Laura Rusche

Assistant Professor of Biochemistry

Research Program

Sir2 deacetylases and their contributions to chromatin structure, transcriptional repression, and genome stability

Sir2 deacetylases, which are found in all kingdoms of life, have a wide range of biological functions and regulate key transitions in the life-cycles of many organisms. In the yeast Saccharomyces cerevisiae, Sir2 is required for assembly of heterochromatin at the telomeres and mating-type loci and for suppressing recombination in the repetitive rDNA array. A closely related deacetylase, Hst1, represses genes required for meiosis and sporulation. We are studying how Sir2 and Hst1 govern the yeast life cycle (mating and sporulation) and how these proteins contribute to the formation of various flavors of repressive chromatin at different locations in the yeast genome.

How is Sir2-mediated heterochromatin restricted to appropriate genomic locations?

A notable property of heterochromatin (condensed, silenced chromatin) is its ability to propagate along a chromosome to form an extended repressive domain. However, this capacity to spread is potentially toxic to a cell if heterochromatin spreads too far and represses essential genes. In S. cerevisiae, the structural components of heterochromatin are the Sir proteins. Sir2 is a conserved histone deacetylase, and Sir3 and Sir4 are nucleosome-binding proteins. (Sir3 and Sir4 are not related to the Sir2 deacetylase family, despite being “Sir” proteins.) Together, these three proteins spread along the chromosome, forming a repressive chromatin structure. DNA sequences termed silencers initiate this process by recruiting Sir proteins to the chromosome. We have investigated the assembly of Sir-silenced chromatin in vivo and discovered that the capacity of Sir proteins to spread is more limited than previously thought, explaining why silenced chromatin rarely assembles in inappropriate locations. We also discovered that, in the face of limitations to spreading, some silencers enhance the association of Sir proteins with nucleosomes over a region of several kilobase pairs, thereby promoting the formation of heterochromatin. We have proposed that this enhancement occurs through the formation of higher-order chromatin loops. Future work will focus on elucidating the architecture of silenced chromatin and how it is shaped by silencers.

How has gene duplication shaped the mechanism of Sir2-mediated silencing?

Approximately 10% of S. cerevisiae genes are retained duplicates from a whole-genome duplication that occurred about 100 million years ago. The deacetylases Sir2 and Hst1 are one such duplicate pair, as are the nucleosome-binding protein Sir3 and the replication protein Orc1. The fact that two of the three proteins that make up heterochromatin in S. cerevisiae have retained duplicates suggests that the whole-genome duplication enabled the specialization of the silencing mechanism. We investigated the functions of the non-duplicated Sir2/Hst1 and Orc1/Sir3 proteins in the yeast Kluyveromyces lactis, which diverged from S. cerevisiae prior to the whole-genome duplication. We discovered that in both cases, the non-duplicated K. lactis protein carries out the functions of both duplicated S. cerevisiae proteins. Therefore, both Sir2 and Orc1 contributed to the formation of heterochromatin prior to duplication, and their silencing functions were partitioned away from other functions after duplication. Sir2 and Orc1 are both conserved throughout eukaryotes, and we speculate that they have an ancient history of cooperation to form special chromatin structures.

What are the links between DNA replication and heterochromatin?

Connections between heterochromatin and the origin recognition complex (ORC), which defines origins of replication, have been reported in a wide variety of organisms, but the functional role of ORC in heterochromatin formation remains poorly understood. The evolution of the nucleosome-binding protein Sir3 from Orc1 suggests that Orc1 has inherent properties that enable it to participate in specialized chromatin structures. In fact, we discovered that in the yeast K. lactis, which diverged from S. cerevisiae prior to the duplication, the single Orc1/Sir3 protein is a structural component of Sir2-silenced chromatin. Future work will investigate how Orc1 contributes to chromatin structures both in K. lactis and S. cerevisiae.

How do Sir2 family deacetylases contribute to the biology of pathogenic yeasts?

Sir2 deacetylases regulate key transitions in the life-cycles of many organisms and have been linked to senescence and aging. In keeping with these observations, in S. cerevisiae, Sir2 and Hst1 repress genes that govern mating and sporulation. Sir2 may also contribute to the ability of yeast to adapt to new environments by forming heterochromatin near telomeres. The subtelomeric regions of yeast genomes contain genes required for utilization of alternate nutrients and for adhesion, which is required by pathogenic yeast for filamentous growth and colonization of host tissues. In principle, Sir2-mediated heterochromatin could regulate the expression of these genes. To gain a broader perspective on how Sir2 deacetylases contribute to the ability of yeasts to adapt to changing environments and complete their life cycles, we are investigating the biological functions of Sir2 in pathogenic yeasts.