Systematic profiling of subtelomeric silencing factors in budding yeast.
Alejandro Juárez-Reyes, J Abraham Avelar-Rivas, Jhonatan A Hernandez-Valdes, Bo Hua, Sergio E Campos, James González, Alicia González, Michael Springer, Eugenio Mancera, Alexander DeLuna
Author Information
Alejandro Juárez-Reyes: Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados, 36824 Irapuato, Guanajuato, Mexico. ORCID
J Abraham Avelar-Rivas: Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados, 36824 Irapuato, Guanajuato, Mexico. ORCID
Jhonatan A Hernandez-Valdes: Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados, 36824 Irapuato, Guanajuato, Mexico.
Bo Hua: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Sergio E Campos: Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados, 36824 Irapuato, Guanajuato, Mexico.
James González: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 Ciudad de México, Mexico.
Alicia González: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 Ciudad de México, Mexico.
Michael Springer: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Eugenio Mancera: Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados, 36824 Irapuato, Guanajuato, Mexico. ORCID
Alexander DeLuna: Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados, 36824 Irapuato, Guanajuato, Mexico. ORCID
Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and ageing. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.
