<p>In <italic>Saccharomyces cerevisiae</italic>, proper maintenance of haploid cell identity requires the SIR complex to mediate the silenced chromatin found at the cryptic mating-type loci, <italic>HML</italic> and <itaic>HMR</italic>. 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 <italic>Saccharomyces</italic> and <italic>Kluyveromyces species</italic>. The histone deacetylase <italic>HST1</italic> is the paralog of <italic>SIR2</italic> and works with the promoter-specific SUM1 complex to repress sporulation and alpha-specific genes. <italic>ORC1</italic> is the paralog of <italic>SIR3</italic> and is an essential subunit of the Origin Recognition Complex and also recruits SIR proteins to the <italic>HM</italic> loci. I have investigated the functions of these proteins in the non-duplicated species <italic>Kluyveromyces lactis</italic> and compared these functions to those found in <italic>S. cerevisiae</italic>. </p>
<p>I have shown that <italic>SIR2</italic> and <italic>HST1</italic> subfunctionalized post-duplication via the duplication, degeneration and complementation mechanism. In <italic>S. cerevisiae</italic>, Sir2 has retained the ability to function like Hst1 when in an <italic>hst1Δ</italic> 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 <italic>K. lactis</italic> can interact with both SIR and SUM1 complexes in <italic>S. cerevisiae</italic>.</p>
<p>Further analysis into the non-duplicated experimental system of <italic>K. lactis</italic> 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 <italic>K. lactis</italic>, and both proteins spread across the <italic>HML</italic> locus and associate with telomeres in a manner similar to <italic>S. cerevisiae</italic>. 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 <italic>KlSUM1</italic> also de-represses <italic>HML</italic> and <italic>HMR</italic>, a phenotype not observed in <italic>S. cerevisiae</italic>. I show by chromatin immunoprecipitation that KlSum1 directly regulates the <italic>HM</italic> 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. </p>
<p>The <italic>SIR3-ORC1</italic> 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 <italic>HML</italic> 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 <italic>HML</italic> 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 <italic>S. cerevisiae</italic>.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/2359 |
| Date | January 2010 |
| Creators | Hickman, Meleah A. |
| Contributors | Rusche, Laura N |
| Source Sets | Duke University |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
| Format | 3897280 bytes, application/pdf |
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