<p>Telomeres are the ends of chromosomes which are composed of repetitive DNA sequence and telomere associated proteins. In C. elegans, the protein F39H2.5 was found to associate with the telomere, regulating both telomere length and genomic integrity. F39H2.5 is a member of the β-CASP family of proteins that are known to possess nuclease activity on DNA substrates. I thus sought to address whether any of the human β-CASP family proteins associated with telomeres. Here I show that hSnm1B localized to the telomere indirectly, via interaction with the double-stranded telomere binding protein TRF2. The terminal 37 amino acids of hSnm1B are necessary and sufficient for binding TRF2, and moreover that binding to TRF2 stabilized hSnm1B protein by preventing ubiquitination. In the absence of exogenous TRF2 this domain acted as a degron, promoting protein instability. I thus termed the domain the Protection And INstability (PAIN) domain. I hypothesize that TRF2 binding ensures that hSnm1B will only accumulate at telomeres by preventing the degradation of hSnm1B. However, hSnm1B stability appears to be further regulated, as telomere specific DNA damage stabilized hSnm1B independent of the PAIN domain. Thus, it appears that the telomere associated protein, hSnm1B, is regulated by protein stability in a manner that is both dependent and independent of the PAIN domain.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 117-143).

Telomerase and telomere play crucial roles in the maintenance of genomic stability. Through its ability to extend chromosome ends with G-rich telomeric sequence, telomerase solves the end-replication problem of linear chromosomes and allows complete replication of the genetic information. Telomere along with its protein partners solves the end-protection problem and guards the chromosome ends against aberrant DNA damage response. In this thesis, I present two single-molecule fluorescence-based studies that determined the functional structure of telomerase RNA within active telomerase holoenzyme and probed the structure of telomere and its dependence on telomere binding proteins. In the first study, we developed a single-molecule Förster resonance energy transfer (FRET) assay to interrogate the structure of telomerase RNA within active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity. FRET signals from individual enzyme molecules during active binding events were then used to determine the organization of telomerase RNA within active telomerase. Using this assay, we have identified an active conformation of telomerase in which the conserved telomerase RNA pseudoknot is properly folded. In the second study, we used super-resolution fluorescence technique STochastic Optical Reconstruction Microscopy (STORM) to probe the structure of mammalian telomere. We showed that previously described telomere loop structures are detected by STORM imaging. Removal of telomere-binding protein TRF2 significantly reduces the fraction of telomeres found in loops. Furthermore, this reduction of telomere loops occurs in the absence of ATM-dependent DNA damage signaling and non-homologous end joining mediated chromosome fusion, suggesting a direct role of TRF2 in the formation or maintenance of telomere loops.

The mechanisms underpinning gene regulation in P. falciparum malaria remain largely elusive, though mounting evidence suggests a major role for epigenetic feedback. Interestingly, long non-(protein)-coding RNAs (lncRNAs) have been found to play a dominant role in initiating and guiding the transcriptional, epigenetic, and post-transcriptional status of specific loci across a broad range of organisms. LncRNAs are uniquely poised to act co-transcriptionally on neighboring loci, and/or to remain physically tethered at their site of origin, and through sequence-specific binding activities can impart temporal and spatial specificity to ubiquitously expressed nuclear protein complexes. Proteins, on the other hand, must be translated in the cytoplasm, and hence lose memory of their transcriptional origins. Encouraged by these features of lncRNAs, we set out to investigate the regulatory capacity of P. falciparum lncRNAs on a genome-wide scale. First, we surveyed transcriptional activity across approximately one quarter of the P. falciparum genome using a custom high-density DNA tiling array. We predicted a set of 60 developmentally regulated intergenic lncRNAs, and found that many of these novel loci neighbored genes involved in parasite survival or virulence pathways. Remarkably, upon further analysis of intergenic lncRNA properties, we discovered a family of twenty-two telomere-associated lncRNAs encoded in the telomere-associated repetitive element (TARE) region of P. falciparum chromosome ends. We found that each lncRNA-TARE was encoded adjacent and divergent to a subtelomeric var virulence gene. Moreover, we found that lncRNA-TARE expression was sharply induced between the parasite DNA replication and cell division cycles, that lncRNA-TARE loci contained numerous transcription factor binding sites only otherwise found in subtelomeric var promoter regions, and that the GC content and evolutionary sequence conservation of lncRNA-TAREs was similar to that of P. falciparum ribosomal RNA. Next, we set out to assemble P. falciparum intergenic lncRNA and antisense RNA transcript structures using state-of-the-art deep sequencing and computational tools. Towards this end, we harvested an unprecedented sample set that finely maps temporal changes across 56 hours of P. falciparum blood stage development, and developed and validated strand-specific, non-polyA-selected RNA sequencing methods. This enabled the annotation of over one thousand high-confidence, bona fide lncRNA transcript models, and their comprehensive global analysis. We discovered an enrichment of negatively correlated, tail-to-tail overlapping sense-antisense transcript pairs, suggesting a conserved role for antisense-mediated transcriptional interference in P. falciparum gene regulation. We also discovered a highly correlated spliced antisense counterpart to a gene required for sexual commitment, that the expression of an intriguing subset of antisense transcripts significantly dropped during parasite invasion, and that lncRNA-TARE and 'sterile' var virulence gene transcription was markedly up-regulated during parasite invasion. Lastly, we predicted over one thousand circular RNAs (circRNAs), and validated six circRNA transcript structures. Importantly, this thesis work represents the first focused investigation of lncRNAs in P. falciparum malaria, with the characterization of a compelling family of telomere-associated lncRNAs and numerous antisense RNAs. The data, methods, and results herein offer exceptional technological advancements coupled with compelling insights into the biology of the devastating human pathogen P. falciparum malaria. It is my hope that this work will facilitate future P. falciparum lncRNA functional studies and the strand-specific profiling of additional P. falciparum samples.

