Characterization of the human nucleolar organizer regions : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand.

Abstract

The short arms of all the five human acrocentric chromosomes contain genomic region known as nucleolar organizer regions (NORs). The NOR is the site of nucleolus formation and therefore play critical role in cell survival. It has two components: a tandem array of ribosomal DNA (rDNA) units and regions surrounding the rDNA tandem array, the rDNA flanking regions. In this work, I have explored both components of the NOR to unravel their genomic and functional features. Aside from their rRNA coding function, the rDNA intergenic spacer (IGS) are thought to contain many non-coding functional elements that are involved in a variety of cellular processes. The repetitive nature of the IGS has made these non-coding elements difficult to identify, therefore I employed phylogenetic footprinting to identify putative functional elements in the human rDNA. To implement phylogenetic footprinting, I performed whole genome assemblies to determine the rDNA sequences of six primate species. These primate rDNA sequences were compared with human rDNA to identify fifty-three conserved regions in the human IGS that correspond to known rDNA functional elements, as well as novel conserved regions with unknown function. The human IGS is known to transcribe noncoding RNAs and therefore, to identify transcripts from the novel conserved regions I performed RNA-seq analysis. Integration of phylogenetic footprinting and RNA-seq analysis results revealed that several conserved regions potentially actively transcribe a number of long poly(A)- transcripts that include a cancerous tissues specific transcript, which is antisense to the pRNA and another transcript from cdc27 pseudogene present in different cell types and a small poly(A)- transcript specific to embryonic cells. The integration of phylogenetic footprinting and IGS chromatin profiling revealed enrichment of active histone modifications and transcription factors in the IGS conserved regions demonstrating that these regions potential act as transcription regulators. Three conserved potential origin of replication sites in the IGS were also identified. Further evidence of Pol II and Pol III association with the human IGS were provided that strongly demonstrate that aside of Pol I other two RNA polymerase machineries potentially transcribe the human IGS. Overall, this work provides an extensive dataset of potential functional conserved regions in the human IGS, and evidence for different functions associated with them. The rDNA flanking regions thought to have role in the nucleolus formation/fusion. However, the genomic characteristic of the regions is unknown as they are missing from the current human genome assembly. Therefore, I characterized the rDNA flanking regions the distal rDNA flanking region (telomere side) and the proximal rDNA flanking region (centromere side) using the sequences of the regions that were identified by our collaborators. The sequences of the flanking regions are highly conserved among the acrocentric chromosomes suggesting that they frequently exchange sequences. The proximal region similar to the pericentromeric regions is highly segmentally duplicated. On the other side, the distal region is merely segmentally duplicated but has two unique features a large inverted repeat region (~227 kb) and a long stretch of CER satellite repeats, potential binding site of a protein of unknown function. These parts of the genome are thought to be heterochromatic, however I employed a gene prediction pipeline that provide evidence for coding potential in both the flanking regions. Finally, it has been reported that the proximal junction point may be variable therefore, I designed a novel bioinformatics mapping technique, which suggests there are at least two distinct proximal junction points. Overall, this work demonstrates that the rDNA flanking regions are not merely heterochromatic wastelands but instead are highly complex and have its own genomic characteristics. Taken together, the results from my work provide a platform for a more comprehensive characterization of the functional elements in the IGS and the rDNA flanking regions. This will lead to a better understanding of the biological processes that are related to NORs and will ultimately help to explore the mechanisms that underlie these processes, which are still far from being completely understood.