Browsing by Author "Rusche, Laura N"

Heterochromatin, or condensed chromatin, is a transcriptionally repressive form of chromatin that occurs in many eukaryotic organisms. At its natural locations, heterochromatin is thought to play important roles in genome organization as well as gene expression. Just as important is the restriction of this repressive form of chromatin to appropriate regions of the genome. In the budding yeast Saccaromyces cerevisiae, domains of condensed, transcriptionally silenced chromatin are found at telomeres and at the silent-mating type cassettes, HML and HMR. At these locations, a complex of Silent Information Regulator (SIR) proteins gets recruited to DNA through discrete silencer elements. Once recruited, the Sir protein complex then spreads along chromosomes in a step-wise manner. This process results in the silencing of gene expression. It is unclear whether silenced chromatin is established in the same manner at different genomic locations. Understanding how silenced chromatin is formed is important for determining how these chromatin structures are regulated.

To better understand how silenced chromatin is established in different genomic contexts, I used chromatin immuoprecipitation to follow the rate of silenced chromatin formation at different locations. The rates of Sir protein assembly were compared at two locations, telomere VI-R and HMR. I discovered that the silencers at these two locations were equally proficient at recruiting Sir proteins. However, the rate of Sir protein assembly onto nucleosomes was far more rapid at HMR than at the telomere VI-R. Furthermore, the rate of Sir protein assembly was more rapid on one side of the HMR-E silencer at HMR than the other. Moreover, insertion of the HMR-E silencer adjacent to the telomere VI-R significantly improved the rate of Sir protein assembly onto nucleosomes. Additionally, observations that the association of Sir protein occurs simultaneously across several kilobases at HMR and that silencing at HMR is insensitive to co-expression of wild-type and catalytically inactive Sir2 proteins suggest that HMR-E enables the assembly of silenced chromatin in a non-linear fashion. These results suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases.

In addition to the HMR-E silencer, HMR is also characterized by the presence of a second auxiliary HMR-I silencer and a tRNA gene that functions as a boundary element to restrict the spread of silenced chromatin. I used chromatin immunoprecipitation to determine how each of these regulatory elements contribute to the steady-state levels of Sir protein association with chromatin. Consistent with a role for HMR-E beyond recruitment, I discovered that the HMR-E silencer alone promoted higher levels of Sir proteins on nucleosomes compared to the telomere VI-R. The levels of Sir protein association with HMR were further elevated by the HMR-I silencer, even though this silencer does not recruit Sir proteins on its own and does not contribute to any of the known functions of silenced chromatin at HMR. Additionally, although the tRNA gene did block the spread Sir proteins, I discovered that the capacity for Sir proteins to spread beyond a few kilobases was severely limited even in the absence of the boundary.

The results of this thesis work provide new insights into the mechanisms of silenced chromatin establishment and regulation in budding yeast. I show here that the capacity of Sir proteins to assemble onto nucleosomes is inherently limited. Additionally, silencers vary in their ability to promote this assembly. I conclude that the silencer is a key factor in determining the relative size, efficiency, and location of silenced chromatin domains in the cell.

In Saccharomyces cerevisiae, proper maintenance of haploid cell identity requires the SIR complex to mediate the silenced chromatin found at the cryptic mating-type loci, HML and HMR. This complex consists of Sir2, a histone deacetylase and the histone binding proteins Sir3 and Sir4. Interestingly, both Sir2 and Sir3 have paralogs from a genome duplication that occurred after the divergence of Saccharomyces and Kluyveromyces species. The histone deacetylase HST1 is the paralog of SIR2 and works with the promoter-specific SUM1 complex to repress sporulation and alpha-specific genes. ORC1 is the paralog of SIR3 and is an essential subunit of the Origin Recognition Complex and also recruits SIR proteins to the HM loci. I have investigated the functions of these proteins in the non-duplicated species Kluyveromyces lactis and compared these functions to those found in S. cerevisiae.

