Fluorescence microscopy methods for examining telomeres during cell aging.

Telomeres are protective structures, composed of nucleic acids and a complex protein mixture, located at the end of the chromosomes. They play an important role in preventing genomic instability and ensuring cell health. Defects in telomere integrity result in cell dysfunction and the development of diseases, including neurodegenerative disorders, cancer and premature aging syndromes, among others. Loss of telomere integrity during normal cell aging also initiates DNA damage signals that culminate in the senescence phenotype. Fluorescence microscopy has allowed researchers to study the dynamics, shape, localization, and co-distribution of telomeres with proteins of interest. The microscopy tools to investigate these structures have evolved, making it possible to understand in greater detail the molecular mechanisms affecting telomeres that contribute to cell aging and the development of age-related diseases. Using human fibroblasts as an example, we will highlight several characteristics of telomeres that can be investigated using three different microscopy systems, including wide-field microscopy, and the two super-resolution techniques called 3D Structured Illumination Microscopy (3D-SIM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). In this review, we will also discuss their limitations and highlight their importance in answering telomere-related scientific questions.

Protocol for the MATCH study (Mindfulness and Tai Chi for cancer health): A preference-based multi-site randomized comparative effectiveness trial (CET) of Mindfulness-Based Cancer Recovery (MBCR) vs. Tai Chi/Qigong (TCQ) for cancer survivors.

PURPOSE: A growing number of cancer survivors suffer high levels of distress, depression and stress, as well as sleep disturbance, pain and fatigue. Two different mind-body interventions helpful for treating these problems are Mindfulness-Based Cancer Recovery (MBCR) and Tai Chi/Qigong (TCQ). However, while both interventions show efficacy compared to usual care, they have never been evaluated in the same study or directly compared. This study will be the first to incorporate innovative design features including patient choice while evaluating two interventions to treat distressed cancer survivors. It will also allow for secondary analyses of which program best targets specific symptoms in particular groups of survivors, based on preferences and baseline characteristics. METHODS AND SIGNIFICANCE: The design is a preference-based multi-site randomized comparative effectiveness trial. Participants (N=600) with a preference for either MBCR or TCQ will receive their preferred intervention; while those without a preference will be randomized into either intervention. Further, within the preference and non-preference groups, participants will be randomized into immediate intervention or wait-list control. Total mood disturbance on the Profile of mood states (POMS) post-intervention is the primary outcome. Other measures taken pre- and post-intervention and at 6-month follow-up include quality of life, psychological functioning, cancer-related symptoms and physical functioning. Exploratory analyses investigate biomarkers (cortisol, cytokines, blood pressure/Heart Rate Variability, telomere length, gene expression), which may uncover potentially important effects on key biological regulatory and antineoplastic functions. Health economic measures will determine potential savings to the health system.

Mindfulness-based cancer recovery and supportive-expressive therapy maintain telomere length relative to controls in distressed breast cancer survivors.

BACKGROUND: Group psychosocial interventions including mindfulness-based cancer recovery (MBCR) and supportive-expressive group therapy (SET) can help breast cancer survivors decrease distress and influence cortisol levels. Although telomere length (TL) has been associated with breast cancer prognosis, the impact of these two interventions on TL has not been studied to date. METHODS: The objective of the current study was to compare the effects of MBCR and SET with a minimal intervention control condition (a 1-day stress management seminar) on TL in distressed breast cancer survivors in a randomized controlled trial. MBCR focused on training in mindfulness meditation and gentle Hatha yoga whereas SET focused on emotional expression and group support. The primary outcome measure was relative TL, the telomere/single-copy gene ratio, assessed before and after each intervention. Secondary outcomes were self-reported mood and stress symptoms. RESULTS: Eighty-eight distressed breast cancer survivors with a diagnosis of stage I to III cancer (using the American Joint Committee on Cancer (AJCC) TNM staging system) who had completed treatment at least 3 months prior participated. Using analyses of covariance on a per-protocol sample, there were no differences noted between the MBCR and SET groups with regard to the telomere/single-copy gene ratio, but a trend effect was observed between the combined intervention group and controls (F [1,84], 3.82; P = .054; η(2) = .043); TL in the intervention group was maintained whereas it was found to decrease for control participants. There were no associations noted between changes in TL and changes in mood or stress scores over time. CONCLUSIONS: Psychosocial interventions providing stress reduction and emotional support resulted in trends toward TL maintenance in distressed breast cancer survivors, compared with decreases in usual care.

hTERT mutations associated with idiopathic pulmonary fibrosis affect telomerase activity, telomere length, and cell growth by distinct mechanisms.

