Investigating the interaction of HP1α with H1.4 in heterochromatin : 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
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2022
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Massey University
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Abstract
Chromatin is a nucleoprotein complex which organises DNA within the cell and is essential for genome fidelity. However, the underlying mechanisms which regulate its formation are not fully understood. Chromatin forms through complex hierarchical folding. DNA wraps around octamers of the four core histones, H2A, H2B, H3 and H4, forming nucleosomes in an array called the 10 nm fibre, which is then condensed by linker histone H1 into the 30 nm fibre. RNA and architectural proteins such as Heterochromatin Protein 1α (HP1α) then fold the chromatin fibre into higher order domains, that ultimately partition the genome into domains of euchromatin and heterochromatin. The histone code model for HP1α induced heterochromatinization is through the chromodomain of HP1α binding to di- and tri-methylated lysine 9 of Histone 3 (H3K9me2/3). The H3K9me2/3 mark however, is present throughout the genome and so lacks the specificity for HP1α targeting. An interaction between HP1α and linker histone H1.4 has been identified previously, and through the establishment of an in vitro pulldown, it has been shown to be mediated by total RNA. To aid in building a model for HP1α targeting to telomeric heterochromatin, it was proposed that the RNA transcribed from this region, which associates with HP1α at the telomeres, could mediate this interaction. Therefore, Telomeric-repeat containing RNA (TERRA), for which HP1α has a high in vitro binding affinity, was tested to determine if it could mediate the interaction. TERRA was shown to mediate the interaction of HP1α with H1.4, indicating that this interaction could potentially be aiding in the targeting of HP1α to telomeric heterochromatin. Work to establish mononucleosomes to explore this interaction in a nucleosomal context has begun, however issues with unbound DNA and heterogeneous mononucleosome populations need to be overcome before these can be utilised. A model for HP1α targeting is proposed for reestablishment of telomeric heterochromatin after the S-phase of the cell cycle, through an interaction between H1.4 and HP1α, mediated by TERRA. These factors are essential in heterochromatin organisation, and their loss results in dysregulation which could lead to cancer and ageing.
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Figure 1.1 (=Fyodorov et al., 2018 Fig 1) was removed for copyright reasons. Figure 1.5 is re-used under a Creative Commons Attribution 4.0 International (CC BY 4.0) license. Other Figures are original or have been adapted from the source.