I have shown that SIR2 and HST1 subfunctionalized post-duplication via the duplication, degeneration and complementation mechanism. In S. cerevisiae, Sir2 has retained the ability to function like Hst1 when in an hst1Δ strain. I have also shown, with a chimeric Sir2-Hst1 protein, that there are distinct specificity domains for Sir2 interaction with the SIR complex and Hst1 interaction with the SUM1 complex that have diverged between Sir2 and Hst1. Trans-species complementation assays show that the non-duplicated Sir2 from K. lactis can interact with both SIR and SUM1 complexes in S. cerevisiae.

Further analysis into the non-duplicated experimental system of K. lactis has revealed that deletion of KlSir2 de-represses the HM loci as well as sporulation and cell-type specific genes. A physical interaction between KlSir2 and the histone binding protein KlSir4 is conserved in K. lactis, and both proteins spread across the HML locus and associate with telomeres in a manner similar to S. cerevisiae. KlSir2 also physically interacts with the DNA-binding protein, KlSum1, to repress sporulation and cell-type specific genes in a promoter-specific manner and recruitment of KlSir2 to these loci is dependent on KlSum1. Surprisingly, deletion of KlSUM1 also de-represses HML and HMR, a phenotype not observed in S. cerevisiae. I show by chromatin immunoprecipitation that KlSum1 directly regulates the HM loci by spreading across these regions in a mechanism that is distinct from its role in repressing sporulation-specific genes. This result indicates that KlSum1 is a key regulator of not only meiotic, but also mating-type transcriptional programming.

The SIR3-ORC1 gene pair has previously been used as an example of neofunctionalization based on accelerated rates of evolution. However, my studies of KlOrc1 show it is distributed across HML and associates with Sir2 and Sir4 at telomeres, indicative of it having Sir3-like capabilities to spread across chromatin. This ability of KlOrc1 to spread is distinct from its functions with ORC, and is entirely dependent on its BAH domain. These findings demonstrate that prior to the genome duplication there was a silencing complex that contained both KlSir2 and KlOrc1. In addition to their functions at HML and the telomeres, KlOrc1 associates with replication origins and KlSir2 and KlSum1 work in complex to repress sporulation genes in a promoter-specific manner. The multiple functions of both KlOrc1 and KlSir2 in K. lactis indicate that after duplication, these properties were divided among paralogs and subsequently specialized to perform the functions that have been characterized in S. cerevisiae.

Chromatin is composed of DNA, histones, and other proteins and contributes to DNA packaging, controlling gene expression and DNA replication. This work focuses on the contributions of histones H3 and H4 to gene regulation in the yeast Saccharomyces cerevisiae. I identified a region of the nucleosome that is critical for three types of long-range transcriptional silencing but not for local repression mediated by some of the same proteins.

In S. cerevisiae, the Sir complex performs long range silencing of the mating type loci, while the promoter specific Sum1 complex represses mid-sporulation genes. Interestingly, the SUM1-1 mutation changes the Sum1 repression complex into a silencing complex capable of long range spreading. Sum1-1 provides a good model to distinguish between properties of nucleosomes important for long-range silencing (common to Sum1-1 and Sir silencing), and specific interactions nucleosomes might make with the Sum1 complex (common to Sum1 and Sum1-1 complexes). Interactions between nucleosomes and silencing proteins are critical to Sir silencing, and the spreading ability of Sum1-1p suggests that a component of the Sum1-1 complex may also interact with nucleosomes. Since the Sum1-1 and Sum1 complex components are shared, histone contacts may also contribute to wild type Sum1 repression.

I investigated the contributions of histones H3 and H4 to Sum1-1 silencing and Sum1 repression using a genetic screen. Interestingly, I found histone mutations that disrupt Sum1-1 silencing and cluster in the LRS/H4 region of the nucleosome, which was previously identified to disrupt silencing at the mating type loci, telomeres, and rDNA. Therefore, this region of the nucleosome is important to silencing mediated by three distinct complexes- Sir, RENT, and Sum1-1. The Sir3p bromo-adjacent homology (BAH) domain binds this region of the nucleosome to facilitate Sir spreading and silencing, and I tested Orc1p, a paralog of Sir3p, to determine if it makes similar contributions to Sum1-1 silencing. Using reporter mating assays and chromatin immunoprecipitation, I found that mutations and deletion of the BAH domain of Orc1p disrupt Sum1-1 silencing. These results suggest that Orc1p may interact with this region of the nucleosome and contribute to Sum1-1 silencing outside of recruitment.

