic locus in Saccharomyces cerevisiae. By disassembling telomeric silent domains without altering the chromatin-mediated silencing machinery, we show that the transcriptional activity of silencer–reporter constructs depends on intranuclear position. This demonstrates that telomeric silent domains are actively involved in transcriptional silencing. Employing fluorescent in situ hybridization (FISH) in combination with genetic assays, we demonstrate that telomeres control the establishment of transcriptional states by reversible partitioning with the perinuclear silencing domains. Anchoring telomeres interferes with their ability to assume an active state, whereas disassembly of silencing domains prevents telomeres from assuming a repressed state. Our data support a model in which domains of enriched transcriptional regulators allow genes to determine transcriptional states by spatial positioning.

Meiotic telomere clustering requires actin for its formation and cohesin for its resolution

In diploid organisms, meiosis reduces the chromosome number by half during the formation of haploid gametes. During meiotic prophase, telomeres transiently cluster at a limited sector of the nuclear envelope (bouquet stage) near the spindle pole body (SPB). Cohesin is a multisubunit complex that contributes to chromosome segregation in meiosis I and II divisions. In yeast meiosis, deficiency for Rec8 cohesin subunit induces telomere clustering to persist, whereas telomere cluster–SPB colocalization is defective. These defects are rescued by expressing the mitotic cohesin Scc1 in rec8Δ meiosis, whereas bouquet-stage exit is independent of Cdc5 pololike kinase. An analysis of living Saccharomyces cerevisiae meiocytes revealed highly mobile telomeres from leptotene up to pachytene, with telomeres experiencing an actin- but not microtubule-dependent constraint of mobility during the bouquet stage. Our results suggest that cohesin is required for exit from actin polymerization–dependent telomere clustering and for linking the SPB to the telomere cluster in synaptic meiosis.

Wild-Type Levels of Spo11-Induced DSBs Are Required for Normal Single-Strand Resection during Meiosis

We have studied the repair of a DNA-DSB created by the VMA1-derived endonuclease in mutants that have different levels of Spo11-DSBs: WT (sae2), few (hop1), and none (spo11-Y135F). In spo11-Y135F and hop1 cells, intrachromosomal repair is more frequent than in WT and sae2 cells. In spo11-Y135F cells there was no chromosome pairing or synapsis and a faster turnover of resected DNA. Compared to WT and sae2 cells, spo11-Y135F and hop1 cells have a greater proportion of long resection tracts. The data suggest that high levels of Spo11-DSBs are required for normal regulation of resection, even at a DSB created by another protein. WT control over resection could be important for directing repair to be interchromosomal, increasing the chance of creating interhomolog connections essential to meiotic segregation.

Increased ploidy and KAR3 and SIR3 disruption alter the dynamics of meiotic chromosomes and telomeres

We investigated the sequence of chromosomal events during meiotic prophase in haploid, diploid and autotetraploid SK1 strains of Saccharomyces cerevisiae. Using molecular cytology, we found that meiosis-specific nuclear topology (i.e. dissolution of centromere clustering, bouquet formation and meiotic divisions) are significantly delayed in polyploid SK1 meiosis. Thus, and in contrast to the situation in plants, an increase in ploidy extends prophase I in budding yeast. Moreover, we found that bouquet formation also occurs in haploid and diploid SK1 meiosis deficient in the telomeric heterochromatin protein Sir3p. Diploid sir3Δ SK1 meiosis showed pleiotropic defects such as delayed centromere cluster resolution in a proportion of cells and impeded downstream events (i.e. bouquet formation, homologue pairing and meiotic divisions). Meiotic telomere clustering occurred in diploid and haploid sir3Δ strains. Using the haploid system, we further show that a bouquet forms at the kar3Δ SPB. Comparison of the expression of meiosis-specific Ndj1p-HA and Zip1p in haploid control and kar3Δ time courses revealed that fewer cells enter the meiotic cycle in absence of Kar3p. Elevated frequencies of bouquets in kar3Δ haploid meiosis suggest a role for Kar3p in regulation of telomere dynamics.

Spatial organisation and behaviour of the parental chromosome sets in the nuclei of Saccharomyces cerevisiae x S. paradoxus hybrids.

We demonstrate that the genomes of Saccharomyces cerevisiae and S. paradoxus are sufficiently divergent to allow their differential labeling by genomic in situ hybridisation (GISH). The cytological discrimination of the genomes allowed us to study the merging of the two genomes during hybrid mating. GISH revealed that in hybrid nuclei the two genomes are intermixed. In hybrid meiosis, extensive intraspectific nonhomologous pairing takes place. GISH on chromosome addition and substitution strains (with chromosomes of S. paradoxus added to or replacing the homoeologous chromosome of an otherwise S. cerevisiae background) was used to delineate individual chromosomes at interphase and to examine various aspects of chromosome structure and arrangement.

Yeast FISH: Delineation of Chromosomal Targets in Vegetative and Meiotic Yeast Cells

Fluorescence in situ hybridization provides an effective means to delineate chromosomes and chromosomal regions of interest, during all stages of the cell cycle, irrespective of chromatin composition and compaction. This property makes FISH particularly useful for studying chromosome behavior in species with minute genomes and/or ill-defined metaphase chromosomes. Genetic model organisms like fission and budding yeast display such cytological characteristics. Consequently, chromosome painting and locus-specific yeast FISH (Scherthan et al. 1992, Scherthan et al. 1994, Uzawa et al. 1992, Guacci et al. 1994, Weiner and Kleckner 1994, Gotta et al. 1996) has become an indispensable tool in the analysis of chromosome dynamics and organization in wild-type and mutant yeast strains.