Author ORCID Identifier
0000-0002-6104-0626
Date of Graduation
5-2019
Document Type
Dissertation (PhD)
Program Affiliation
Biomedical Sciences
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Francesca Cole
Committee Member
Swathi Arur
Committee Member
Richard Wood
Committee Member
Taiping Chen
Committee Member
Kevin McBride
Abstract
Meiotic recombination is required for parental chromosomes to find each other (pairing/synapsis) and to exchange genetic information thus allowing faithful segregation of chromosomes and the production of haploid gametes. At the start of meiotic prophase I, meiotic chromosomes organize into loop arrays that extrude out of the chromosome axis. Then, a large number of programmed double-strand breaks (DSBs) are formed at specific chromosomal locations or “hotspots” on parental chromosomes, which are repaired by homologous recombination (HR). HR produces either crossovers, which result in the exchange of flanking markers between homologs, or noncrossovers, which are short regions ofgene conversion to the donor genotype. Crossover formation is critical for proper chromosome segregation and crossovers arise from crossover precursors that form at a subset of DSBs that are designated to become future crossovers. Our current understanding of meiotic progression in mammals is largely derived from cytological observation.Many semi-redundant HR pathways can repair meiotic DSBs; however, the time at which different pathways are active, how the pathways interact, and the relative contribution of each pathway towards maintaining germline genomic integrity are poorly understood in vivoat endogenous sites, especially in a mammalian system. More importantly, how germline genome integrity is ensured at both the DNA level by recombination activity and by higher order chromosome structural changes has not been defined. Failure to maintain germline genome integrity can lead to aneuploidy, genetic disorders, birth defects and miscarriages.
To define and dissect the temporal dynamics of different HR pathways and chromosome organization in vivo, I have established a novel and robust system to synchronize mouse spermatogenesis in F1 hybrid mice using the inhibitor WIN 18,446. My synchronizationprotocol allows the isolation of cells at specific stages of meiotic prophase I by flow cytometry, allowing me to analyze recombination outcomes at two meiotic hotspots and perform genome-wide Hi-C, a chromosome conformation capture method combined with high-throughput sequencing to investigate changes in higher order chromosome architecture during prophase I.
Here, I provide the first direct molecular evidence that HR pathways that lead to to distinct meiotic outcomes aretemporally regulated. I have identified two novel classes of noncrossover pathways: 1) one that likely regulates the pairing/synapsis of parental chromosomes during early prophase I; and 2) one that derives from the crossover/noncrossover decision during mid-prophase I. My data show that crossover formation is suppressed until full synapsis is achieved at mid-prophase I, suggesting a previously unknown mechanism that prevents deleterious premature recombination. In addition, I show that alternative repair pathways are not activated until late prophase I, thus preventing designated crossover precursors from inappropriately forming noncrossovers.
Furthermore, the Hi-C data I present provides evidence for dynamic genome reorganization during meiotic prophase I. There is evidence for loop array formation and loop extrusion as chromosomes condense. While topologically associating domains disappear at the onset of meiotic prophase I, chromosome compartments are well maintained. Most meiotic DSBs occur within a gene-dense open compartment A, suggesting that higher order chromosome structure plays an important role in meiotic recombination. Finally, interhomolog interactions and specialized chromosomal architecture in regions of pairing and synapsis could be inferred. Taken together, my data reveals that both chromosome recombination and chromosome structure are highly regulated to ensure chromosome pairing and segregation. These results provide important, novel insights to the field of meiosis and our understanding of germline genomic integrity and mammalian reproductive health.
Keywords
Meiosis, homologous recombination, DNA, spermatocytes, Hi-C, higher order chromosome organization, molecular pathway, temporal analysis, crossover, noncrossover