Rusche Lab Research Highlights

The Rusche lab studies chromatin and its impact on gene expression and chromosome function. We also study how protein functions shift over evolutionary time through mechanisms including gene duplication and rewiring of transcriptional circuits. In our studies, we gain an evolutionary perspective by comparing multiple yeast species, taking advantage of the genome editing and comparative genomic approaches available for these species.

One arm of our research program focuses on Sir2 proteins (sirtuins), which deacetylate histones to repress transcription. Because sirtuins require NAD+  for activity, they are thought to link the processes they regulate with nutrient availability. We are investigating how the functions of yeast sirtuins have shifted over the course of evolution to enable species to develop distinct responses to low NAD+ stress.

Another arm of our research focuses on the nucleosome-binding protein Sir3, which partners with Sir2 to form heterochromatin. We are reconstructing the steps by which Sir3 evolved from the conserved replication protein Orc1 through gene duplication, subfunctionalization, and specialization.

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Rusche lab, 2019 Back row: Chris Rupert, Guolei Zhao, Tianyi Zhou, Haniam Maria Front row: Shivali Kapoor, Lynn Sidor, Laura Rusche .

Rusche Lab, 2019 Group Photo (left to right) Back row: Chris Rupert, Guolei Zhao, Tianyi Zhou, Haniam Maria. Front row: Shivali Kapoor, Lynn Sidor, Laura Rusche.

Network of genes regulated by yeast sirtuin deacetylases

  • Evolution of distinct responses to low NAD+ stress by rewiring the Sir2 deacetylase network in yeasts.
    Humphrey KM, Zhu L, Hickman MA, Hasan S, Maria H, Liu T, Rusche LN. (2020) DOI: 10.1534/genetics.120.303087
    In this study, we identified the genes regulated by the NAD+-dependent deacetylase Sir2/Hst1 in two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis. We found distinct sets of sirtuin-regulated genes in the two species. K. lactis has a larger set of regulated genes, some of which are involved in biological functions beyond those observed in the S. cerevisiae gene set. We also found that both regulated gene sets were depleted for broadly conserved genes, consistent with sirtuins regulating processes restricted to a few species. These results support a model in which sirtuins serve as rewiring points that allow species to evolve distinct responses to low NAD+ stress.
     
  • The Sir2-Sum1 repressor complex uses promoter-specific and long-range mechanisms to regulate cell identity and sexual cycle in the yeast Kluyveromyces lactis.
    Hickman MA and Rusche LN. (2009) DOI: 10.1371/journal.pgen.1000710
    In this study, we characterized the deacetylase Sir2 in the yeast Kluyveromyces lactis. KlSir2 is orthologous to the Saccharomyces cerevisiae proteins Sir2 and Hst1, which arose through gene duplication. We found that KlSir2 is multifunctional, resembling both ScSir2 and ScHst1. This result indicates that Sir2 and Hst1 subfunctionalized after duplication. We also found that the K. lactis Sir2-Sum1 complex employs both long-range and promoter-specific mechanisms to repress transcription, revealing that a single repressive complex can act through two distinct mechanisms.
     

Sirtuin deacetylases in fungal pathogens

  • Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans.
    Zhao G and Rusche LN. (2021) DOI: 10.1128/mSphere.00053-21
    In this study, we examined the roles of three sirtuin deacetylases - Sir2, Hst1, and Hst2 - in the filamentous growth of the fungal pathogen Candida albicans. We found that formation of hyphae was reduced when Sir2 was absent or catalytically inactive, but it was unaffected by the loss of HST1 or HST2. Because Sir2 requires NAD+ for deacetylase activity, it could serve as a sensor to reduce filamentous growth when intracellular NAD+ is low.

  • Sporadic gene loss after duplication is associated with functional divergence of sirtuin deacetylases among Candida yeast species.
    Rupert CB, Heltzel JM, Taylor DJ, Rusche LN. (2016) DOI:  10.1534/g3.116.033845
    In this study, we traced duplications and losses of the SIR2/HST1 genes in the CTG Candida clade of yeasts, which includes pathogenic species such as Candida albicans. We found that a single duplication of the SIR2/HST1 gene occurred prior to the emergence of the CTG clade. This ancient duplication was followed by at least two independent losses of SIR2. In contrast, HST1 was uniformly retained. Functional characterization of Sir2 and Hst1 in three species revealed that these proteins have not maintained consistent functions since the duplication, supporting the model that sirtuin deacetylases can regain ancestral functions to compensate for gene loss.

Duplication and subfunctionalization of the deacetylase Sir2

  • The duplicated deacetylases Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations.
    Froyd CA and Rusche LN. (2011) DOI: 10.1128/MCB.05175-11
    In this study, we examined how the paralogous deacetylases Sir2 and Hst1 subfunctionalized after duplication. To do so, we compared the duplicated deacetylases from Saccharomyces cerevisiae with a non-duplicated ortholog from Kluyveromyces lactis. These deacetylases interact with adaptor proteins that target them to specific genomic loci. We found that the interaction domains for these adaptor proteins have been retained over the course of evolution and can be disrupted by simple amino acid substitutions. Therefore, Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations in these interaction domains.

