Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.

Telomeres and telomerase: three decades of progress.

Many recent advances have emerged in the telomere and telomerase fields. This Timeline article highlights the key advances that have expanded our views on the mechanistic underpinnings of telomeres and telomerase and their roles in ageing and disease. Three decades ago, the classic view was that telomeres protected the natural ends of linear chromosomes and that telomerase was a specific telomere-terminal transferase necessary for the replication of chromosome ends in single-celled organisms. While this concept is still correct, many diverse fields associated with telomeres and telomerase have substantially matured. These areas include the discovery of most of the key molecular components of telomerase, implications for limits to cellular replication, identification and characterization of human genetic disorders that result in premature telomere shortening, the concept that inhibiting telomerase might be a successful therapeutic strategy and roles for telomeres in regulating gene expression. We discuss progress in these areas and conclude with challenges and unanswered questions in the field.

Telomere extension occurs at most chromosome ends and is uncoupled from fill-in in human cancer cells.

Telomeres are thought to be maintained by the preferential recruitment of telomerase to the shortest telomeres. The extension of the G-rich telomeric strand by telomerase is also believed to be coordinated with the complementary synthesis of the C strand by the conventional replication machinery. However, we show that under telomere length-maintenance conditions in cancer cells, human telomerase extends most chromosome ends during each S phase and is not preferentially recruited to the shortest telomeres. Telomerase rapidly extends the G-rich strand following telomere replication but fill-in of the C strand is delayed into late S phase. This late C-strand fill-in is not executed by conventional Okazaki fragment synthesis but by a mechanism using a series of small incremental steps. These findings highlight differences between telomerase actions during steady state versus nonequilibrium conditions and reveal steps in the human telomere maintenance pathway that may provide additional targets for the development of anti-telomerase therapeutics.

Telomere position effect: regulation of gene expression with progressive telomere shortening over long distances.

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.

Catalysis-dependent inactivation of human telomerase and its reactivation by intracellular telomerase-activating factors (iTAFs).

Human telomerase maintains genome stability by adding telomeric repeats to the ends of linear chromosomes. Although previous studies have revealed profound insights into telomerase functions, the low cellular abundance of functional telomerase and the difficulties in quantifying its activity leave its thermodynamic and kinetic properties only partially characterized. Employing a stable cell line overexpressing both the human telomerase RNA component and the N-terminally biotinylated human telomerase reverse transcriptase and using a newly developed method to count individual extension products, we demonstrate here that human telomerase holoenzymes contain fast- and slow-acting catalytic sites. Surprisingly, both active sites became inactive after two consecutive rounds of catalysis, named single-run catalysis. The fast active sites turned off ∼40-fold quicker than the slow ones and exhibited higher affinities to DNA substrates. In a dimeric enzyme, the two active sites work in tandem, with the faster site functioning before the slower one, and in the monomeric enzyme, the active sites also perform single-run catalysis. Interestingly, inactive enzymes could be reactivated by intracellular telomerase-activating factors (iTAFs) from multiple cell types. We conclude that the single-run catalysis and the iTAF-triggered reactivation serve as an unprecedented control circuit for dynamic regulation of telomerase. They endow native telomerase holoenzymes with the ability to match their total number of active sites to the number of telomeres they extend. We propose that the exquisite kinetic control of telomerase activity may play important roles in both cell division and cell aging.

2D gel electrophoresis reveals dynamics of t-loop formation during the cell cycle and t-loop in maintenance regulated by heterochromatin state.

Linear chromosome ends are capped by telomeres that have been previously reported to adopt a t-loop structure. The lack of simple methods for detecting t-loops has hindered progress in understanding the dynamics of t-loop formation and its function in protecting chromosome ends. Here, we employed a classical two-dimensional agarose gel method (2D gel method) to innovatively apply to t-loop detection. Briefly, restriction fragments of genomic DNA were separated in a 2D gel, and the telomere sequence was detected by in-gel hybridization with telomeric probe. Using this method, we found that t-loops are present throughout the cell cycle, and t-loop formation tightly couples to telomere replication. We also observed that t-loop abundance positively correlates with chromatin condensation, i.e. cells with less compact telomeric chromatin (ALT cells and trichostatin A (TSA)-treated HeLa cells) exhibited fewer t-loops. Moreover, we observed that telomere dysfunction-induced foci, ALT-associated promyelocytic leukemia bodies, and telomere sister chromatid exchanges are activated upon TSA-induced loss of t-loops. These findings confirm the importance of the t-loop in protecting linear chromosomes from damage or illegitimate recombination.

