ABSTRACT
The ability to rapidly assess chromosome instability (CIN) has enabled profiling of most yeast genes for potential effects on genome stability. The A-like faker (ALF) assay is one of several qualitative and quantitative marker loss assays that indirectly measure loss or conversion of genetic material using a counterselection step. The ALF assay relies on the ability to count spurious mating events that occur upon loss of the MATα locus of haploid Saccharomyces cerevisiae strains. Here, we describe the deployment of the ALF assay for both rapid and simple qualitative, and more in-depth quantitative analysis allowing determination of absolute ALF frequencies.
Subject(s)
Chromosomal Instability , Chromosomes, Fungal , Genetic Testing , Yeasts/genetics , Genetic Testing/methods , Genome, Fungal , Genomic Instability , Saccharomyces cerevisiae/geneticsABSTRACT
Sgs1, the orthologue of human Bloom's syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription-replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1Δ reveals copy number changes flanked by repetitive regions with high R-loop-forming potential. Analysis of BLM in Bloom's syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop-associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop-mediated genome instability.
Subject(s)
Bloom Syndrome/genetics , DNA/chemistry , Genomic Instability , RecQ Helicases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Bloom Syndrome/metabolism , Bloom Syndrome/pathology , Cell Line, Transformed , Cell Line, Tumor , DNA/genetics , DNA/metabolism , DNA Repair , DNA Replication , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Dosage , Gene Expression Regulation , Histones/genetics , Histones/metabolism , Humans , Nucleic Acid Conformation , Protein Binding , RNA/genetics , RNA/metabolism , RecQ Helicases/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
BACKGROUND: The transcription factor, Sox2, is central to the behaviour of neural stem cells. It is also one of the key embryonic stem cell factors that, when overexpressed can convert somatic cells into induced pluripotent cells. Although generally studied as a transcriptional activator, recent evidence suggests that it might also repress gene expression. RESULTS: We show that in neural stem cells Sox2 represses as many genes as it activates. We found that Sox2 interacts directly with members of the groucho family of corepressors and that repression of several target genes required this interaction. Strikingly, where many of the genes activated by Sox2 encode transcriptional regulators, no such genes were repressed. Finally, we found that a mutant form of Sox2 that was unable to bind groucho was no longer able to inhibit differentiation of neural stem cells to the same extent as the wild type protein. CONCLUSIONS: These data reveal a major new mechanism of action for this key transcription factor. In the context of our understanding of endogenous stem cells, this highlights the need to determine how such a central regulator can distinguish which genes to activate and which to repress.
