Browsing by Subject "Telomere"
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Item Assessing the Relationship Between Telomere Length and Adipose Tissue Distribution(2018-01-23) Bleiberg, Benjamin; Ayers, Colby; Neeland, Ian J.BACKGROUND: A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration. Telomere shortening, a surrogate marker of cellular aging, may accelerate from the inflammatory stressors of obesity. The association between adipose tissue depots and telomere length is unknown. METHODS: Data were analyzed from 2,551 participants in the Dallas Heart Study, a prospective multiethnic cohort. The sample composition was 41% male, 59% female, 48% African American, 35% Caucasian, 15% Hispanic and participants had a mean age of 51 years with 23.4% >60 years of age. Leukocyte telomere length (LTL) was determined using qPCR on DNA isolated from circulating leukocytes. Visceral (VAT) and subcutaneous (SAT) abdominal fat masses were measured by MRI, lower body fat (LBF) by dual x-ray absorptiometry, and liver fat by MR spectroscopy. Linear regression was used to evaluate the association between LTL and body fat depots. RESULTS: In univariate analysis, shorter LTL was associated with higher VAT (p=.0002) and less LBF (p=.02). Shorter telomeres were also associated with older age, male sex, hypertension, diabetes, smoking, decreased kidney function and decreased physical activity (p<0.05 for all). Adjustment for age and sex attenuated the relationships between LTL and VAT, SAT, LBF, and liver fat. No significant interactions were seen by stratification within age groups or by severity of obesity. Variable Beta unadjusted p unadjusted Beta adjusted p adjusted VAT -0.072 0.0002* 0.009 0.6 SAT 0.021 0.29 0.016 0.38 Lower Fat 0.048 0.02* 0.024 0.19 Liver Fat 0.03 0.41 0.032 0.4 Adjustments for age and sex and * p<0.05 Standardized β coefficients = estimated unit change in 1-SD of the log-transformed variable for a 1-SD increase in the telomere parameter CONCLUSIONS: While LTL is associated with pathogenic patterns of adipose tissue, this association is confounded by the close relationship between LTL and temporal aging. These findings suggest that cellular aging is not independently linked to variation in adipose tissue distribution patterns.Item Development of a T-Loop Assay to Investigate T-Loop Dynamics and Structure(2014-03-27) Mak, Sin Man; Burma, Sandeep; Shay, Jerry W.; Wright, Woodring E.; Yu, HongtaoAn attractive target in cancer therapy development has been the study of telomeres, which are repetitive sequences at the ends of chromosomes critical in maintaining genomic stability. T-loops, formed by the 3’ overhang inserting into the double strand region of the telomere, are thought to protect the ends from being recognized as double-strand breaks. An almost universal marker for cancer, telomerase, is a promising therapeutic target since its inhibition results in critically short telomeres, compromising t-loop structures. The clinical application has yet to be fully realized due to the lag phase between the times in which telomerase is inhibited and the times that telomeres become sufficiently short that the tumor undergoes apoptosis. Thus, improvements are needed including a greater understanding in t-loop dynamics and the cooperative interaction with telomerase to provide the strategy in which the lag phase can be shortened. Unfortunately, the study of t-loops has been challenging due to the difficulty of isolating and visualizing DNA with intact loop structures. Thus, we have developed novel methods to isolate DNA such that biochemical assays and microscopic visualizations for authentic t-loops are now possible. Digestion of proteins that stabilize t-loop and significant melting at the ends of DNA allow the 3’overhang to migrate out of the double strand region, thus unfolding t-loop and exposing the overhang. DNA isolated with typical procedures (Proteinase K at 55°C for 4 hrs, phenol/chloroform extraction) are thus linear and telomeric overhangs are susceptible to digestion by a 3’-5’ exonuclease (ExoI). The “overhangs” in t-loop structures should be resistant to ExoI. If we lower the temperature of Proteinase K digestion to 4°C to reduce the amount of DNA melting that can occur, we find that the ends are in fact resistant to ExoI digestion. A consistent ~2 fold higher overhang signal in isolated t-loops compared to linear telomeres was observed to distinguish between the two samples. Heating these 4°C samples to 37°C and 55°C caused unfolding of t-loops, resulting in sensitivity of the overhangs to ExoI to the same extent as normal DNA preparations. To validate the t-loop assay, transmission electron microscopy (TEM), a method with powerful magnification and extremely high resolution, is used to visualize DNA isolated at 55°C (linear structures) and 4°C (t-loop structures). The assay was then used to investigate t-loop dynamics throughout cell cycle, and we found that t-loops remain in a folded conformation throughout S phase, confirming the hypothesis that t-loops would unfold a second time for late S/G2 C-strand fill in. We also found that an overhang size