Project description:FACT mediates cohesin function on chromatin Cohesin is a key regulator of genome architecture with roles in sister chromatid cohesion and the organisation of higher-order structures during interphase and mitosis. The recruitment and mobility of cohesin complexes on DNA are restricted by nucleosomes. Here we show that cohesin role in chromosome organization requires the histone chaperone FACT. Depletion of FACT in metaphase cells affects cohesin stability on chromatin reducing its accumulation at pericentric regions and binding on chromosome arms. Using Hi-C, we show that cohesin-dependent TAD (Topological Associated Domains)-like structures in G1 and metaphase chromosomes are disrupted in the absence of FACT. Surprisingly, sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our results uncover a role for FACT in genome organisation by facilitating cohesin dependent compartmentalization of chromosomes into loop domains.
Project description:To address the mechanisms of suppression, we analyzed time course of mRNA expression of four suppressed smc2-8 mutant strains. We addressed the question of genomic robustness by systematically screening genomic open reading frames, when induced for high-level expression, for their ability to suppress 55 conditional lethal mutations in yeast, and have discovered 636 suppressor genes participating in 822 novel dosage suppressor interactions. The suppressor genes are functionally broad and are enriched for overlapping open reading frames where mutually overlapping genes tend to be co-suppressors. Studies on suppressors of defects in chromosome condensation, telomere stability, and RNA polymerase II function suggest that adding interactions, by making significant connections where only weak or undetectable interactions were present (rewiring of gene regulatory pathways, and interaction within and between protein complexes) are frequent mechanisms of dosage suppression.
Project description:Chromosomes are packaged and organized in the nucleus in an ordered, non-random manner. This organization influences many nuclear processes such as transcription, gene silencing and mitosis. While transfer RNA genes (tDNAs) are essential for the generation of tRNAs these gene loci are also binding sites for transcription factors and chromosomal architectural proteins. In the yeast Saccharomyces cerevisiae, tDNAs are dispersed along all sixteen chromosomes. In this study, we investigated the role of tDNAs in genomic organization and nuclear function by editing a chromosome so that it lacks any tDNAs. Our analyses of this tDNA-less chromosome show that loss of tDNAs affect nucleosome positioning, binding of SMC proteins, centromere clustering, long-range chromosome folding and epigenetic gene silencing. We propose that these effects are primarily mediated via changes in local interactions between tDNAs and other regulatory sequences that then manifest as alterations in long-range chromosome architecture with effects on gene regulation over large distances.
Project description:Condensins are broadly conserved chromosome organizers that function in chromatin compaction and transcriptional regulation, but to what extent these two functions are linked has remained unclear. Here, we analyzed the effect of condensin inactivation on genome compaction and global gene expression in the yeast Saccharomyces cerevisiae. Spike-in-controlled 3C-seq analysis revealed that acute condensin inactivation led to a global decrease in close-range chromosomal interactions as well as more specific losses of homotypic tRNA gene clustering. In addition, we identified a condensin-rich topologically associated domain between the ribosomal DNA and the centromere on chromosome XII that is lost upon condensin inactivation. Unexpectedly, these large-scale changes in chromosome architecture were not associated with global changes in transcript levels as determined by spike-in-controlled mRNA-seq analysis. Our data suggest that the global transcriptional program of S. cerevisiae is resistant to condensin inactivation and the associated profound changes in genome organization.
Project description:To address the mechanisms of suppression, we analyzed time course of mRNA expression of four suppressed smc2-8 mutant strains. We addressed the question of genomic robustness by systematically screening genomic open reading frames, when induced for high-level expression, for their ability to suppress 55 conditional lethal mutations in yeast, and have discovered 636 suppressor genes participating in 822 novel dosage suppressor interactions. The suppressor genes are functionally broad and are enriched for overlapping open reading frames where mutually overlapping genes tend to be co-suppressors. Studies on suppressors of defects in chromosome condensation, telomere stability, and RNA polymerase II function suggest that adding interactions, by making significant connections where only weak or undetectable interactions were present (rewiring of gene regulatory pathways, and interaction within and between protein complexes) are frequent mechanisms of dosage suppression. RNA samples isolated from two individual biological replicates, four time points each (0, 45, 90 and 180 min), of smc2-8 mutant strains harboring pGAL:SMC2, pGAL:UME1, pGAL:MEK1, pGAL:HTA2, pGAL:SNU66, and the negative control MORF plasmid (pGAL:negative, BG1766) and hybridized Affymetrix microarrays.
Project description:Natural genetic variation can cause significant differences in gene expression, but little is known about the polymorphisms that affect gene regulation. We analyzed regulatory variation in a cross between laboratory and wild strains of Saccharomyces cerevisiae. Clustering and linkage analysis defined groups of coregulated genes and the loci involved in their regulation. Most expression differences mapped to trans-acting loci. Positional cloning and functional assays showed that polymorphisms in GPA1 and AMN1 affect expression of genes involved in pheromone response and daughter cell separation, respectively. We also asked whether particular classes of genes were more likely to contain trans-regulatory polymorphisms. Notably, transcription factors showed no enrichment, and trans-regulatory variation seems to be broadly dispersed across classes of genes with different molecular functions Keywords: other