Early and late steps in telomere overhang processing in normal human cells: the position of the final RNA primer drives telomere shortening.

Telomere overhangs are essential for telomere end protection and telomerase extension, but how telomere overhangs are generated is unknown. Leading daughter strands synthesized by conventional semiconservation DNA replication are initially blunt, while lagging daughter strands are shorter by at least the size of the final RNA primer, which is thought to be located at extreme chromosome ends. We developed a variety of new approaches to define the steps in the processing of these overhangs. We show that the final lagging RNA primer is not terminal but is randomly positioned ~70-100 nucleotides from the ends and is not removed for more than an hour. This identifies an important intrinsic step in replicative aging. Telomeric termini are processed in two distinct phases. During the early phase, which occupies 1-2 h following replication of the duplex telomeric DNA, several steps occur on both leading and lagging daughters. Leading telomere processing remains incomplete until late S/G2, when the C-terminal nucleotide is specified-referred to as the late phase. These observations suggest the presence of previously unsuspected complexes and signaling events required for the replication of the ends of human chromosomes.

Regulation of the Human Telomerase Gene TERT by Telomere Position Effect-Over Long Distances (TPE-OLD): Implications for Aging and Cancer.

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.

Processive and distributive extension of human telomeres by telomerase under homeostatic and nonequilibrium conditions.

Specific information about how telomerase acts in vivo is necessary for understanding telomere dynamics in human tumor cells. Our results imply that, under homeostatic telomere length-maintenance conditions, only one molecule of telomerase acts at each telomere during every cell division and processively adds ∼60 nt to each end. In contrast, multiple molecules of telomerase act at each telomere when telomeres are elongating (nonequilibrium conditions). Telomerase extension is less processive during the first few weeks following the reversal of long-term treatment with the telomerase inhibitor Imetelstat (GRN163L), a time when Cajal bodies fail to deliver telomerase RNA to telomeres. This result implies that processing of telomerase by Cajal bodies may affect its processivity. Overexpressed telomerase is also less processive than the endogenously expressed telomerase. These findings reveal two major distinct extension modes adopted by telomerase in vivo.

Alternative lengthening of telomeres can be maintained by preferential elongation of lagging strands.

Alternative lengthening of telomeres (ALT) is a telomerase independent telomere maintenance mechanism that occurs in ∼15% of cancers. The potential mechanism of ALT is homology-directed telomere synthesis, but molecular mechanisms of how ALT maintains telomere length in human cancer is poorly understood. Here, we generated TERC (telomerase RNA) gene knockouts in telomerase positive cell lines that resulted in long-term surviving clones acquiring the ALT pathway but at a very low frequency. By comparing these ALT cells with parental telomerase positive cells, we observed that ALT cells possess excessively long telomeric overhangs derived from telomere elongation processes that mostly occur during S phase. ALT cells exhibited preferential elongation of the telomeric lagging strands, whereas telomerase positive cells exhibited similar elongation between leading and lagging strands. We propose that the ALT pathway preferentially occurs at telomeric lagging strands leading to heterogeneous telomere lengths observed in most ALT cancers.

SORBS2 transcription is activated by telomere position effect-over long distance upon telomere shortening in muscle cells from patients with facioscapulohumeral dystrophy.

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.

Alternative splicing regulation of telomerase: a new paradigm?

Alternative splicing affects approximately 95% of eukaryotic genes, greatly expanding the coding capacity of complex genomes. Although our understanding of alternative splicing has increased rapidly, current knowledge of splicing regulation has largely been derived from studies of highly expressed mRNAs. Telomerase is a key example of a protein that is alternatively spliced, but it is expressed at very low levels and although it is known that misregulation of telomerase splicing is a hallmark of nearly all cancers, the details of this process are unclear. Here we review work showing that hTERT expression is in part regulated by atypical alternative splicing, perhaps due to its exceptionally low expression level. We propose that these differential regulatory mechanisms may be widely applicable to other genes and may provide new opportunities for the development of cancer therapeutics.

Impaired telomere maintenance in Alazami syndrome patients with LARP7 deficiency.

BACKGROUND: Loss of function in genes required for telomere maintenance result in disorders known as telomeropathies, which are characterized by a pattern of symptoms including generalized and specific lymphocytopenias as well as very short telomere length and disease anticipation. METHODS: Because human LARP7 is the most likely ortholog of the Tetrahymena p65 protein, which is required for telomerase activity in that organism, we investigated the effects of LARP7 silencing in human cells as well as in two distinct families with Alazami syndrome (loss of function of LARP7). RESULTS: Depletion of LARP7 caused a reduction in telomerase enzymatic activity and progressively shorter telomeres in human cancer cell lines. Alazami syndrome patients from two separate cohorts exhibited very short lymphocyte telomeres. Further, wild-type offspring of LARP7 mutant individuals also had very short telomeres, comparable to what is observed in telomerase (hTERT) mutant cohorts. CONCLUSIONS: Together, these experiments demonstrate that in addition to the readily apparent developmental disorder associated with LARP7 deficiency, an underlying telomeropathy exists even in unaffected siblings of these individuals.

