While global chromatin conformation studies are emerging, very little is known about the chromatin conformation of human telomeres. Most studies have focused on the role of telomeres as a tumor suppressor mechanism. Here we describe how telomere length regulates gene expression long before telomeres become short enough to produce a DNA damage response (senescence). We directly mapped the interactions adjacent to specific telomere ends using a Hi-C (chromosome capture followed by high-throughput sequencing) technique modified to enrich for specific genomic regions. We demonstrate that chromosome looping brings the telomere close to genes up to 10 Mb away from the telomere when telomeres are long and that the same loci become separated when telomeres are short. Furthermore, expression array analysis reveals that many loci, including noncoding RNAs, may be regulated by telomere length. We report three genes (ISG15 [interferon-stimulated gene 15 kd], DSP [Desmoplakin], and C1S [complement component 1s subcomplement]) located at three different subtelomeric ends (1p, 6p, and 12p) whose expressions are altered with telomere length. Additionally, we confirmed by in situ analysis (3D-FISH [three-dimensional fluorescence in situ hybridization]) that chromosomal looping occurs between the loci of those genes and their respective telomere ends. We term this process TPE-OLD for ''telomere position effect over long distances.'' Our results suggest a potential novel mechanism for how telomere shortening could contribute to aging and disease initiation/progression in human cells long before the induction of a critical DNA damage response.

Telomerase is expressed in early human development and then becomes silenced in most normal tissues. Because ~90% of primary human tumors express telomerase and generally maintain very short telomeres, telomerase is carefully regulated, particularly in large, long-lived mammals. In the current report, we provide substantial evidence for a new regulatory control mechanism of the rate limiting catalytic protein component of telomerase (hTERT) that is determined by the length of telomeres. We document that normal, young human cells with long telomeres have a repressed hTERT epigenetic status (chromatin and DNA methylation), but the epigenetic status is altered when telomeres become short. The change in epigenetic status correlates with altered expression of TERT and genes near to TERT, indicating a change in chromatin. Furthermore, we identified a chromosome 5p telomere loop to a region near TERT in human cells with long telomeres that is disengaged with increased cell divisions as telomeres progressively shorten. Finally, we provide support for a role of the TRF2 protein, and possibly TERRA, in the telomere looping maintenance mechanism through interactions with interstitial TTAGGG repeats. This provides new insights into how the changes in genome structure during replicative aging result in an increased susceptibility to age-related diseases and cancer prior to the initiation of a DNA damage signal.

Purpose-The purpose of this study was to examine the relationship of exercise energy expenditure (EEE) with both telomere length and telomerase activity in addition to accounting for hTERT C-1327T promoter genotype.Methods-Sixty-nine (n = 34 males; n = 35 females) participants 50-70 yr were assessed for weekly EEE level using the Yale Physical Activity Survey. Lifetime consistency of EEE was also determined. Subjects were recruited across a large range of EEE levels and separated into quartiles: 0-990, 991-2340, 2341-3540, and 93541 kcalIwk j1 . Relative telomere length and telomerase activity were measured in peripheral blood mononuclear cells (PBMC).Results-The second EEE quartile exhibited significantly longer telomere lengths [1.12 T 0.03 relative units (RU)] than both the first and fourth EEE quartiles (0.94 T 0.03 and 0.96 T 0.03 RU, respectively; P G 0.05) but was not different from the third quartile. Telomerase activity was not different among the EEE quartiles. An association was observed between telomerase enzyme activity and hTERT genotype with the TT genotype (1.0 • 10 j2 T 4.0 • 10 j3 attomoles (amol) per 10,000 cells; n = 19) having significantly greater telomerase enzyme activity than both the CT (1.3 • 10 j3 T 3.2 • 10 j3 ; n = 30) and CC groups (5.0 • 10 j4 T 3.9 • 10 j3 ; n = 20; P = 0.01).Conclusion-These results indicate that moderate physical activity levels may provide a protective effect on PBMC telomere length compared with both low and high EEE levels. Keywords TELOMERE BIOLOGY; EXERCISE ENERGY EXPENDITURE; hTERT GENOTYPE; GENETICS; DNAPhysical activity (PA) and increased physical fitness are known to decrease the likelihood of morbidity and mortality from a variety of causes (e.g., reduced cardiovascular disease (CVD), insulin resistance, and hypertension) (6), with concomitant increases in longevity (21). Whereas the reduction of disease end points will necessarily increase longevity, whether PA also directly affects cellular aging remains unclear for either rodents or humans (7). Telomere length is a primary biomarker of cellular aging that has recently been associated with CVD (1), insulin resistance and hypertension (14), and morbidity and mortality (9). In the present study, we explored the correlation of PA levels with telomere length and telomerase enzyme activity.Address for correspondence: Stephen M. Roth, Ph.D., 2134 SPH Bldg, Department of Kinesiology, University of Maryland, College Park, MD 20742-2611; E-mail: sroth1@umd.edu.. Telomeres are found on the ends of linear chromosomes and act as a mitotic clock (18), which shortens with every cell division until cellular senescence. Thus, telomeres are considered an important aging biomarker (2,4). Telomeres and their length are not, however, static entities but rather are a dynamic system (4). In certain cells, the ribonucleoprotein, telomerase, maintains and lengthens telomeres, allowing continued mitotic activity without progression to senescence (5). In cells with telomerase activity, telomere length can be maintain...

