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.

The strengths and limitations of the major methods developed to measure telomere lengths (TLs) in cells and tissues are presented in this review. These include Q-PCR (uantitative olymerasehain eaction), TRF (erminal estrictionragment) analysis, a variety of Q-FISH (uantitative luorescencen ituybridization) methods, STELA (ingle lomereength nalysis) and TeSLA (lomere hortestength ssay). For each method, we will cover information about validation studies, including reproducibility in independent laboratories, accuracy, reliability and sensitivity for measuring not only the average but also the shortest telomeres. There is substantial evidence that it is the shortest telomeres that trigger DNA damage responses leading to replicative senescence in mammals. However, the most commonly used TL measurement methods generally provide information on average or relative TL, but it is the shortest telomeres that leads to telomere dysfunction (identified by TIF,elomere dysfunction nducedoci) and limit cell proliferation in the absence of a telomere maintenance mechanism, such as telomerase. As the length of the shortest telomeres is a key biomarker determining cell fate and the onset of senescence, a new technique (TeSLA) that provides quantitative information about all the shortest telomeres will be highlighted.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.

Improved methods to measure the shortest (not just average) telomere lengths (TLs) are needed. We developed 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. TeSLA improves the specificity and efficiency of TL measurements that is facilitated by user friendly image-processing software to automatically detect and annotate band sizes, calculate average TL, as well as the percent of the shortest telomeres. Compared with other TL measurement methods, TeSLA provides more information about the shortest telomeres. The length of telomeres was measured longitudinally in peripheral blood mononuclear cells during human aging, in tissues during colon cancer progression, in telomere-related diseases such as idiopathic pulmonary fibrosis, as well as in mice and other organisms. The results indicate that TeSLA is a robust method that provides a better understanding of the shortest length of telomeres.

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.

It is generally recognized that the function of the immune system declines with increased age and one of the major immune changes is impaired T‐cell responses upon antigen presentation/stimulation. Some “high‐performing” centenarians (100+ years old) are remarkably successful in escaping, or largely postponing, major age‐related diseases. However, the majority of centenarians (“low‐performing”) have experienced these pathologies and are forced to reside in long‐term nursing facilities. Previous studies have pooled all centenarians examining heterogeneous populations of resting/unstimulated peripheral blood mononuclear cells (PBMCs). T cells represent around 60% of PBMC and are in a quiescence state when unstimulated. However, upon stimulation, T cells rapidly divide and exhibit dramatic changes in gene expression. We have compared stimulated T‐cell responses and identified a set of transcripts expressed in vitro that are dramatically different in high‐ vs. low‐performing centenarians. We have also identified several other measurements that are different between high‐ and low‐performing centenarians: (a) The amount of proliferation following in vitro stimulation is dramatically greater in high‐performing centenarians compared to 67‐ to 83‐year‐old controls and low‐performing centenarians; (b) telomere length is greater in the high‐performing centenarians; and (c) telomerase activity following stimulation is greater in the high‐performing centenarians. In addition, we have validated a number of genes whose expression is directly related to telomere length and these are potential fundamental biomarkers of aging that may influence the risk and progression of multiple aging conditions.