of ~30 nts is too short to maintain stabilized t-loops compared to ~90 nts in BJ cells. In summary, we have significant evidence that we are able to prepare and analyze t-loops. Using this assay to determine t-loop structure and timing of t-loop repackage following replication and telomerase action will exceedingly add to our understanding of telomere biology. These are the key steps in setting the stage for many additional future studies, such as what factors contribute in generation of t-loops, how t-loop folding varies with telomere length, and what is the timing of t-loop folding and unfolding throughout cell cycle. All of which will provide critical information for the discovery of new improvements in anti-telomerase therapeutics.Item An Imaging Approach to Examine Telomere Dynamics and Regulation of Gene Expression with Aging(2020-08-01T05:00:00.000Z) Zhang, Ning; Xie, Yang; Danuser, Gaudenz; Shay, Jerry W.; Jaqaman, Khuloud; Siegwart, Daniel J.Telomeres are repetitive non-coding nucleotide sequences (TTAGGG)n capping the ends of chromosomes. Improved methods to measure the shortest (not just average) telomere lengths (TLs) are needed. Progressive telomere shortening with increasing age has been associated with shifts in gene expression through models such as the telomere position effect (TPE), which suggests reduced interference of the telomere with transcriptional activity of increasingly more distant genes. A modification of the TPE model, referred to as Telomere Position Effects over Long Distance (TPE-OLD), explains why some genes 1-10 MB from a telomere are still affected by TPE, but genes closer to the telomere are not. Therefore, demonstrating the regulatory roles of telomere length shortening on genes with accurate TL measurement will improve our understanding to the 3D genomic DNA landscape including telomeres. In this doctoral dissertation, I developed a user-friendly software for automatic electrophoresis gel quantification and contributed to developing the Telomere Shortest Length Assay (TeSLA), a technique that detects telomeres from all chromosome ends from <1 kb to 18 kb using small amounts of input DNA. Using cells with more TL information provided by TeSLA, I conducted an imaging approach to systematically examine the occurrence of TPE-OLD at the single cell level. Compared to existing methods, the pipeline allows rapid analysis of hundreds to thousands of cells, which is necessary to establish TPE-OLD as an acceptable mechanism of gene expression regulation. I examined two human genes, for which TPE-OLD has been described before, ISG15 (Interferon Stimulated Gene 15) and TERT (TElomerase Reverse Transcriptase). For both genes I found less interaction with the telomere on the same chromosome in old cells compared to young cells. Experimentally elongated telomeres in old cells rescued the level of telomere interaction for both genes. However, the dependency of the interactions on the age progression from young to old cells varied. One model for the differences between ISG15 and TERT may relate to the markedly distinct interstitial telomeric sequence arrangement in the two genes. Overall, this provides a strong rationale for the role of telomere length shortening in the regulation of gene expression.Item [Southwestern News](2001-06-15) Morrison, SusanItem Telomere and Telomerase Dynamics in Human T Lymphocytes(2017-06-28) Huang, Ejun; Zhu, Hao; Shay, Jerry W.; Wright, Woodring E.; Burma, Sandeep; Brekken, Rolf A.Advanced cancer is characterized by a phenotype that permits cells to divide indefinitely while cellular aging is characterized by cells ceasing to divide. It is believed that senescence may have evolved as an anti-cancer protection mechanism in long-lived species such as humans. Therefore, cancer and aging are essentially opposite ends of the same problem. Telomeres are the cellular aging-clock that determines a cell's capability of proliferation. When normal cells divide, telomeres gets progressively shorter. When telomeres reach a critical short length, cells stop dividing. To compensate for the loss of telomeres, telomerase is a ribonucleoprotein enzyme complex that elongates telomeres, but its expression is restricted to certain subsets of cell types. Investigating how telomerase is regulated in normal cells may provide insights and new approaches to block telomerase activity in cancer cells or to re-activate telomerase in aging cells. Immune cells play central roles in defending humans from pathogens, infections, and malignant cells. Immunotherapy has shown great potential in cancer treatment and has gained increasing public attention. Thus, a better understanding of how to increase the proliferation efficiency of normal immune cells, especially in older individuals whose immune stem-like cells may becoming less efficient is important. Due to the accessibility of blood from volunteers, immune cells are a model system to investigate healthy aging. For my doctoral research, I decided to study telomeres and telomerase dynamics in immune cells. Using novel techniques in telomere length analysis and telomerase activity measurements developed in