Centromere identification and inactivation on a neo-Y chromosome fusion in threespine stickleback fish (Gasterosteus aculeatus)
Cech, Jennifer Nell
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Centromeres are the primary constriction observed on many chromosomes, and they are required for normal cell division. Having one and only one centromere per chromosome is essential for proper chromosome segregation during both mitosis and meiosis. Chromosomes containing two centromeres (dicentric) often mis-segregate during cell division, resulting in aneuploidy or chromosome breakage. Dicentric chromosomes can be stabilized by centromere inactivation, a process which re-establishes monocentric chromosomes. There are two proposed mechanisms of centromere inactivation: a solely epigenetic mechanism involving loss of the centromeric histone, also called centromere protein A (CENP-A), or a genetic mechanism involving deletion or mutation of centromeric DNA. However, little is known about this process in naturally occurring dicentric chromosomes. For my dissertation, I characterized the mechanism of centromere inactivation on a Y chromosome-autosome fusion (referred to as a neo-Y chromosome) that has been fixed in Japan Sea threespine stickleback fish (Gasterosteus nipponicus). In order to characterize the Japan Sea neo-Y chromosome, I first needed to identify the threespine stickleback centromeric DNA sequence. Centromere sequences exist as gaps in many genome assemblies due to their repetitive nature. Thus, I took an unbiased approach utilizing CENP-A chomatin immunoprecipitation followed by high-throughput sequencing to identify the centromeric repeat sequence in the closely related Pacific Ocean threespine stickleback fish (Gasterosteus aculeatus). A 186-bp, AT-rich repeat was validated as centromeric using both fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on interphase nuclei and metaphase spreads. This repeat (GacCEN) hybridizes strongly to the centromere on all chromosomes, with the exception of weak hybridization to the Y chromosome. To test whether epigenetic or genetic inactivation has occurred on the Japan Sea neo-Y chromosome, I used a combination of GacCEN FISH and CENP-A immunofluoresence on metaphase chromosome spreads. I demonstrated that there has been epigenetic inactivation of the centromere derived from the Y chromosome on the Japan Sea neo-Y chromosome. Furthermore, my data suggest that there may be genetic changes to the centromere derived from the ancestral Y chromosome, potentially contributing to its inactivation. Together, my work provides the first validated sequence information for the threespine stickleback centromere. Additionally, the Japan Sea stickleback neo-Y is one of the few examples of a naturally-occurring and stable dicentric chromosome involving two functionally important chromosomes that shows evidence for centromere inactivation. It is also one of the first examples showing centromere inactivation as a potential mechanism used to maintain a chromosome fusion that may play a role in the process of speciation between the Pacific Ocean and Japan Sea sticklebacks.