Towards understanding the mechanisms of gene expression in Tetrahymena thermophila

Candidate: Alexandra Petrova
Supervisor: Dr. Jeffrey Fillingham

ABSTRACT

Gene regulation in eukaryotes is highly complex, and our understanding of many mechanisms remains limited, particularly in divergent eukaryotes. One of the key proteins in eukaryotic transcription is RNA polymerase II (RNAPII), an enzyme that transcribes genes to produce a functional product. Regulation of transcription is accomplished through many mechanisms, for instance, by utilizing histone variants to control recruitment of the transcriptional machinery and its access to DNA-binding sites. Histone variants H3.3 and H2A.Z are typically associated with active transcription. H3.3 deposition is usually observed in promoters and gene bodies, whereas H2A.Z is typically found in well-positioned nucleosomes around the transcription start sites.

This project aimed to establish DNA-binding patterns of the third-largest RNAPII subunit, Rpb3, and H3.3 in a model ciliate and divergent eukaryote Tetrahymena thermophila by utilizing next-generation sequencing and computational approaches. While Rpb3 research is still ongoing, I examined H3.3 deposition patterns and found the enrichment to be particularly strong in gene bodies and around transcription termination sites of genes involved in essential cellular processes. H3.3 also had an unexpected effect on the expression of DNA replication and chromatin organization genes, which requires further investigation. Furthermore, I examined the binding patterns of bromodomain-containing protein Ibd2, which was thought to have a role in H2A.Z removal by a chromatin-associated complex INO80. The analysis revealed that Ibd2 exclusively localized to H2A.Z-enriched genes, which provides further evidence for the current model of H2A.Z deposition pathway. Collectively, these findings contributed to a better understanding of gene regulation in protists. Furthermore, Tetrahymena shares a lot of conserved gene regulation mechanisms with higher eukaryotes. Therefore, these findings may be applicable to metazoans, including humans.