Characterising changes in chromatin structure across ribosomal DNA and genome-wide during malignant transformation
| dc.contributor.author | Udumanne, Thejaani | |
| dc.date.accessioned | 2022-08-09T10:58:38Z | |
| dc.date.available | 2022-08-09T10:58:38Z | |
| dc.date.issued | 2022 | |
| dc.description.abstract | Ribosomal RNA (rRNA) gene transcription by RNA Polymerase I (Pol-I) occurs in the nucleolus, the site of ribosome biogenesis, and governs proliferation and cell growth in all organisms. rRNA genes are part of ribosomal DNA (rDNA) repeats along with non-coding intergenic spacers. While it is estimated that there are ~200 copies of rDNA repeats per haploid genome, remarkably less than 50% are typically transcribed in normal cells. It is well documented that elevated rRNA transcription is a common feature in malignancy, and that tumour cells compared to normal cells, are more sensitive to drugs targeting Pol-I transcription. Malignant transformation is accompanied by the activation of pseudo-silent rDNA repeats (rDNA class switching), due to increased loading of Pol-I specific transcription factor, upstream binding factor (UBF) onto the rDNA repeats. However, the exact molecular mechanisms behind this process are not yet understood. Intriguingly, previous studies have shown that rDNA class switching is required for malignant B-cell survival independent of changes in rRNA synthesis suggesting a broader role for rDNA chromatin in malignancy. Thus, the central hypothesis of this thesis is that; characterisation of rDNA chromatin landscape in the context of rest of the genome, during malignant transformation, will provide new understanding of the epigenetic mechanisms by which rRNA genes modulate the malignant phenotype and ultimately open new avenues for treating cancer. To test this hypothesis, we utilised a MYC-driven mouse model of B-cell lymphoma (Eu-Myc), and first developed and tested novel bioinformatics approaches. Chromatin Immunoprecipitation Sequencing (ChIP-seq) and RNA-seq data analysis pipelines were optimised to incorporate external spike-in normalisation. To account for the highly repetitive nature of rDNA loci, a customised manual masked reference genome was created, and ChIP-seq pipeline was further modified to correct for the higher coverage across rDNA. Using the modified bioinformatics pipeline, we discovered that the transition from wildtype to pre-malignant and to malignant B-cells is associated with marked changes in rDNA chromatin. A significant increase in UBF loading across regulatory and coding regions was observed aligning with previous studies. Linker histone H1 occupancy in contrast was reduced across the rDNA repeat, exhibiting a mutually exclusive enrichment pattern with UBF. For bivalent histone marks, we observed a notable depletion of H3K27me3 (repressive), without any substantial changes in H3K4me3 (active) enrichment across the promoter. H3K9ac (active) enrichment at the enhancer region increased significantly in malignant cells, while histone variant H2A.Z occupancy was reduced. The levels of total histone H3 and H3K9me3 (repressive) were also moderately decreased. Overall, a more accessible rDNA chromatin structure is found to be established during the transition to malignancy. MYC-driven malignant transformation is also associated with genome-wide changes in the epigenetic landscape and transcriptional programmes. Integration of ChIP-seq and RNA-seq data demonstrated that epigenetic alterations during malignancy correlated with the observed transcriptional changes, notably a significant upregulation of Pol-II dependent genes related to translation and biosynthesis. In summary, this thesis identifies the epigenetic alternations that promote a more accessible rDNA chromatin state during malignant transformation, which could facilitate the increase in UBF loading and consequent rDNA class switching in MYC-driven malignancies. The customised bioinformatics pipelines developed in this study provide a robust, comparative analysis approach for rDNA repeats, and genome-wide, in a disease progression model. | |
| dc.identifier.uri | http://hdl.handle.net/1885/270321 | |
| dc.language.iso | en_UK | |
| dc.title | Characterising changes in chromatin structure across ribosomal DNA and genome-wide during malignant transformation | |
| dc.type | Thesis (PhD) | |
| local.contributor.affiliation | John Curtin School of Medical Research, ANU College of Science, The Australian National University | |
| local.contributor.authoremail | u6567349@anu.edu.au | |
| local.contributor.supervisor | Hein, Nadine | |
| local.contributor.supervisorcontact | u1001977@anu.edu.au | |
| local.description.embargo | 2025-08-16 | |
| local.identifier.doi | 10.25911/TQM0-B737 | |
| local.identifier.proquest | No | |
| local.identifier.researcherID | GPF-5150-2022 | |
| local.mintdoi | mint | |
| local.thesisANUonly.author | a1808841-36f5-4fef-b453-a5e9b0b9876d | |
| local.thesisANUonly.key | 813c0ee1-3160-ddbe-fd0e-975ffc7fd9f1 | |
| local.thesisANUonly.title | 000000019450_TC_1 |