Telomerase is a ribonucleoprotein reverse transcriptase (RT) that synthesizes specific DNA repeats, or telomeric DNA, at the ends of chromosomes. Telomerase is minimally composed of a protein subunit, TERT, and an RNA component, TR. Aberrant telomerase activity has been associated with most human cancers and several premature aging diseases, such as idiopathic pulmonary fibrosis (IPF), a chronic, progressive, and fatal lung disease characterized by alveolar epithelial cell damage and fibrosis. Our study focuses on three hTERT mutations that were identified in a subset of patients with IPF, in which these patients also exhibited shorter telomeres compared with age-matched controls. We characterized how three IPF-associated hTERT mutations, V144M, R865C, and R865H, affected telomerase function both in vitro and in human cells. We demonstrated that the R865 residue is crucial for repeat addition processivity and thus telomere synthesis in telomerase-positive 293 cells and telomerase-negative BJ cells, consistent with its location in the hTERT nucleotide-binding motif. In contrast, while the V144M mutant did not exhibit any biochemical defects, this mutant was unable to elongate telomeres in human cells. As a result, our studies have identified hTERT V144 and R865 as two critical residues required for proper telomerase function in cells. Together, this may explain how inherited hTERT mutations can lead to shortened telomeres in patients with IPF and, thus, provide further insight into the role of naturally occurring telomerase mutations in the pathophysiology of certain age-related disease states.

InTERTpreting telomerase structure and function.

The Nobel Prize in Physiology or Medicine was recently awarded to Elizabeth Blackburn, Carol Greider and Jack Szostak for their pioneering studies on chromosome termini (telomeres) and their discovery of telomerase, the enzyme that synthesizes telomeres. Telomerase is a unique cellular reverse transcriptase that contains an integral RNA subunit, the telomerase RNA and a catalytic protein subunit, the telomerase reverse transcriptase (TERT), as well as several species-specific accessory proteins. Telomerase is essential for genome stability and is associated with a broad spectrum of human diseases including various forms of cancer, bone marrow failure and pulmonary fibrosis. A better understanding of telomerase structure and function will shed important insights into how this enzyme contributes to human disease. To this end, a series of high-resolution structural studies have provided critical information on TERT architecture and may ultimately elucidate novel targets for therapeutic intervention. In this review, we discuss the current knowledge of TERT structure and function, revealed through the detailed analysis of TERT from model organisms. To emphasize the physiological importance of telomeres and telomerase, we also present a general discussion of the human diseases associated with telomerase dysfunction.

Human telomerase reverse transcriptase (hTERT) Q169 is essential for telomerase function in vitro and in vivo.

BACKGROUND: Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE: We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention.

The human telomerase RNA component, hTR, activates the DNA-dependent protein kinase to phosphorylate heterogeneous nuclear ribonucleoprotein A1.

Telomere integrity in human cells is maintained by the dynamic interplay between telomerase, telomere associated proteins, and DNA repair proteins. These interactions are vital to suppress DNA damage responses and unfavorable changes in chromosome dynamics. The DNA-dependent protein kinase (DNA-PK) is critical for this process. Cells deficient for functional DNA-PKcs show increased rates of telomere loss, accompanied by chromosomal fusions and translocations. Treatment of cells with specific DNA-PK kinase inhibitors leads to similar phenotypes. These observations indicate that the kinase activity of DNA-PK is required for its function at telomeres possibly through phosphorylation of essential proteins needed for telomere length maintenance. Here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a direct substrate for DNA-PK in vitro. Phosphorylation of hnRNP A1 is stimulated not only by the presence of DNA but also by the telomerase RNA component, hTR. Furthermore, we show that hnRNP A1 is phosphorylated in vivo in a DNA-PK-dependent manner and that this phosphorylation is greatly reduced in cell lines which lack hTR. These data are the first to report that hTR stimulates the kinase activity of DNA-PK toward a known telomere-associated protein, and may provide further insights into the function of DNA-PK at telomeres.

Multiple factors influence the normal and UV-inducible alternative splicing of PIG3.

In addition to normal alternative splicing events (those that take place in untreated cells), there are also those that are inducible in response to environmental stimuli. We previously reported that the alternative splicing of p53-inducible gene 3 (PIG3) exon 4 is modulated in response to UV radiation. Here, we investigate the mechanisms mediating the alternative splicing of this exon. Through the use of various minigene constructs our results reveal that numerous factors influence the normal alternative splicing of PIG3 exon 4, and that these ultimately affect its UV-inducible alternative splicing. Included among these are sequence elements located within exon 4 itself as well as adjacent sequences required for intron definition (the pyrimidine tract, 3'- and 5'-splice sites). Within exon 4, we identified a novel hnRNP A1-dependent exonic splicing silencer (ESS) whose mutation inhibited the alternative splicing of a PIG3 minigene. Although previously implicated in the UV-inducible alternative splicing of other transcripts, RNAi-mediated silencing of hnRNP A1/A2 or hSlu7 did not prevent the UV-enhancement of exon 4 skipping. Overall, our results suggest that numerous factors contribute to the normal alternative splicing of PIG3 exon 4 and that UV-inducible increases in this process require that the splicing of this exon be maintained in a sufficiently weakened state under normal conditions.

Discovery and characterization of a novel splice variant of the GM-CSF receptor alpha subunit.