  • Substitution as a mechanism for genetic robustness: The duplicated deacetylases Hst1p and Sir2p in Saccharomyces cerevisiae. 
    Hickman MA and Rusche LN. (2007) DOI: 10.1371/journal.pgen.0030126
    In this study, we examined the sirtuin deacetylases Sir2 and Hst1, which are products of gene duplication and have non-overlapping functions in Saccharomyces cerevisiae. We found that these paralogs can provide genetic robustness against inactivating mutations through a substitution mechanism – Sir2 can fill in for Hst1 in the SUM1C repressor complex. This ability likely results from a retained but reduced affinity for the SUM1C that is a consequence of subfunctionalization via the duplication, degeneration, and complementation mechanism. These results suggest that the evolutionary path of duplicate gene preservation may be an important indicator for the ability of duplicated genes to contribute to genetic robustness.

Evolution of heterochromatin protein Sir3 from DNA replication protein Orc1

  • The transcriptional silencing functions of the yeast protein Orc1/Sir3 subfunctionalized after gene duplication.
    Hickman MA and Rusche LN. (2010) DOI: 10.1073/pnas.1006436107
    In this study, we examined how the heterochromatin protein Sir3 arose from the replication protein Orc1. We found that a non-duplicated Orc1 from the yeast Kluyveromyces lactis contributes to heterochromatin formation at telomeres and a mating-type locus, much as Sir3 does in Saccharomyces cerevisiae. Moreover, the ability of KlOrc1 to spread across a silenced locus depends on its nucleosome-binding BAH domain and the deacetylase Sir2. These findings demonstrate that Orc1 functioned in silencing before duplication and suggest that Orc1 and Sir2, both of which are broadly conserved among eukaryotes, may have an ancient history of cooperating to generate chromatin structures.

  • The yeast heterochromatin protein Sir3 experienced functional changes in the AAA+ domain after gene duplication and subfunctionalization.
    Hanner AS and Rusche LN. (2017) DOI: 10.1534/genetics.117.300180
    In this study, we investigated whether Sir3 evolved new or optimized properties after it diverged from Orc1 through duplication and subfunctionalization. To identify regions of Sir3 that may have evolved new properties, we created chimeric proteins of Sir3 from Saccharomyces cerevisiae and nonduplicated Orc1 from Kluyveromyces lactis. We identified the AAA+ base subdomain of KlOrc1 as insufficient for heterochromatin formation in S. cerevisiae. In Orc1, this subdomain is intimately associated with other ORC subunits, enabling ATP hydrolysis. Thus, once Sir3 was no longer constrained to assemble into the ORC complex, its heterochromatin-forming potential likely evolved through changes in the AAA+ base subdomain.

Spreading of heterochromatin proteins along the chromosome

  • A silencer promotes the assembly of silenced chromatin independently of recruitment. 
    Lynch PJ and Rusche LN. (2009) DOI: 10.1128/MCB.00983-08
    In this study, we examined the kinetics of Sir protein recruitment and spreading at two heterochromatic loci, the mating-type locus HMR and a telomere. We found that, although the Sir proteins were recruited to both loci at equivalent rates, they spread away from the recruitment site considerably more rapidly at HMR than the telomere. Additionally, insertion of the recruitment sequence (silencer) from HMR into the telomere increased the rate of Sir protein spreading. This ability of a silencer to promote assembly of silenced chromatin over several kilobases is likely an important mechanism for maintaining what would otherwise be unstable chromatin.

  • Evolution of a new function through a single amino acid change in the yeast repressor Sum1p.
    Safi A, Wallace KA, and Rusche LN. (2008) DOI: 10.1128/MCB.01785-07
    In this study, we examined a case in which a single amino acid change results in a new function – the Sum1 DNA binding repressor gains the ability to form an extended chromatin structure. We found that a threonine to isoleucine change reduced the DNA binding affinity of Sum1 and caused it to self-associate. These findings suggest that interaction domains may be hotspots for gain-of-function mutations because alterations in such domains have the potential to redirect a protein to new sets of binding partners.

Chromosomal features in yeast species

  • Conservation of a DNA replication motif among phylogenetically distant budding yeast species.
    Maria H, Kapoor S, Liu T, and Rusche LN. (2021) DOI: 10.1093/gbe/evab137
    In this study, we identified and characterized the origins of DNA replication in the yeast Torulaspora delbrueckii, which belongs to a clade of yeast that did not undergo a whole-genome duplication. We found that a DNA sequence motif associated with these origins is nearly identical to the known motif in the duplicated species Saccharomyces cerevisiae. Thus, the DNA replication motif arose prior to the whole-genome duplication and has been maintained for over 100 million years.

  • Regional centromeres in the yeast Candida lusitaniae lack pericentromeric heterochromatin.
    Kapoor S, Zhu L, Froyd C, Liu T, and Rusche LN. (2015) DOI: 10.1073/pnas.150874911
    In this study, we identified and characterized the centromeres in the yeast Clavispora lusitaniae, which is related to the emerging pathogen Candida auris. We identified a distinct type of regional centromere that is epigenetically inherited and lacks pericentromeric heterochromatin.

Rusche Lab Research Areas