The promoters of genes have a stereotypical chromatin architecture

characterized by a nucleosome-free region (NFR), which contain DNA motifs that

are involved in gene transcription. Additionally, at least one of the nucleosomes

flanking a NFR tends to contain the histone H2A variant H2A.Z. The

mechanisms behind NFR formation and H2A.Z deposition have not been well

characterized. This dissertation encompasses approaches towards an

understanding of these mechanisms. A specific sequence of DNA isolated from

a yeast promoter was shown to be sufficient to program NFR formation and

H2A.Z deposition. The remodeling activity of this sequence of DNA required the

activities of the transcription factor Reb1 and chromatin remodeling complex

RSC. The involvement of Reb1, RSC, and another transcription factor, Abf1, in

nucleosome positioning at other gene promoters was assayed, and RSC was

determined to play a significant role in the proper positioning of nucleosomes at

promoters. Lastly, because H2A.Z deposition is not required for general NFR

formation, the hypothesis that H2A.Z deposition requires prior NFR formation

was tested.

Qsp1 is a peptide of unknown function secreted by the fungal meningitis pathogen Cryptococcus neoformans. We identified QSP1 as a target of three transcription factors required for virulence. Indeed, mutants lacking Qsp1 are attenuated for infection and growth within macrophages. Qsp1 signaling modulates the activities of secreted proteases and promotes cell wall function at high cell densities. Production of the peptide requires its release from a secreted precursor by a cell- associated protease, while sensing of Qsp1 requires an oligopeptide importer. The effects of cytoplasmic expression of Qsp1 indicate that it can function intracellularly. These features closely mirror the quorum sensing systems of gram-positive bacteria in which peptide precursors are exported, processed and then imported to bind cytoplasmic receptors. Despite these remarkable similarities, the components of the respective systems are not ancestrally related, implying convergent evolution. Our studies reveal a bacterial-like signaling system that promotes the virulence of a eukaryotic pathogen.

Cells must integrate signals from their environment and respond accurately in order to grow and divide properly. However, the chemical reactions within single cells do not occur with deterministic certainty. Rather, they occur with a probability due, in part, to the abundance of molecular components and their rates of synthesis and degradation. Therefore, while an entire population of cells may respond appropriately to environmental cues, single cells may vary wildly in their output responses. This phenomenon is called biological noise, and increasingly has been reported to be an important determinant for population heterogeneity, fitness, and development. Although biological noise has been characterized in a variety of systems and cellular pathways, mechanisms that regulate cell-to-cell variability remain poorly understood. In this work, we investigated the role of biological noise in the transcriptional output of the mating pathway, a canonical MAP kinase pathway in S. cerevisiae. We identified a role in noise suppression for Dig1, a negative regulator of the transcription factor Ste12. The removal of Dig1 causes an increase in intrinsic and extrinsic noise in the expression of Ste12 target genes. In conjunction, Dig1 inhibits long-range interactions between Ste12 target genes in vivo. Finally, we demonstrate a link between fitness defects and the increased gene expression noise in cells lacking Dig1. These studies suggest that gene expression noise is an evolvable trait and that, when necessary, mechanisms can arise to modulate cell-to-cell variability.

As more genome sequences become available with every passing day, it is apparent that not all of it codes for useful proteins. Much of eukaryotic genomes are composed of transposons, parasitic elements that propagate in a cell. Their success impacts host genome integrity and shapes gene expression. However, genetic analysis reveals that most transposons are quiescent and non-duplicating. This is achieved by silencing pathways that manage to keep transposons in an inactive state under normal conditions. Chief among them is RNA interference, a small RNA guided mechanism that suppresses the expression of complementary transcripts. RNAi acts as a nucleic acid based immune system against viruses and repetitive sequences that might otherwise cause genome instability. The diversity of RNAi directed silencing pathways is matched only by the diversity of transposons. In many organisms, RNAi cooperates with heterochromatin factors to target repeats clustered in specific genomic regions like centromeres for small RNA generation leading to RNAi-mediated transcriptional silencing. In this work, we report two mechanisms of post-transcriptional silencing of transposable elements that are independent of the heterochromatin machinery as well as the preliminary characterization of two new RNAi-related components using the model fungus, Cryptococcus neoformans.

Chapter 2 of this thesis focuses on the ‘multi-point lock’ involving mRNA degradation and translation silencing effected by RNAi on an active DNA element in C. neoformans. This sheds light on a transposon suppression pathway that is capable of severely limiting transposon mobilization in a manner independent of genomic location.

Chapter 3 discusses an attempt to discover additional components of the RNAi-mediated transposon silencing pathway through the application of a trans-kingdom insertional mutagenesis screen in a transposon activity reporter. We outline the identification of Sqs1, a spliceosomal discard related factor as a strong positive hit in the screen such that small RNA accumulation is completely abolished in the mutant. Further genetic and proteomic studies hint at the involvement of the spliceosome release machinery in the coupling of the two important biological processes during small RNA biogenesis.

In Chapter 4, we characterize an Argonaute-associated factor that might play a role in the bi-fold silencing pathway described in Chapter 2. Argonaute proteins are essential components of the RNA induced silencing complex (RISC). We isolated Ago1 binding proteins in C. neoformans and identified a protein called Gwo1 that contains numerous GW/WG dipeptide repeats common to Ago-associated proteins in higher organisms. It bears other hallmarks of GW-proteins playing a role in the miRNA pathway such as P-body localization but is absolutely required for transposon silencing. Thus, Gwo1 might act as the missing link connecting Ago1 to mRNA degradation/ translational silencing of transposable elements.

These studies uncover novel pathways and proteins that are critical for RNAi-mediated silencing of transposable elements. This might offer insight into similar strategies that are operating in other eukaryotes.