Human CST promotes telomere duplex replication and general replication restart after fork stalling.

Mammalian CST (CTC1-STN1-TEN1) associates with telomeres and depletion of CTC1 or STN1 causes telomere defects. However, the function of mammalian CST remains poorly understood. We show here that depletion of CST subunits leads to both telomeric and non-telomeric phenotypes associated with DNA replication defects. Stable knockdown of CTC1 or STN1 increases the incidence of anaphase bridges and multi-telomeric signals, indicating genomic and telomeric instability. STN1 knockdown also delays replication through the telomere indicating a role in replication fork passage through this natural barrier. Furthermore, we find that STN1 plays a novel role in genome-wide replication restart after hydroxyurea (HU)-induced replication fork stalling. STN1 depletion leads to reduced EdU incorporation after HU release. However, most forks rapidly resume replication, indicating replisome integrity is largely intact and STN1 depletion has little effect on fork restart. Instead, STN1 depletion leads to a decrease in new origin firing. Our findings suggest that CST rescues stalled replication forks during conditions of replication stress, such as those found at natural replication barriers, likely by facilitating dormant origin firing.

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.

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.

Exome Sequencing of Normal and Isogenic Transformed Human Colonic Epithelial Cells (HCECs) Reveals Novel Genes Potentially Involved in the Early Stages of Colorectal Tumorigenesis.

BACKGROUND: We have generated a series of isogenically derived immortalized human colonic epithelial cell (HCEC 1CT and HCEC 2CT) lines, including parental un-immortalized normal cell strains. The CDK4 and hTERT immortalized colonic epithelial cell line (HCEC 1CT) is initially karyotypically normal diploid and expresses a series of epithelial cell markers including stem cell markers. Under stressful tissue culture conditions, a spontaneous aneuploidy event occurred in the HCEC 1CT line, resulting in a single chromosomal change leading to a stable trisomy 7 cell line (1CT7). Trisomy 7 occurs in about 40% of all benign human adenomas (polyps) and thus this specific chromosomal change in diploid HCEC 1CT cells appears to be non random. In addition, we have partially transformed the HCEC 1CT line by introducing stable knockdown of wild type APC and TP53, and ectopically introducing a mutant Krasv12 and a mutant version of APC (A1309), all commonly found mutations in colorectal cancer (CRC). METHODS: Whole exome sequencing and bioinformatic analyses were performed to comprehensively examine the genetic background of these isogenic cell lines. RESULTS: Exome sequencing of these experimentally progressed cell lines recapitulates a list of genes previously reported to be involved in CRC tumorigenesis. In addition, sequencing revealed a collection of novel genes specifically detected in 1CT7 and A1309 cells but not normal diploid 1CT cells. CONCLUSION: This study demonstrates the utility of using isogenic experimentally derived HCEC lines as a model to recapitulate CRC initiation and progression. Exome sequencing reveals a collection of novel genes that may play important roles in CRC tumorigenesis.

Tissue culture as a hostile environment: identifying conditions for breast cancer progression studies.

The cell culture environment (substrate, atmosphere, and medium) can have a significant influence on the characteristics of cells that propagate from clinical samples. In this issue of Cancer Cell, Ince and colleagues report improved conditions for the culture of primary human breast epithelial cells. They demonstrate that, when cells cultured using the new conditions are experimentally transformed, they are more tumorigenic, form tumor xenografts that closely resemble human breast ductal adenocarcinoma, and are more metastatic compared to cells cultured under standard conditions similarly transformed. This suggests that pre-existing differences in cell culture can modulate the tumor phenotype.

