Date Available

2-29-2024

Year of Publication

2024

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department/School/Program

Biology

First Advisor

Jeramiah Smith

Abstract

During early embryonic development, the sea lamprey (Petromyzon marinus) undergoes programmatic elimination of DNA from somatic progenitor cells in a process termed programmed genome rearrangement (PGR). Eliminated DNA eventually becomes condensed into micronuclei, which are then physically degraded and permanently lost from the cell. Previous studies indicated that many of the genes eliminated during PGR have mammalian homologs that are bound by polycomb repressive complex (PRC) in embryonic stem cells. To test whether PRC components play a role in the faithful elimination of germline-specific sequences, we used a combination of CRISPR/Cas9 and lightsheet microscopy to investigate the impact of gene knockouts on early development and the progression through stages of DNA elimination. Analysis of knockout embryos for the core PRC2 subunits EZH, SUZ12, and EED show that disruption of all three genes results in an increase in micronucleus number, altered distribution of micronuclei within embryos, and an increase in micronucleus volume in mutant embryos. While the upstream events of DNA elimination are not strongly impacted by loss of PRC2 components, this study suggests that PRC2 plays a role in the later stages of elimination in relation to micronucleus condensation and degradation. These findings also suggest that other genes/epigenetic pathways may work in parallel during DNA elimination to mediate chromatin structure, accessibility, and the ultimate loss of germline-specific DNA.

This dissertation describes our efforts to more precisely understand the genetic and cellular mechanisms that contribute to programmed DNA loss in the sea lamprey, as well as the development of the tools and strategies utilized. In the first chapter, I introduce and describe programmed DNA elimination in sea lamprey and other eliminating taxa, and briefly summarize the role of the polycomb genes during vertebrate development. In the second chapter, I present a published work describing a functional analysis of three core components of the PRC2 complex, a class of epigenetic modifiers, during programmed DNA loss. In the third chapter, I present a study that focuses on the loss of the KMT genes, a different class of epigenetic modifiers, and discuss their potential function during the later stages of PGR in sea lamprey. In the fourth chapter, I detail the process of developing a novel approach for visualizing, quantifying and measuring the subcellular structures associated with programmed DNA loss and describe how this approach led to new insights into the spatiotemporal aspects of programmed DNA loss that were hitherto unknown. In chapter five, I discuss improvements to the approach discussed in chapter four, and the advantages and new potential it provides for future studies. Finally, in chapter six I discuss how my work fits into the larger body of literature on DNA loss and how my findings have “moved the needle” with respect to our overall understanding of the phenomenon of programmed DNA elimination in sea lamprey and potentially other species.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.28

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