Massey Documents by Type
Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294
Browse
3 results
Search Results
Item Untangling the interaction between HP1α and the TERRA G-quadruplex : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2023) Roach, RubyOver 50 years ago the DNA double-helix structure was solved, revealing the code in which life is written. This set of instructions equates to three billion base-pairs, amounting to two metres in length, fitting into a human nucleus just six microns across. The stable yet dynamic structure that allows for the functional organisation of the genome is chromatin: a complex formed between DNA and proteins. Chromatin is delineated into two microscopically defined compartments: the less dense gene-rich euchromatin and the structurally compact gene-poor heterochromatin. Protective heterochromatin is constitutively maintained for telomere protection, chromosome segregation, DNA repair, and suppression of transposon activity. Essential for the propagation and maintenance of heterochromatin is Heterochromatin Protein 1α (HP1α), which is comprised of a chromodomain and chromoshadow domain separated by a disordered hinge. The histone code model proposes that HP1α is recruited to regions of heterochromatin by its chromodomain-mediated recognition of silent heterochromatin mark trimethylated lysine 9 of histone H3 (H3K9me3); however, this does not account for the specificity of HP1 paralogs, location-specific recruitment, or the contribution of RNA to heterochromatin formation. Here, Telomeric Repeat-containing RNA (TERRA), transcribed from the telomeres and shown to be involved in telomeric maintenance and stability, is investigated in its interaction with HP1α. The interaction with TERRA is proposed as a means for recruitment of HP1α to telomeres for chromosome end protection. Due to its guanine (G)-rich sequence, TERRA folds into a G quadruplex (G4), a topology distinctly different from canonical nucleic acid structures. The interaction between HP1α and TERRA is therefore investigated to establish the means of interaction between HP1α and a G4, and to examine the HP1α specificity towards TERRA G4s. This work showed that binding to TERRA is dependent upon multiple positively charged patches within the disordered hinge of HP1α, and is also affected by mimicking N-terminal phosphorylation, which alters the structure of HP1α. While the hinge of HP1α binds to a myriad of nucleic acid structures, the globular chromoshadow domain provided the specificity for the parallel G4 topology evident in TERRA. Solution structures of HP1α in complex with TERRA show that HP1α undergoes a conformational shift, becoming less flexible. These results show that noncanonical nucleic acid structures such as those formed by TERRA may act as determinants of HP1α function, serving as signposts in the genome for formation of protective heterochromatin. This biophysical study also further implicates non-canonical structures such as G4s as formidable regulators of genomic function.Item Loss of HP1α alters nuclear integrity to promote cellular invasion : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2019) Solomon, RaoulThe onset of invasion is a key step towards the development of metastatic cancer. For a cell to invade through interstitial spaces in the tissue requires a reduction in nuclear rigidity as the cell needs to deform to squeeze through small spaces. Heterochromatin Protein 1α (HP1α) is a protein that defines domains of heterochromatin, the highly compact regions of the genome, and is essential for maintaining the appropriate patterns of gene expression and genome stability. Loss or reduction of HP1α has been correlated with an increase in invasive potential in human tumours. Using an established model of Drosophila melanogaster epithelial cell invasion, the causative role HP1α plays in suppressing cellular invasive is confirmed within an epithelial tissue microenvironment. This model also demonstrates that loss of the Drosophila melanogaster HP1 homologue synergistically promotes cellular invasion in conjunction with an activated malignant signalling pathway. Importantly, human HP1α is shown to rescue this highly invasive Drosophila phenotype and demonstrates the relevance of this model to human disease, and its use for exploring protein interactions in a cellular microenvironment. As loss of nuclear integrity has been linked to a reduction in peripheral heterochromatin, the biophysical mechanisms by which HP1α acts as a suppressor of invasive potential were explored in the poorly invasive MCF7 breast cancer cell line with constitutive HP1α knock-down. These cells with reduced HP1α expression had a significant loss of nuclear membrane integrity and stiffness. The underlying nuclear lamina meshwork and associated peripheral heterochromatin was disrupted. This was associated with an increased solubility of lamina proteins, particularly lamin A, as well as the altered localisation of a number of peripheral nuclear proteins. In summary, this work established the important contribution of HP1α to the mechanical integrity of the nucleoskeleton and the role HP1α plays in suppressing malignant signalling pathways that promote cell invasion.Item Characterising a biologically relevant protein-G4 interaction : HP1α and TERRA : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2019) Roach, RubyOur genetic material is intricately folded and protected through the formation of a compact nucleoprotein complex, termed heterochromatin. In addition to controlling the expression of genes, heterochromatin formation is important for the structural integrity of our genome, specifically for the centromeres, the central attachment point of our chromosomes, and also the telomeres, the ends of our chromosomes. The way in which the heterochromatin in these areas is formed and maintained is though the recruitment and binding of the pivotal chromatin regulator, Heterochromatin Protein 1α (HP1α). The current model that explains how and why HP1α is recruited to, and maintained at, regions of constitutive heterochromatin is simple: HP1α binds post-translational modifications on histones (eg. H3K9me3). However, this binding is not high affinity, therefore may not be the sole determinant in HP1α localisation. At the centromeres, it has been shown that a long non-coding RNA transcribed from the peri-centromeres is responsible for recruiting HP1α to this crucial region. At the telomeres, it is proposed that the long non-coding RNA transcribed from the telomeric DNA is responsible for this same purpose. Because of its guanine-rich sequence, it is able to form a non-canonical nucleic acid structure, the G-quadruplex, which may provide the specificity for heterochromatin formation at telomeres. This TElomeric Repeat-containing RNA (TERRA) has been implicated in telomeric elongation pathways, relating it to the immortalisation of cancer cells. It was found that HP1α can specifically recognise the parallel topology of TERRA, binding with high affinity through HP1α’s unstructured hinge. HP1α was also shown to bind other G-quadruplexes of parallel topology, specifically DNA present in promoter and regulatory regions of many proto-oncogenes, implicating HP1α in potential G-quadruplex-dependent gene expression regulatory mechanisms. The interaction shown here between HP1α and TERRA shows a novel mechanism of telomeric heterochromatin formation, providing crucial insights into telomere maintenance and health in human cells, and furthering our understanding of the role of G-quadruplexes in the epigenome.