Purpose-Previous studies have linked an insertion/deletion polymorphism in the angiotensinconverting enzyme (ACE) gene with variability in muscle strength responses to strength training (ST), though conclusions have been inconsistent across investigations. Moreover, most previous studies have not investigated the influence of sex on the association of ACE I/D genotype with muscle phenotypes. The purpose of this study was to investigate the association of ACE genotype with muscle phenotypes before and after ST in older men and women.Methods-Eighty-six inactive men and 139 inactive women, ages 50-85 yr (mean: 62 yr), were studied before and after 10 wk of unilateral knee extensor ST. The one-repetition maximum (1RM) test was used to assess knee extensor muscle strength, and computed tomography was used to measure quadriceps muscle volume (MV). Differences were compared among ACE genotype groups (II vs ID vs DD).Results-Across the entire cohort at baseline, ACE genotype was significantly associated with total lean mass and body weight, with higher values in DD genotype carriers (both P < 0.05). At baseline, DD genotype carriers exhibited significantly greater MV compared with II genotype carriers for both the trained leg (men: 1828 ± 44 vs 1629 ± 70; women: 1299 ± 34 vs 1233 ± 49; P = 0.02) and untrained leg (men: 1801 ± 46 vs 1559 ± 72; women: 1268 ± 36 vs 1189 ± 51; P = 0.01), with no significant genotype × sex interaction. No ACE genotype associations were observed for the 1RM or MV adaptations to ST in either men or women.Conclusions-In the present study, ACE genotype was associated with baseline differences in muscle volume, but it was not associated with the muscle hypertrophic response to ST. Keywords ANGIOTENSIN-CONVERTING ENZYME; GENETICS; MUSCLE MASS; MUSCLE SIZE; SKELETAL MUSCLEMuscle strength and mass are heritable phenotypes, with a heritability range of 14-80% for strength (1,21,28,30) and 20-85% for muscle mass (1,14,24,28). Although the heritability of the adaptation of these muscle phenotypes to strength training (ST) has not been well studied, the adaptive response also appears to have a genetic component (26,27 The purpose of the present study was to investigate the association of ACE genotype with muscle phenotypes before and after ST in older men and women. Though the literature is inconclusive, the biological rationale suggests an advantage for the D allele with regard to muscle phenotypes; thus, we hypothesized that the D allele would be associated with higher values for muscle phenotypes before ST, and greater increases in muscle phenotypes in response to ST. As most studies have investigated only men, we investigated men and women to determine possible sex differences. METHODS SubjectsParticipants in the study consisted of 243 inactive, healthy volunteers between the ages of 50 and 85 yr. For this investigation, "inactive" was operationally defined as a person who performs less than 20 min of vigorous activity per week. Subjects were required to be nonsmokers with no significant car...

DNA is organized into complex three-dimensional chromatin structures, but how this spatial organization regulates gene expression remains a central question. These DNA/chromatin looping structures can range in size from 10-20 kb (enhancers/repressors) to many megabases during intra-and inter-chromosomal interactions. Recently, the influence of telomere length on chromatin organization prior to senescence has revealed the existence of long-distance chromatin loops that dictate the expression of genes located up to 10 Mb from the telomeres (Telomere Position Effect-Over Long Distances [TPE-OLD]). Here, we demonstrate the existence of a telomere loop at the 4q35 locus involving the sorbin and SH3 domain-containing protein 2 gene, SORBS2, a skeletal muscle protein using a modification of the chromosome conformation capture method. The loop reveals a cis-acting mechanism modifying SORBS2 transcription. The expression of this gene is altered by TPE-OLD in myoblasts from patients affected with the age-associated genetic disease, facioscapulohumeral muscular dystrophy (FSHD1A, MIM 158900). SORBS2 is expressed in FSHD myoblasts with short telomeres, while not detectable in FSHD myoblasts with long telomeres or in healthy myoblasts regardless of telomere length. This indicates that TPE-OLD may modify the regulation of the 4q35 locus in a pathogenic context. Upon differentiation, both FSHD and healthy myotubes express SORBS2, suggesting that SORBS2 is normally up-regulated by maturation/differentiation of skeletal muscle and is misregulated by TPE-OLD-dependent variegation in FSHD myoblasts. These findings provide additional insights for the complexity and age-related symptoms of FSHD.

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer.

Performance on the Sternberg working memory task, and MEG cortical response on a variation of the Sternberg task were examined in middle-aged carriers and non-carriers of the APOE epsilon4 allele. Physical activity was also assessed to examine whether exercise level modifies the relationship between APOE genotype and neurocognitive function. Regression revealed that high physical activity was associated with faster RT in the six- and eight-letter conditions of the Sternberg in epsilon4 carriers, but not in the non-carriers after controlling for age, gender, and education (N=54). Furthermore, the MEG analysis revealed that sedentary epsilon4 carriers exhibited lower right temporal lobe activation on matching probe trials relative to high-active epsilon4 carriers, while physical activity did not distinguish non-carriers (N=23). The M170 peak was identified as a potential marker for pre-clinical decline as epsilon4 carriers exhibited longer M170 latency, and highly physically active participants exhibited greater M170 amplitude to matching probe trials.