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An investigation into the chromatin structure of human telomeres

Norris, Kevin 2013. An investigation into the chromatin structure of human telomeres. PhD Thesis, Cardiff University.
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Telomeres cap the end of eukaryotic chromosomes and prevent the natural end of a chromosome from being recognised as a double-stranded DNA break. Dysfunctional telomeres may trigger replicative senescence, or fuse with other telomeres or with non-telomeric DNA breaks. The length of a telomere plays a key role in telomere function. Relatively little is known about how telomeric chromatin influences telomere length and function. A number of studies in mammalian cells have identified a handful of chromatin remodelling proteins and the chromatin marks they deposit in telomere length regulation. However such examples at human telomeres are scarce. The primary aim of this thesis was to investigate whether the chromatin structure of a telomere is a determinant of its length in human cells. Two approaches were taken to address this issue: Firstly, the chromatin structure of telomeres of differing lengths were directly analysed by measuring enrichment of histone modifications known to be prominent at telomeres in other model organisms. Secondly, selected chromatin remodelling proteins were studied to determine whether they play a role in telomeric chromatin structure and telomere length. Single Telomere Length Analysis (STELA) provides a high resolution method to measure telomere length distributions at individual chromosome ends. STELA assays were previously designed for the 2p, 9p, 11q, 12q, 16p, 17p and 18q telomeres. An allele‐specific STELA assay has also been designed for the XpYp chromosome end. In this study novel telomere and telomeric allele‐specific qPCR assays were developed for the same chromosome ends. These qPCR assays, when used in conjunction with ChIP provide a tool for analysing telomeric chromatin structure at individual chromosome ends. Applying this ChIP‐qPCR approach alongside STELA allows any correlations between telomeric chromatin structure and telomere length to be identified. This approach suggested differences in telomeric chromatin structure between telomeres of different lengths in telomerase‐positive HT1080 fibrosarcoma cells. Shorter HT1080 telomeres were less abundant in H3 and TRF1 and also had lower levels of H4K20me3 and, to a lesser extent, H3K4me3 compared to longer telomeres. Differences in chromatin structure were not observed between telomeres of different lengths in telomerase negative MRC5 fibroblasts. Changes in chromatin structure were observed at individual telomeres/telomere alleles were observed between actively proliferating cells and in cells undergoing senescence. Telomeric enrichment of H3 and TRF1 as well as the histone methylation marks H3K4me3, H3K9me3 and H4K20me3 were reduced in senescent cells. The degree of chromatin structural change as the cells entered senescence differed between chromosome ends. This highlights the benefits of using the telomere-specific ChIP-qPCR approach over the more traditional ChIP-dot blot assays which would not be able to differentiate between the chromatin structure of different chromosome ends. To identify roles for chromatin remodelling proteins in telomere length maintenance siRNA mediated knockdown of selected chromatin remodelers was performed in a clonal population of HT1080 cells followed by STELA analysis. RNAi-depletion of the histone methyltransferase (HMTase) III EHMT2 resulted in an increase in very short 17p telomeres whereas loss of another HMTase, DOT1L caused a divergence in the 17p telomere length distribution suggesting the presence of two subpopulations of cells each with differing telomere length distributions. Subtle changes in mean telomere length was observed after siRNA mediated knockdown of the HMTases MLL and EZH2, the histone deacetylases (HDACs) HDAC1 and SIRT6, the ATP dependent chromatin remodelling complex subunit BAF155 and the H3.3 histone chaperone DAXX. However due to certain limitations of the RNAi screen the validity of these observations is questionable and more work would have to be performed to confirm whether these chromatin remodelers have an effect on telomere length. Finally, dramatic telomere shortening was observed in a keratinocyte holoclone population after siRNA mediated knockdown of DAXX at number of chromosome ends. Prolonged depletion of DAXX also caused an increase in telomere‐to‐telomere fusions. A similarly dramatic loss in telomere length was seen in these cells after knockdown of EHMT2.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Subjects: Q Science > QH Natural history > QH426 Genetics
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Date of First Compliant Deposit: 30 March 2016
Last Modified: 04 Jun 2017 05:48

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