Statement of Problem Telomere dynamics in the three-dimensional (3D) space of the mammalian nucleus plays an important role in the maintenance of genomic stability. However, the telomere distribution in 3D nuclear space of normal and tumor cells was unknown when the study was initiated. Methods Telomere fluorescence in situ hybridization (FISH) and 3D molecular imaging, deconvolution, and analysis were used to investigate telomere organization in normal, immortalized and tumor cells from mouse and human cell lines, and primary tissues. Results Telomeres are organized in a non-overlapping manner and in a cell-cycle dependant fashion in normal cells. In the late G2 phase of cell cycle, telomeres are assembled into a flattened sphere that is termed the telomeric disk In contrast, the telomeric disk is disrupted in the tumor cells. Moreover, telomeric aggregates (TAs) are found in tumor cells. Conditional c-Myc over-expression induces telomeric aggregation leading to the onset of breakage-bridge-fusion cycles and subsequent chromosomal abnormality. Conclusions Telomeres are distributed in a nonrandom and dynamic fashion in the 3D space of a normal cell. Telomeric aggregates are present in cells with genomic instability such as tumor cells and cells with deregulation of c-Myc. Consequently, TA can be a useful biomarker for research in cancer and other disease processes.

Statement of Problem Telomere dynamics in the three-dimensional (3D) space of the mammalian nucleus plays an important role in the maintenance of genomic stability. However, the telomere distribution in 3D nuclear space of normal and tumor cells was unknown when the study was initiated. Methods Telomere fluorescence in situ hybridization (FISH) and 3D molecular imaging, deconvolution, and analysis were used to investigate telomere organization in normal, immortalized and tumor cells from mouse and human cell lines, and primary tissues. Results Telomeres are organized in a non-overlapping manner and in a cell-cycle dependant fashion in normal cells. In the late G2 phase of cell cycle, telomeres are assembled into a flattened sphere that is termed the telomeric disk In contrast, the telomeric disk is disrupted in the tumor cells. Moreover, telomeric aggregates (TAs) are found in tumor cells. Conditional c-Myc over-expression induces telomeric aggregation leading to the onset of breakage-bridge-fusion cycles and subsequent chromosomal abnormality. Conclusions Telomeres are distributed in a nonrandom and dynamic fashion in the 3D space of a normal cell. Telomeric aggregates are present in cells with genomic instability such as tumor cells and cells with deregulation of c-Myc. Consequently, TA can be a useful biomarker for research in cancer and other disease processes.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

This thesis details the development of two computational tools, Telomerecat and Parabam, as well as their applications to whole genome sequencing (WGS) data. Telomerecat is a tool for estimating telomere length from WGS data. The strength of Telomerecat lies in its applicability. This applicability is due to a number of advantages over previous attempts to estimate telomere length from WGS. Chief amongst these advantages is that it makes no assumption about the underlying chromosome count or size of the genome within input samples. This means that Telomerecat lends itself well to analysing cancer samples where such assumptions are unfounded. This also means it is applicable to non-human samples, a first for tools of its kind. Furthermore, a novel method for filtering reads derived from interstitial telomere sequences means that it does not rely on previously applied analyses, a source of bias. The other tool described in this thesis is Parabam. Parabam is the first tool of its kind to allow users to apply a function to all of the reads in sequence alignment files, in parallel. Furthermore, Parabam includes a novel method for iterating over index sorted sequence files as if they were name sorted. We provide evidence that Parabam is a quicker way to create complex subsets and statistics from sequence alignment files. In the latter half of the thesis we detail two applications of Telomerecat to large scale WGS projects. The first application, to the Prostate ICGC UK cohort, unveils hitherto uncovered associations between telomere length and previously identified molecular subtypes as well as cancer stage. In the second application, to the NIHR BioResource - Rare Disease cohort, we discover a previously unidentified variant in DKC1 that we propose is directly linked to short telomeres and an immunodeficient phenotype.