Browsing by Author "Roach, Ruby"

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    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, Ruby
    Our 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.
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    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, Ruby
    Over 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.