Surprisingly, Sum1 repression was not disrupted by histone mutations. I conducted in vitro binding assays to identify a region in Sum1p that may interact with histones and account for the spreading ability of Sum1-1p. Consistent with results that histones do not contribute to Sum1 repression, I did not find evidence of Sum1p binding to histone peptides. Therefore, interactions with histones H3 and H4 are important to Sir and Sum1-1 silencing and not Sum1 repression. These interactions with histones may facilitate the formation of higher order chromatin structures necessary for long range silencing complexes.

I also identified mutations in the H3 tail that disrupt Sum1-1 silencing. Surprisingly, these mutations did not disrupt the enrichment of Sum1-1p. Similar observations have been made for Sir proteins in the absence of the H3 tail, and the H3 tail may contribute to chromatin compaction and silencing after the assembly of silencing proteins. Therefore, the Sir and Sum1-1 complexes may share several features that facilitate silencing. The use of the LRS/H4 region of the nucleosome may be a common interaction surface with silencing proteins, and the H3 tail may assist in the formation of a specialized chromatin structure. These interactions may also be utilized in the formation of heterochromatin in higher eukaryotes.

Gene duplication is an important evolutionary tool for fostering diversification and expanding gene families. However, while this concept is well understood and accepted in a theoretical capacity, the particular changes that lead to the functional diversification of gene duplicates are less well understood and documented. Additionally, little work has been done to understand how functions are gained or lost, which leads to the diversification of orthologous genes. The Sir2 family of NAD+-dependent deacetylases is an excellent gene family to study questions of duplication and diversification as it is ubiquitous throughout all kingdoms of life, and it has expanded through a number of gene duplications so that while most bacteria have a single sirtuin/species, mammals have seven sirtuins/species. Sirtuins also have a wide array of biological functions and targets, but some of these functions are conserved in eukaryotes.

In this study, Sir2 is used to investigate the principles behind gene duplication and functional diversification in a molecular context. Sir2 function is studied in multiple species of budding yeast, the model organism Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida lusitaniae using a combination of genetic, biochemical, and high-throughput methods. Sir2 and its paralog Hst1 from S. cerevisiae were used with their non-duplicated ortholog Sir2 from K. lactis to examine the type of molecular changes that occur after gene duplication and lead to subfunctionalization. Then Sir2 from the more divergent C. lusitaniae was used to study how functions are gained or lost.

To study the molecular mechanism of subfunctionalization in the duplicated deacetylases ScSir2 and ScHst1 with the non-duplicated KlSir2 used as a proxy for the ancestral state, we hypothesized that the basis for subfunctionalization in this case was in the interaction domains. ScSir2 and ScHst1 act in distinct complexes that target them to the genomic loci they regulate. KlSir2 interacts with the same complexes as both ScSir2 and ScHst1. Therefore, we first identified the minimal regions of ScSir2 and ScHst1 necessary for each to interact with its respective complex. Then we identified mutations in those interaction domains that eliminated those interactions. Those mutations were then tested in KlSir2 for their impact on its interactions with the same complexes. We found that the interaction domains in ScSir2 and ScHst1 were conserved in KlSir2, demonstrating that Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations in these interaction domains.

To understand better how Sir2 has gained or lost functions, we studied the Sir2 function in C. lusitaniae to serve as an intermediate between the fission yeast Schizosaccharomyces pombe Sir2, whose functions have been identified, and K. lactis and S. cerevisiae. Interestingly, ClSir2 was localized to the rDNA, which is also the case in S. pombe, K. lactis, and S. cerevisiae, but not at the telomeres, which is another locus at which Sir2 is found in other yeast. Additionally, ClSir2 was not found to have an impact on gene expression unlike Sir2 and Hst1 in other yeast where they repress transcription.