Indiana University-Purdue University Indianapolis (IUPUI) / Ends of linear chromosomes are maintained by specialized structures known as telomeres. These structures are protected by a number of essential protein complexes including the shelterin complex and CST (CTC1 – STN1 – TEN1) complex. CST is an RPA-like ssDNA binding protein that is vital for telomere length maintenance via inhibition of telomerase and stimulation of DNA polymerase α -primase during C-strand fill-in synthesis. CST is also known to possess additional genome-wide roles in regulating DNA replication and repair including helping facilitate replication re-start at stalled forks, activating checkpoint signaling at double-strand breaks, and promoting replication origin firing. Proper and efficient repair of DNA is critical in order to protect the integrity of the genome and prevent extreme mutagenesis. Telomeres have a strong predisposition to oxidative DNA damage in the form of 8-oxoguanine caused by exposure to reactive oxygen species and free radicals. These oxidative lesions are repaired by the base-excision repair (BER) pathway. Previous work has implicated telomeric proteins such as the shelterin complex in mediating BER. Here we show for the first time that the CST complex and individual subunits robustly stimulate a myriad of proteins involved in the BER pathway including Pol β, APE1, FEN1, and LIGI. CST’s ability to augment these BER-associated proteins could be instrumental in promoting efficient DNA repair. Additionally, we find that CTC1 and STN1 are able to significantly enhance the polymerase activity of Pol δ and Pol α on both random-sequence and telomeric-sequence DNA substrates in vitro. What is more, we establish the ability of CST to resolve G4 structure and promote Pol δ synthesis, which we predict is a key feature of CST’s involvement in DNA replication at telomeres, which are known to form replication-inhibiting G4’s. Our results define important mechanistic insight into CST’s role in DNA replication and repair, and provide a strong foundation for future studies relating defective telomere maintenance to aging disorders and cancers which impact human health.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19958号 / 理博第4225号 / 新制||理||1607(附属図書館) / 33054 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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T cells in chronic viral infections are featured by premature aging with accelerated telomere erosion, but the mechanisms underlying telomere attrition remain unclear. Here, we employed human CD4 T cells treated with KML001 (a telomere-targeting drug) as a model to investigate the role of telomere integrity in remodeling T cell senescence. We demonstrated that KML001 could inhibit cell proliferation, cytokine production, and promote apoptosis via disrupting telomere integrity and DNA repair machineries. Specifically, KML001-treated T cells increased dysfunctional telomere-induced foci (TIF), DNA damage marker γH2AX, and topoisomerase cleavage complex (TOPcc) accumulation, leading to telomere attrition. Mechanistically, KML001 compromised telomere integrity by inhibiting telomeric repeat binding factor 2 (TRF2), telomerase, topoisomerase I and II alpha (Top1/2a), and ataxia telangiectasia mutated (ATM) kinase activities. Importantly, these KML001-induced telomeric DNA damage and T cell senescent phenotype and machineries recapitulated our findings in patients with clinical HCV or HIV infection in that their T cells were also senescent with short telomeres and thus more vulnerable to KML001-induced apoptosis. These results shed new insights on the T cell aging network that is critical and essential in protecting chromosomal telomeres from unwanted DNA damage and securing T cell survival during cell crisis upon genomic insult.

Background: T cells play a key role in controlling viral infections; however, the underlying mechanisms regulating their functions during human viral infections remain incompletely understood. Here, we used CD4 T cells derived from individuals with chronic viral infections or healthy T cells treated with camptothecin (CPT) - a topoisomerase I (Top 1) inhibitor - as a model to investigate the role of DNA topology in reprogramming telomeric DNA damage responses (DDR) and remodeling T cell functions. Results: We demonstrated that Top 1 protein expression and enzyme activity were significantly inhibited, while the Top 1 cleavage complex (TOP1cc) was trapped in genomic DNA, in T cells derived from individuals with chronic viral (HCV, HBV, or HIV) infections. Top 1 inhibition by CPT treatment of healthy CD4 T cells caused topological DNA damage, telomere attrition, and T cell apoptosis or dysfunction via inducing Top1cc accumulation, PARP1 cleavage, and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. Moreover, T cells from virally infected subjects with inhibited Top 1 activity were more vulnerable to CPT-induced topological DNA damage and cell apoptosis, indicating an important role for Top 1 in securing DNA integrity and cell survival. Conclusion: These findings provide novel insights into the molecular mechanisms for immunomodulation by chronic viral infections via disrupting DNA topology to induce telomeric DNA damage, T cell senescence, apoptosis and dysfunction. As such, restoring the impaired DNA topologic machinery may offer a new strategy for maintaining T cell function against human viral diseases.