Targeting of Nrf2 induces DNA damage signaling and protects colonic epithelial cells from ionizing radiation.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for antioxidant and anti-inflammation enzymes that binds to its endogenous inhibitor protein, Kelch-like ECH (erythroid cell-derived protein with CNC homology)-associated protein 1, in the cytoplasm under normal conditions. Various endogenous or environmental oxidative stresses, such as ionizing radiation (IR), can disrupt the Nrf2-Kelch-like ECH-associated protein 1 complex. This allows Nrf2 to translocate from the cytoplasm into the nucleus to induce transcription of heme oxygenase-1 and other cytoprotective enzymes through binding to antioxidant responsive elements. However, how Nrf2 protects cells from IR-induced damage remains unclear. Here, we report that Nrf2 activation by the synthetic triterpenoids, bardoxolone methyl (BARD) and 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide, protects colonic epithelial cells against IR-induced damage, in part, by enhancing signaling of the DNA damage response. Pretreatment with BARD reduced the frequency of both G1 and S/G2 chromosome aberrations and enhanced the disappearance of repairosomes (C-terminal binding protein interacting protein, Rad51, and p53 binding protein-1 foci) after IR. BARD protected cells from IR toxicity in a Nrf2-dependent manner. The p53 binding protein-1 promoter contains three antioxidant responsive elements in which Nrf2 directly binds following BARD treatment. In addition, 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide provided before exposure to a lethal dose of whole-body irradiation protected WT mice from DNA damage and acute gastrointestinal toxicity, which resulted in improved overall survival. These results demonstrate that Nrf2 activation by synthetic triterpenoids is a promising candidate target to protect the gastrointestinal tract against acute IR in vitro and in vivo.

Branching morphogenesis of immortalized human bronchial epithelial cells in three-dimensional culture.

While mouse models have contributed in our understanding of lung development, repair and regeneration, inherent differences between the murine and human airways requires the development of new models using human airway epithelial cells. In this study, we describe a three-dimensional model system using human bronchial epithelial cells (HBECs) cultured on reconstituted basement membrane. HBECs form complex budding and branching structures on reconstituted basement membrane when co-cultured with human lung fetal fibroblasts. These structures are reminiscent of the branching epithelia during lung development. The HBECs also retain markers indicative of epithelial cell types from both the central and distal airways suggesting their multipotent potential. In addition, we illustrate how the model can be utilized to understand respiratory diseases such as lung cancer. The 3D novel cell culture system recapitulates stromal-epithelial interactions in vitro that can be utilized to understand important aspects of lung development and diseases.

Receptor-interacting protein kinase 2 promotes triple-negative breast cancer cell migration and invasion via activation of nuclear factor-kappaB and c-Jun N-terminal kinase pathways.

INTRODUCTION: Metastasis is the main cause of breast cancer morbidity and mortality. Processes that allow for tumor cell migration and invasion are important therapeutic targets. Here we demonstrate that receptor-interacting protein kinase 2 (RIP2), a kinase known to be involved in inflammatory processes, also has novel roles in cancer cell migration and invasion. METHODS: A total of six breast cancer expression databases, including The Cancer Genome Atlas, were assessed for RIP2 expression among various clinical subtypes and its role as a prognostic biomarker. mRNA fluorescence in situ hybridization (FISH) for RIP2 was performed on 17 stage III breast cancers to determine if there was a correlation between RIP2 expression and lymph node involvement. RNA-interference was used to knock-down RIP2 expression in MDA-MB-231, Htb126, SUM149PT, MCF7, T47D, and HCC1428 cells. Cell migration and invasion were measured in vitro by scratch/wound healing and transwell migration assays. A xenograft mouse model was used to assess tumor growth and chemosensitivity to docetaxel in vivo in MDA-MB-231 cells with and without RIP2 small hairpin RNA knockdown. Western blot and immunofluorescence imaging were used to evaluate protein expressions. RESULTS: Interrogation of expression databases showed that RIP2 expression is significantly over-expressed in triple-negative breast cancers (TNBC: estrogen-receptor (ER) negative, progesterone-receptor (PR) negative, Her2/neu- (Her2) negative), compared to other clinical subtypes. High RIP2 expression correlates with worse progression-free survival using a combined breast cancer expression array dataset consisting of 946 patients. Multivariate analysis shows RIP2 as an independent prognostic biomarker. Knock-down of RIP2 significantly decreases migration in both scratch/wound healing and transwell migration assays in MDA-MB-231, Htb126, SUM149PT, MCF7, and T47D cells and is correlated with decreased Nuclear Factor-kappaB and c-Jun N-terminal kinase (JNK) activation. Finally, RIP2 knock-down leads to increased sensitivity to docetaxel and decreased tumor mass and lung metastases in a xenograft mouse model. CONCLUSION: These results highlight RIP2 as a pro-metastasis kinase in patients with advanced breast cancer. These results also illustrate a novel role for this kinase in addition to its known role in inflammation, and suggest that targeting RIP2 may improve outcomes in advanced breast cancer patients, in which it is overexpressed.