Exploring Centromere Epigeneticity to Facilitate Genetic Tool Development in Yeasts

Centromeres (CENs) are the chromosomal regions promoting kinetochore formation for faithful chromosome segregation. In yeasts, CENs have been recognized as the essential elements for extra-chromosomal DNA stabilization. However, the epigeneticity of CENs makes their localization on individual chromosomes very challenging, especially in many not well-studied nonconventional yeast species. We recently developed a library-based strategy that integrates in silico GC₃ chromosome scanning and high-throughput functional screening, which enabled the isolation of all eight Scheffersomyces stipitis centromeres with a 16, 000-fold reduction in sequence very efficiently. Further identification of a 125-bp CEN core sequence that appears multiple times on each chromosome but all in the unique signature GC3-valley indicates that CEN location might be accurately discerned by their local GC3 percentages in an epigenetic manner.

The essential role played by epigeneticity in modulating the function of centromeres was also recognized in Yarrowia lipolitica. The current Yarrowia plasmids are notoriously unstable. We identified that a previously ignored sequence located between an origin of replication and a CEN in the genome context impacts the plasmid stability, most likely through an epigenetic manner. Inserting this sequence into one of the most popular Yarrowia plasmids significantly improved the plasmid stability, increasing the GFP-positive population from 37% to 72%. These two examples illustrated the importance of understanding the epigenetic feature of CENs when developing the genetic tools for not-well studied eukaryotic hosts.