Biology and Biomedical Sciences: Molecular Genetics and Genomics
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Douglas L Chalker
Pdd1 is a developmentally expressed HP1-like protein of Tetrahymena thermophila that is required during conjugation, when a copy of the cell's transcriptionally silent germline micronucleus differentiates into an active somatic macronucleus. Differentiation of these somatic chromosomes involves genome-wide fragmentation and amplification. These DNA rearrangements are facilitated by an RNAi mechanism, in which small RNAs target silencing histone modifications, H3K9 and H3K27 methylation, to Internal Eliminated Sequences: IESs), which are bound by Pdd1 and later excised from the genome. Pdd1 features two chromodomains, one of which shares homology with that of HP1, and a C-terminal chromoshadow domain. In this study, we endeavored to determine the necessity and function of these domains during sexual reproduction by mutating each through point mutation and/or deletion. We show that chromodomain 1: CD1) is critical for conjugation, with either deletion of the entire domain or amino acid substitutions of two key aromatic residues: W97, 100A) causing protein mislocalization, preventing the establishment of H3K9me2, and abolishing IES excision. Chromodomain 2: CD2) is necessary for producing viable progeny, and loss of this domain disrupts but does not abrogate, cellular processes. Loss of the entire chromoshadow domain: CSD) prevents nuclear targeting of Pdd1, while mutation of a single residue within the domain, I504D, reduces progeny production to ~5% and prevents Pdd1 foci formation and the recruitment of excision factors to an ectopic IES. Finally, we show the disruptive effect of the Pdd1 C-terminal CFP tag on mature foci formation and examine abnormal foci seen in RNAi mutants.
Schwope, Rachel, "Mutations of Pdd1 Chromo- and Chromoshadow Domains Reveal Critical Functions for Each During Development of Tetrahymena thermophila" (2013). All Theses and Dissertations (ETDs). 1183.