T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases.

<p> Comprehensive mastery of modern genetics involves a myriad of data processing and analytic techniques; these approaches vary because some genetic conditions are the result of single gene mutations that alter protein function, while other more complex diseases and traits are influenced by many genes. This dissertation will undertake investigation of the heritability and genetic risk of cranial cruciate ligament rupture in Labrador Retrievers, a congenital hypomyelinating polyneuropathy in four Golden Retrievers, and the potential usage of telomere length as a biomarker of welfare in dogs housed in commercial breeding facilities. </p> <p>In the first disease studied, 333 Labrador Retrievers with known torn or healthy cranial cruciate ligament(s) were genotyped via SNP array. Heritability of this polygenic trait was calculated using a variety of programs and including different fixed effects. Overall, heritability was high, ranging from 0.550 to 0.893, with sex and sterilization at a young age (≤12 months) strongly influencing risk of cranial cruciate ligament rupture. Neither genome-wide association analyses using this novel dataset of 333 Labrador Retrievers, nor additional analyses combining this data with publicly-available data, identified any significantly associated SNPs. However, the most associated SNPs were located near biologically relevant genes, such as <em>COL1A2</em> (a collagen gene) and <em>ITGA11</em> (a protein that binds to collagen), as well as genes encoding sex hormone receptors, such as <em>FSHR </em>and <em>LHCCGR</em>. Splitting the data in an attempt to predict phenotypes based on genotype was unsuccessful. Future work focused on parsing out genetic influencers of cranial cruciate ligament rupture risk should continue to collect sex, sterilization status, and age at sterilization data, and larger collaborations and use of publicly-available data will be required to increase the data robustness.</p> <p>For the second study, DNA from four unrelated Golden Retrievers diagnosed with congenital hypomyelinating polyneuropathy via neurological examination, electrodiagnostic evaluation, and peripheral nerve pathology were explored for genetic causes. Whole-genome sequencing was performed on all four dogs to identify potential causative variants. When compared to WGS from >1,000 other dogs who were presumably unaffected by this rare disease, likely causative variants were identified in all four dogs. Two cases shared a homozygous <em>MTMR2 </em>splice donor site variant, with a stop codon introduced within six codons following the inclusion of the intron. One case had a heterozygous <em>MPZ</em> missense mutation leading to an isoleucine to threonine substitution. The last case had a homozygous <em>SH3TC2</em> nonsense variant predicted to truncate approximately one-half of the protein. Haplotype analyses using 524 Golden Retrievers indicated that these variants emerged recently. Each of these variants occurred in genes that are associated with the human Charcot-Marie-Tooth group of heterogeneous peripheral nervous system diseases. Testing a population of unrelated Golden Retrievers (n > 200) did not identify any other dogs with these variants, though breeders should be cautious to avoid propagating these alleles.</p> <p>Finally, the last study within this dissertation investigates the relationship between telomere length and metrics such as age, breed, environment, average breed lifespan, parity, and response to a mild social stressor in a population of dogs housed at commercial breeding facilities. FIDO scores (behavioral phenotypes) were collected for all dogs as a measurement of response to a social stressor. This study is not yet complete; many more dogs remain to be recruited in the near future. Telomere lengths were measured using qPCR and compared to a single-copy gene, <em>36B4</em>, for 309 dogs representing 37 breeds or breed crosses. Age was not significantly associated with telomere length after making appropriate corrections (p-value = 0.077). Breed and facility were significantly associated with telomere length after corrections (p-value = 0.010 and <2.2E-16, respectively). Neither parity nor average breed lifespan were associated with telomere length, however, response to a mild social stressor was, with dogs who responded positively having significantly longer telomeres than dogs who responded negatively across all analyses. This preliminary data indicates that, within this population, breed, environment, and response to stress have strong influences on telomere length, while parity and average breed lifespan did not. As this work continues, increased sample sizes will lead to increased power for detecting associations. Future work should examine these identified relationships in other populations of dogs.</p> <p>Taken together, these studies encompass phenotypes of various complexity, and each study encompassed different methodologies utilized in modern canine genetics. The overall goal for this work was to improve canine health, with the potential for translational implications to human health. The identification of genetic markers associated with or causative of disease, or indicative of health and welfare traits, is necessary for reducing the prevalence of disease and increasing the knowledge of welfare metrics in canines, respectively.</p>