OBJECTIVE: To characterize a novel splice variant of the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMRalpha), which we discovered in human neutrophils. METHODS: We used reverse transcriptase polymerase chain reaction to identify, characterize, and examine the expression of a novel splice variant of the GMRalpha transcript. At the protein level, surface plasmon resonance was used to measure the affinity of a recombinant soluble form of the novel GMRalpha protein for GM-CSF ligand. The full-length novel GMRalpha protein was expressed in a recombinant cell culture system, and its expression and localization were examined using Western blotting, I(125) GM-CSF binding assays, flow cytometry, and a soluble GMRalpha enzyme-linked immunosorbent assay. RESULTS: The novel GMRalpha transcript identified herein contains a previously undescribed exon of the GMRalpha gene; this exon derives from an Alu DNA repeat element, and is alternatively spliced in the novel GMRalpha transcript. Inclusion of this 102 nucleotide exon results in translation of a protein product, which we have named Alu-GMRalpha. Alu-GMRalpha is identical to cell surface GMRalpha, but additionally contains a 34 amino-acid insert in the juxtamembrane region of the extracellular domain of GMRalpha. Functionally, the Alu-GMRalpha-specific epitope does not modify the ability of the protein to bind GM-CSF, but rather appears to be preferentially targeted by ectodomain proteases to mediate the release of a third soluble GM-CSF receptor into the extracellular space. CONCLUSIONS: This study provides the first example of a cytokine receptor system in which soluble receptors are produced by three distinct mechanisms. Our results highlight the importance of soluble GMRalpha proteins in regulation of GM-CSF signaling.

Characterization of physical and functional anchor site interactions in human telomerase.

Telomerase is a ribonucleoprotein reverse transcriptase (RT) that processively synthesizes telomeric repeats onto the ends of linear chromosomes to maintain genomic stability. It has been proposed that the N terminus of the telomerase protein subunit, telomerase RT (TERT), contains an anchor site that forms stable interactions with DNA to prevent enzyme-DNA dissociation during translocation and to promote realignment events that accompany each round of telomere synthesis. However, it is not known whether human TERT (hTERT) can directly interact with DNA in the absence of the telomerase RNA subunit. Here we use a novel primer binding assay to establish that hTERT forms stable and specific contacts with telomeric DNA in the absence of the human telomerase RNA component (hTR). We show that hTERT-mediated primer binding can be functionally uncoupled from telomerase-mediated primer extension. Our results demonstrate that the first 350 amino acids of hTERT have a critical role in regulating the strength and specificity of protein-DNA interactions, providing additional evidence that the TERT N terminus contains an anchor site. Furthermore, we establish that the RT domain of hTERT mediates important protein-DNA interactions. Collectively, these data suggest that hTERT contains distinct anchor regions that cooperate to help regulate telomerase-mediated DNA recognition and elongation.

Human Ku70/80 interacts directly with hTR, the RNA component of human telomerase.

Maintenance of telomere integrity requires the dynamic interplay between telomerase, telomere-associated proteins and DNA repair proteins. These interactions are vital to suppress DNA damage responses and changes in chromosome dynamics that can result in aneuploidy or other transforming aberrations. The interaction between the DNA repair protein Ku and the RNA component of telomerase (TLC1) in Saccharomyces cerevisiae has been shown to be important for maintaining telomere length. Here, we sought to determine whether this interaction was conserved in higher eukaryotes. Although there is no sequence similarity between TLC1 and the RNA component (hTR) of human telomerase, we show that human Ku70/80 interacts with hTR both in vitro and in a cellular context. Specifically, Ku70/80 interacts with a 47 nt region of the 3' end of hTR, which resembles the stem-loop region of the yeast Ku70/80 binding domain on TLC1. Furthermore, utilizing immunoprecipitation/RT-PCR experiments, we show that Ku interacts with hTR in cell lines deficient in the human telomerase reverse transcriptase protein (hTERT), suggesting that this interaction does not require hTERT. These data suggest that Ku interacts directly with hTR, independent of hTERT, providing evidence for the conservation of the interaction between Ku and telomerase RNA among various species and provide significant insight into how Ku is involved in telomere maintenance in higher eukaryotes.

UV-dependent alternative splicing uncouples p53 activity and PIG3 gene function through rapid proteolytic degradation.

The p53-inducible gene 3 (PIG3) is a transcriptional target of the tumor suppressor protein p53 and is thought to play a role in apoptosis. In this report, we identify a novel alternatively spliced product from the PIG3 gene that we call PIG3AS (PIG3 alternative splice). PIG3AS results from alternative pre-mRNA splicing that skips exon 4 of the five exons included in the PIG3 transcript. The resulting protein product shares its first 206 amino acids with PIG3 but has a unique 42-amino acid C terminus. In unstressed cells and after most DNA damage conditions that induce transcription from the PIG3 gene, production of the PIG3 transcript dominates. However, in response to UV light, pre-mRNA splicing shifts dramatically in favor of PIG3AS. Unlike the PIG3 protein, the PIG3AS protein is rapidly degraded with a short half-life and is stabilized by proteasome inhibition. Our results illustrate the first example of an endogenous, UV-inducible, alternative splicing event and that control of the splicing machinery is involved in the cellular DNA damage response. They also suggest that rapid proteolytic degradation represents a cellular mechanism for uncoupling p53 activity from PIG3 gene activation that is independent of promoter selectivity.