Precision chromosome engineering to study nucleolar dynamics in human cells

Nucleoli, sites of ribosome biogenesis, form around ribosomal gene (rDNA) arrays, nucleolar organiser regions (NORs). NORs are located on the p-arms of each of the five acrocentric chromosomes 13, 14, 15, 21, and 22, surrounded by a variety of satellite repeats and segmental duplications. The default status of NORs in non-transformed cell appears to be active. The one to three nucleoli typically present in most human cells can be considered multivalent, each involving multiple acrocentric p-arms. In some cells, as many as 10 acrocentric p-arms can contribute to a single large nucleolus. Here, I describe the use mega-base scale chromosome engineering to reveal, for the first time, the chromosomal requirements for this major genome organisational event. Precise deletion of rDNA from individual acrocentric p-arms reveals that association of acrocentric p-arms occurs independent of rDNA. Deletion of sequences from telomere to NOR interval, or from NOR to centromere interval, individually, have only a marginal effect on nucleolar association of the targeted chromosome. Finally, deletion of an entire acrocentric p-arm while leaving the centromere function intact, effectively destroys the nucleolar association potential of that chromosome. These findings lead me to conclude that formation of multivalent nucleoli is not a nucleolar fusion event per se, rather it is driven by the chromosomal context in which NORs are located. Furthermore, this targeting information is redundant being localised both distal and proximal to NORs, but not involving centromeres.

Publisher

University of Galway

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CC BY-NC-ND

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University of Galway Theses (PhD Theses)