Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xxxiii, 284 pages) : illustrations (chiefly color)

Dissertation/Thesis Chair

Joan Curcio

Committee Members

Marlene Belfort, Randall Morse, Doug Conklin, Prashanth Rangan

Keywords

Asymmetric cell division, LTR retrotransposon, microscopy, molecular genetics, Spindle Pole body, Ty1, Centrosomes, Cell cycle, Cellular control mechanisms, Eukaryotic cells, Growth factors, Mobile genetic elements, Transposons, Saccharomyces cerevisiae

Subject Categories

Genetics | Molecular Biology | Virology

Abstract

In 1969, the enrichment of mobile element repeat sequences led Britton and Davidson to propose the hypothesis that gene expression in higher eukaryotes is regulated through the exaptation of mobile elements. In this work, I have explored the hypothesis that mobile elements can also be harnessed by the host to regulate asymmetric cell division, thereby determining cell fate. Retrotransposons are ubiquitous eukaryotic mobile elements that transpose through an RNA intermediate. They are often active in cell types that divide asymmetrically to yield daughter cells with different fates, such as embryonic stem cells, germline stem cells, neuronal stem cells and the budding yeast, Saccharomyces cerevisiae. Intriguingly, these same cell types often have an asymmetric pattern of centrosome inheritance. Centrosomes, the microtubule organizing centers, duplicate conservatively prior to mitosis. Asymmetric centrosome inheritance occurs when the young and old centrosome segregate to the nascent daughter cells in a canonical, age-dependent pattern that underlies the fate of each daughter cell. Using budding yeast as a model, I asked whether activation of a domesticated family of retrotransposons alters the asymmetric inheritance of the new and old spindle pole body (SPB), the equivalent of the centrosome, by the mother cell and bud, respectively. Here, I show that inducing the activity of the Ty1 long-terminal repeat retrotransposon by growth of cells at low temperature results in non-canonical SPB inheritance in one-quarter of all cell divisions. This perturbation of SPB fate requires the Ty1 transcription factor, Tec1, but not the retromobility host factor, Loc1, which represses expression of a self-encoded Ty1 restriction factor. The temperature and Tec1-dependence and Loc1-independence suggest that the mature form of the Ty1 nucleocapsid protein, p45-Gag, which accumulates to high levels at low temperature, is the retrotransposition intermediate responsible for altering SPB fate. My data further demonstrate that non-canonical SPB segregation in one-quarter of mitoses results from a Ty1 intermediate randomizing SPB fate in one-half of cell divisions. The Ty1 intermediate interferes with an extrinsic pathway of SPB fate determination in which the spindle pole inheritance network (SPIN) maintains old SPB identity by modifying the SPB protein Nud1 (homolog of centriolin), which in turn functions as a platform for the mitotic exit network (MEN). MEN promotes the association of Kar9 (homolog of adenomatous polypopsis coli, APC) with the old SPB and directs it into the bud. The Ty1 intermediate acts in the opposite half of the dividing cell population as the SPIN factor, Swe1 and has no effect in the absence of Kar9. Together these findings demonstrate that Ty1 activity perturbs SPB fate in cells that inherited the old SPB in the previous generation, giving rise to cellular heterogeneity in half the population while leaving the other half unperturbed. Using dominant, gain-of-function mutations in a genomic Ty1 element in which an open reading frame (ORF) is fused to the GAG gene at the proteolytic processing site, I discovered that Gag:ORF fusion proteins associate with SPB proteins, Spc42 and Nud1, forming a stable cytoplasmic structure that represses retrotransposition in trans and promotes the massive accumulation of Ty1 nucleocapsid particles. In wild-type cells at low temperature, the Ty1 nucleocapsid assembly site sporadically localizes next to the SPB, where Nud1 co-localizes with Ty1 RNA and Gag; moreover, Nud1 represses Ty1 retrotransposition. Together, my findings support a model in which cross-talk between p45-Gag and Nud1 at low-temperature impairs Nud1’s role in the old SPB as the platform for MEN and results in loss of SPB identity in cells that inherit the old SPB. Overall, my findings demonstrate for the first time that retrotransposon activity can link an environmental cue to the controlled perturbation of centrosome fate, giving rise to cellular plasticity that may underly cell fate and cell function and adaptability to environmental change.

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