Nucleoli form around rDNA arrays at nucleolar organiser regions (NORs) located on the five acrocentric chromosome p-arms, providing positional cues for 10 of the 46 chromosomes in diploid human cells. Thus, nucleoli are a paradigm for two general features of genome organisation; specific associations between multiple chromosome territories and internal organisation of nuclear bodies involving multiple chromosomes. Exploration of this organisational paradigm is complicated by the shared DNA-sequence composition of acrocentric p-arms. Recently we have devised a methodology for genetic manipulation and functional testing of individual p-arms/NORs. Efficient ‘scarless’ genome editing of rDNA is achieved on ‘poised’ human NORs within mouse mono-chromosomal cell-hybrids. Subsequent transfer to human cells introduces ‘active’ NORs yielding readily discernible functional customised ribosomes. We propose exploiting this transformative methodology to identify p-arm elements that drive acrocentric co-location at large nucleoli. We further aim to determine how rDNA arrays from multiple NORs partition within nucleoli. NOR genome-editing will also facilitate exploration of rDNA genome-stability and links with cellular senescence. Finally, we propose using top-down chromosome-engineering to construct minimal NOR-bearing chromosomes as a platform for future research. These studies will advance our understanding of the relationship between acrocentric chromosomes and the largest functional sub-domain in the human nucleus.