our lab, I discovered that a subset of CD28+ T cells show robust telomerase activity and the ability to maintain telomere lengths during stimulated cell proliferation. In a centenarian study, T cells from a subset of centenarians showed stronger telomerase activation compared with many younger individuals. RNA-seq analysis revealed distinct differences between high performance centenarians and other age groups. Finally, we developed novel techniques using ddPCR for quantitating mitochondrial DNA numbers per cell and validated the differences we found in centenarian samples. Altogether, my doctoral project has added value to our current knowledge of how telomerase is regulated during immune responses and revealed its importance in longevity studies.Item Telomere Length, Uncommon Genetic Variation, and Depression(2015-07-20) Wignall, Nicholas David; Brown, E. Sherwood; Cohen, Jonathan C.; Jeon-Slaughter, Haekyung; Evans, Harry M.; Deschner, MartinPrevious research attempting to better understand the genetic basis of depression has demonstrated few clear findings. The present study employed a cross-sectional design to analyze the association between leukocyte telomere length and depression in 2,710 participants from the Dallas Heart Study (DHS), as well as the potentially moderating influence of ethnicity/race, APOE allele variation, and average self-reported hours of sleep per night. Furthermore, in an exploratory exome-wide association study, we analyzed associations between a subset of 2,949 participants from the DHS with depression data available and 247,870 genetic variants. Scores from the primary measure of depression--The Quick Inventory of Depressive Symptomatology (QIDS)--showed a significant negative association with telomere length but only in the Caucasian subset of the sample. Scores from the secondary measure of depression--The DHS's "felt depressed" question--showed a significant negative association with telomere length across the whole sample, as well as in the African American subset, but not within the Caucasian or Hispanic subsets. Neither APOE allele variation nor average nightly sleep was a significant moderator of the association between depression and telomere length. No significant associations between depressions scores and specific genetic variants were found in the exome-wide association study. These finding help clarify the relationship between depression and telomere length by highlighting the importance of ethnicity/race in the relationship between depression and telomere length. Future research involving depression and telomere length should account for ethnicity/race in their analyses, while future exome-wide association studies of depression should seek more severe and well-defined cases of depression.Item Telomere Specific Homologous Recombination in the Alternative Lengthening of Telomeres(2013-01-16) Whitington, Aric J.; Shay, Jerry W.; Wright, Woodring E.Over twenty years have passed since the discovery of telomerase-independent telomere maintenance, yet the precise details of the ALT mechanism remain a mystery. A growing body of evidence suggests that ALT cells maintain telomeres by homologous recombination (Reddel 2003 for Review). Groundbreaking work by Oliver Bechter demonstrated that ALT cells and telomerase-positive cells show no difference in the rate of general HR. This study fundamentally shaped our current concept of the ALT mechanism, implying that it involves preferential recombination of telomeric repeats. Since ALT seems to require proteins involved in normal HR, it follows that this telomeric recombination must be suppressed in telomerase positive or normal cells. However, to date there has been no direct evidence to support this hypothesis. Seeking to investigate the rates of telomere specific recombination, previous work in the Shay and Wright lab utilized the Tel-Tel vector. However, due to the method of integration only a limited number of clones could be analyzed and no statistically significant conclusions could be made. My work has focused on remedying this limitation. I have developed a strategy for integrating the Tel-Tel vector into a variety of host cell lines and generating a large number of distinct clones for each line. Using this strategy I was able to measure the average rates of telomeric HR for each cell line and provide the first direct evidence that ALT cells show distinctly elevated levels of increased telomere-specific HR. Additionally, I have constructed a control vector which functions in the same manner as Tel-Tel, differing only in that the telomeric repeats are replaced by non-telomeric repeat sequence. Using this vector (referred to as Mut-Mut) and the same incorporation method, I have demonstrated that there is no significant difference in the rates of general HR in ALT and telomerase positive cells. Finally, I used this novel ALT reporter as well as previously established methods to investigate some proteins that may play a central role in the ALT mechanism.Item [UT Southwestern Medical Center News](2009-09-01) McKenzie, AlineItem [UT Southwestern Medical Center News](2006-02-02) McKenzie, Aline