Silenced heterochromatin influences all nuclear processes including chromosome structure, nuclear organization, transcription, replication, and repair. Proteins that mediate silencing affect all of these nuclear processes. Similarly proteins involved in replication, repair, and chromosome structure play a role in the formation and maintenance of silenced heterochromatin. In this chapter we describe a handful of simple tools and methods that can be used to study the atypical role of proteins in gene silencing.

Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms.

The genome is packaged and organized in an ordered, nonrandom manner, and specific chromatin segments contact nuclear substructures to mediate this organization. tRNA genes (tDNAs) are binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the roles of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacked any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromatin architecture or chromosome tethering and mobility. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long-range HML-HMR heterochromatin clustering with concomitant effects on gene silencing. We propose that the tDNAs primarily affect local chromatin structure, which results in effects on long-range chromosome architecture.

The genome is packaged and organized in an ordered, nonrandom manner, and specific chromatin segments contact nuclear substructures to mediate this organization. tRNA genes (tDNAs) are binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the roles of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacked any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromatin architecture or chromosome tethering and mobility. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long-range HML-HMR heterochromatin clustering with concomitant effects on gene silencing. We propose that the tDNAs primarily affect local chromatin structure, which results in effects on long-range chromosome architecture.