The genomic architecture of nucleolar organiser regions on the short arms of human acrocentric chromosomes

Nucleolar Organiser Regions (NORs) are comprised of ribosomal gene (rDNA) arrays and adjacent sequences. Nucleoli, the sites of ribosome biogenesis and key regulators of cellular growth and proliferation, form around NORs. In humans, NORs are positioned on the short arms of the five acrocentric chromosomes (13, 14, 15, 21 and 22). These chromosome arms are not included in the human reference genome and have only recently started to be mapped and characterised. This thesis has focussed on contributing to the characterisation and extension of these underexplored genomic regions. Previous work had suggested that as many as one third of rDNA repeats are rearranged. These could impact on nucleolar and ribosomal formation and protein synthesis. By performing next generation sequencing on DNA extracted from purified nucleoli, I demonstrated that there is no evidence for rearranged rDNA repeats in human cell lines. This conclusion was emphasised by a detailed analysis of more recent long read DNA sequence data sets. The second objective of this thesis was to describe the spatial organisation of sequences distal to the clusters of rDNA repeats. These sequences exhibit a euchromatic-like chromatin organisation, are transcriptionally active and appear to function as an anchor for the linked rDNA array during interphase. In the post genomic age, much effort now focuses on describing the chromatin status and 3D organization of the genome in a variety of human cell types and it is common practice to make the raw sequencing data from these genome-wide studies publicly available. Exploiting Hi-C data sets designed to capture genome organisation revealed the existence of a transcription dependent stem-loop structure encompassing over 200 kb of NOR distal sequence that may play a role in NOR regulation. The third objective was to extend the sequences distal to NORs and characterise them. Using a combination of nucleolar sequencing reads and Hi-C data, this region was extended by 180 kb. Analysis of data from the ENCODE project suggests that this region is transcriptionally active and marks the beginning of interchromosomal variability on the short arms of acrocentric chromosomes. These results provide a platform for investigating the role of NORs in nucleolar formation and maintenance and serve as a starting point for the identification and characterisation of the unknown regions of the p-arms of acrocentric chromosomes.2017-11-2