Mutations in the telomere length maintenance pathway can lead to a spectrum of diseases called telomere syndromes, however, the pathway is not fully understood and there may still be unknown components. We designed a forward genetic screen to identify new genes involved in telomere length maintenance. Of the top ranked genes, ZNF827, a zinc finger protein, is the most promising candidate gene. The possible discovery of a new component involved in telomere length maintenance increases our understanding of the pathway and opens new avenues of research. Recent advances in molecular biology techniques, such as the use of RNA-guided nuclease CRISPR associated protein 9 (Cas9), have made screens like this possible. Cas9 is a nuclease that uses a guide RNA(gRNA) to direct its endonuclease activity. The use of Cas9 has revolutionized the field of genome engineering, providing scientists with more efficient methods to knockout and modify genomes. We sought to optimize CRISPR-Cas9 genome editing to make it as widely accessible as possible. We compared plasmid, ribonucleoprotein (RNP), and RNA only lipid-mediated transfection of CRISPR-Cas9 into cell lines using a novel reporter system to measure genome editing efficiency. All methods were successful to some extent, however, RNP lipofection was the most efficient and has many advantages over other methods. We also found that short homology arms of 30-35bp on donor templates was able to mediate site specific editing. These methods should broaden the accessibility of CRISPR-Cas9 genome editing.

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<p>TRF1, a shelterin protein, is a negative mediator of telomere length maintenance. Phosphorylation has been shown to play an important role in modulating TRF1 function. T137 and S249 of TRF1 have been indentified to be candidate phosphorylation sites in vivo, and one of my thesis objectives was to examine their role in regulating TRF1 function. Both T137 and S249 have each been changed to either alanine (nonphosphorylatable) or phosphomimic mutation. The TRF1 mutants were introduced into a TRF1-depleted cell line. Southern analysis revealed that neither T137 nor S249 of TRF1 is involved in telomere length maintenance. Immunoprecipitation studies showed that T137 and S249 are not required for TRF1 interaction with TIN2. In vitro gel-shift assays indicated that T137 and S249 are not important for TRF1 binding to telomeric DNA. Taken together, these results suggest that T137 and S249 may not be required for TRF1 function in telomere length maintenance. Human Est1A has been suggested to play a role in telomere length maintenance. To identify the domain of hEst1A involved in telomere length maintenance, a number of deletion constructs were generated and retrovirally introduced into HT1080 cells. Southern analysis revealed that the RID domain may positively regulate telomere length maintenance whereas the first 220 amino acids at the N-terminus may be a negative mediator of telomere length maintenance. In S. cerevisiae, Est1 recruits telomerase to telomeres in a Tel1- (homolog of ATM) and MRX-dependent manner. To assess whether atm-1 and smg-6 may function in the same genetic pathway of regulation of telomere length in C.elegans, the single mutant strain atm-1(gk186) was crossed with three of smg-6 mutant strains (tm1308, ok1794 and r896) to generate double mutants. Southern analysis revealed that deletion of ATM-1 or SMG-6 (tm1308) results in telomere shortening, suggesting that atm-1 and smg-6 may function in the same genetic pathway to regulate telomere length maintenance.</p> / Master of Science (MSc)