Spt6 is a conserved eukaryotic transcription factor, known to interact with both nucleosomes and RNA polymerase II (RNAPII) to control transcription. We have initiated study of Spt6 in S. pombe in order to identify both novel and conserved roles in regulation of transcription and chromatin. We first constructed and analyzed spt6 mutants by several approaches. As Spt6 is known to be required for histone H3K36 methylation in both Saccharomyces cerevisiae and human cells, we examined the global levels of several histone modifications; we found that in S. pombe, Spt6 is required for both H3K4 and H3K36 trimethylation. We examined the chromatin state at two highly expressed genes, \(act1^+\) and \(pma1^+\), and found that there is a defect in recruitment of the methyltransferases responsible for those marks, Set1 and Set2, respectively. We also observed loss of nucleosomes, as well as a decrease in histone H2B monoubiquitylation. These results suggest that Spt6 plays an important role in chromatin regulation during transcription. We also conducted transcriptional analysis of an spt6 mutant by both microarray and high-throughput sequencing (RNA-seq) and discovered that Spt6 plays a critical role in maintaining the integrity of transcription genome-wide. We found that Spt6 is required to repress antisense transcription, with nearly 70% of genes having antisense transcripts increased by at least two-fold in an spt6 mutant. We also found that transcription of most long terminal repeats (LTRs) is derepressed. Finally, we found that a major class of transcripts elevated in the spt6 mutant is derived from heterochromatin, which is normally silenced. To study the heterochromatic silencing defect in greater detail, we analyzed the chromatin state of the pericentric repeats and found a decrease in H3K9 trimethylation, elevated levels of H3K14 acetylation, reduced recruitment of several known silencing factors and a loss of siRNA production. We also see a very modest increase in RNAPII recruitment. Based on this combination of phenotypes, Spt6 is likely to contribute to both transcriptional and post-transcriptional silencing mechanisms. Taken together, we have found that Spt6 plays several important roles to control transcription in both euchromatin and heterochromatin in S. pombe.

Chromatin structure is crucial to regulate access to the genome for processes such as a transcription, recombination, DNA repair, and DNA replication. Spt6, a key factor involved in regulating chromatin structure, is conserved throughout eukaryotes. Spt6 has been shown to function in many aspects of gene expression, including nucleosome assembly, transcription initiation and elongation, and mRNA processing and export. In addition, Spt6 has several conserved domains; however, little is known about their functions. I have performed a structure-function analysis of Spt6 using three separate approaches. First, I employed a random insertion mutagenesis that has identified sixty-seven mutants. While these mutants did not provide information regarding known domains, some have phenotypes that may prove useful for future study. Second, in a collaborative project with Romier lab, I studied the functional roles of the Spt6 SH2 domains. I have shown that deletion of the region of Spt6 encoding the SH2 domains causes severe mutant phenotypes without affecting Spt6 protein levels, demonstrating the importance of the SH2 domains of Spt6. Third, in an additional collaboration with the Romier lab, I showed that mutations that alter the region of Spt6 that interacts with the conserved transcription factor Spn1 impair Spt6 functions in vivo. Overall, this multi-pronged structure-function analysis of Spt6 has provided new insights into the tandem SH2 domains of Spt6, the Spt6-Spn1 interaction, and the uses and limitations of insertion mutagenesis. In addition, I have attempted to explore a possible role for Spt6 in transcription-associated mutagenesis. After employing several types of in vivo assays, I conclude that a possible role for Spt6 in transcription-associated mutagenesis is uncertain, as the results (with respect to a role for Spt6) reproducibly vary depending on the assay used. Thus, understanding this aspect of Spt6 biology awaits better assays and understanding of transcription-associated mutagenesis. Overall, the work in this dissertation will serve to further elucidate the mechanisms of Spt6 in chromatin regulation, transcription, and DNA damage repair.

Les expériences de séquençage à haut débit ont permis de démontrer que la transcription ne se limite pas aux régions codantes et qu’une grande partie du génome est transcrite en ARN non-codants (ARNnc). Parmi eux, les transcrits cryptiques sont initiés à l’intérieur des régions codantes. Des études faites chez la levure Saccharomyces cerevisiae, ont pu identifier plusieurs facteurs qui répriment la transcription cryptique. Un de ces facteurs est Spt6, une chaperonne d’histones requise pour le maintien d’un bon niveau de nucléosomes le long des gènes transcrits. Lorsque Spt6 est muté, on observe une déplétion des nucléosomes conduisant à l’activation des promoteurs cryptiques. Cependant, le mécanisme par lequel ces transcrits cryptiques sont régulés n’est pas encore clair. Dans ce mémoire, nous présentons un travail dans lequel nous avons développé une méthode probabiliste dans le but de caractériser les transcrits cryptiques à partir de données de RNA-Seq. Cette méthode est basée sur une cumulation des données et permet de tenir compte des variations dans l’expression et dans la longueur des gènes, grâce à une étape de randomisation des données. Les résultats démontrent que notre méthode est au moins aussi efficace que les méthodes précédemment décrites dans la littérature et offre un bon compromis entre le taux de faux positifs et de faux négatifs. Enfin, le plus important est que cette méthode permet de prédire les régions génomiques où les transcrits cryptiques sont initiés. Nous avons mis en évidence la présence de transcrits cryptiques sur les brins sens et antisens par rapport au gène. Nous avons également montré que les promoteurs cryptiques sens et antisens sont enrichis en motif TATA et que les transcrits cryptiques sont polyadénylés, ce qui suggère qu’ils peuvent être régulés par les mêmes mécanismes qui régulent les gènes. Alors que les transcrits cryptiques sur le brin sens se terminent à la même position que les gènes dont ils sont issus, les transcrits cryptiques sur le brin antisens terminent préférablement aux extrémités 3’ des gènes situés en amont. Nous proposons donc que les terminateurs chez S. cerevisiae ont évolué pour terminer la transcription de manière bidirectionnelle afin d’empêcher une transcription aberrante qui pourrait envahir les gènes voisins. / High throughout sequencing experiments have shown that transcription in not limited to coding regions and that most of the genome is transcribed into non-coding RNA (ncRNA). Among them, cryptic transcripts are aberrantly initiated from within the coding regions. Several studies in Saccharomyces cerevisiae have identified many factors that suppress cryptic transcription. One such factor is Spt6, a histone chaperone required for maintaining appropriate nucleosome levels on transcribed genes. In Spt6 mutant cells, nucleosomes are depleted, leading to activation of cryptic promoters. However, the mechanism by which these cryptic transcripts are regulated remains unclear. In this thesis, we present the development of a probabilistic method for the characterization of cryptic transcripts from RNA-Seq data. The method is used to characterize cryptic transcription in spt6-1004 cells. The method is based on a cumulative distribution function, thus taking into account variations in gene expression and gene length thanks to a data randomization step. Results show that our method is at least as good as previously published methods and provides a good compromise between false positives and false negatives. Importantly, this method allows for the prediction of genomic regions where cryptic transcripts are initiated. We have demonstrated the presence of cryptic transcripts running on the sense and antisense strands relative to genes. We also showed that, both sense and antisense cryptic promoters are enriched for TATA-like sequences and that cryptic transcripts are polyadenylated, suggesting that they may be regulated by the same mechanism that occurs on genes. While the cryptic transcripts on the sense strand terminate at the same position as the genes from which they are derived, cryptic transcripts on the antisense strand preferentially terminate at the 3’-end of upstream genes. We therefore propose that S. cerevisiae terminators have evolved to terminate transcription bidirectionally in order to prevent an aberrant transcription that could invade neighboring genes.