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7.7: Telomeres - Biology

7.7: Telomeres - Biology


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If there is a mechanism for recognizing loose ends of DNA, what about the ends of every eukaryotic chromosome? They are linear chromosomes, so they have ends, right? What prevents the double-strand-break repair systems from mis-recognizing them all as broken DNA and concatenating all of the chromosomes together? Interestingly, the answer to this question is intimately tied up with the answer to the problem of end- replication, which was very briefly alluded to in our description of replication.

The end-replication problem is one that affects all linear chromosomes. It boils down to one simple fact: an RNA primer is needed to start any DNA replication. So on the 5’ end of each strand is an RNA primer (Figure (PageIndex{24}) in yellow) that gets removed by the error-correction process. Thus with every round of replication, information is lost from the 5’ end of each strand of each chromosome.

Eventually, crucial genes are lost and the cell will die; most likely many cellular functions will be compromised long before that happens. The solution to the end-replication problem might be considered more a treatment of symptoms than a cure, to use an analogy to medicine. In short, during the very early stages of an organism’s life, a lot of non-coding DNA is added onto the ends of the DNA so that as the cell and its progeny continue to reproduce, the nucleotides do not affect any functional genes. This process is catalyzed by the enzyme telomerase.

Telomerase is a large holoenzyme that acts as a reverse transcriptase, reading a self-contained RNA template to add on telomere sequence to the 3’ ends of linear chromosomes. Note that this does not add onto the 5’ ends - as mentioned above, there is nothing to be done about the 5’ ends directly. However, while telomerases are active, the 3’ ends can be extended, and thus when the 5’ end primer is removed, the sequence lost (forever) from that strand of DNA is only a telomeric repeat and not something more useful. The repeats are well conserved across eukaryotes, and almost completely conserved across mammalian species (shown in Figure (PageIndex{25})).

In metazoans, telomerase activity is high in the embryonic stages of life, but is virtually non-existent in adults except in cell types that must constantly proliferate (e.g. blood and epithelial cells). The activity of telomerase is primarily regulated by expression of the TERT gene (telomerase reverse transcriptase) although construction of the full telomerase also requires the expression of the TERC gene (the telomerase RNA, also abbreviated TR), and dyskerin. Roughly speaking, the number of telomeric repeats that are placed on a chromosome in early development determines the number of DNA replications and cell divisions that the cell can undergo before succumbing to apoptosis (programmed cell death). Experiments on cells in culture demonstrate a strong correlation between telomere length and longevity, and it is known that cells taken from people with the premature aging disease, progeria, have relatively short telomeres.

Conversely, cancer cells almost universally have unregulated expression of telomerase. Given that a defining characteristic of cancer cells is the ability to proliferate rapidly and indefinitely, turning telomerase back on is, not surprisingly, an important aspect of carcinogenesis. It is therefore a target for anti-cancer treatments; however, to date no telomerase-targeting therapies have proven effective.

Now that we know about telomeres, the question that started this section becomes even more problematic: with these repeated sequence overhangs, how are chromosomes prevented from connecting end-to-end through a double-strand repair-like process? In part due to their repeated sequences, telomeres are able to form end-caps and protect chromosomal ends. The telomeres protect the ends of each chromosome by binding to protective proteins and by forming complex structures. Telomere end binding proteins (TEBP) bind to the 3’ overhanging end of the telomere. Other capping proteins, such as the mammalian TRF1 and TRF2 (telomere repeat binding factors) not only bind the telomere, but help to organize it into large looped structures known as T-loops (Figure (PageIndex{26})).

Finally, the T-loop ends are further stabilized by the formation of G-quartets (Figure (PageIndex{27})). G-quartets are a cyclic tetramers that can form in sequences with four consecutive guanine residues, which hydrogen-bond to each other to make a linked square shape stabilized by a metal ion in the center. Furthermore in cases like the telomere, in which such sequences are repeated, the G-quartets can stack and associate three-dimensionally, increasing their stability.


Save Your Telomeres

Since their discovery more than 75 years ago by the Nobel Laureate geneticist Hermann Müller, telomeres have attracted worldwide attention among scientists investigating the aging process.

Telomeres are the protective caps on the ends of chromosomes composed of short DNA sequences protecting our DNA and genetic material from damage. Another Nobel Laureate, Elizabeth Blackburn, likened telomeres to the little plastic caps on the ends of shoelaces (aglets).

Under normal conditions, when a cell divides, telomeres shorten. If they grow too short, they reach what’s known as the Hayflick limit (named after the esteemed gerontologist Leonard Hayflick), and the protective capacity of the telomere decreases. Real-world relevance of telomere shortening can be observed during the aging process in humans when comparing the length of telomeres from newborns (8,000 base pairs) to adults (3,000 base pairs) to elderly individuals (1,500 base pairs).

Therefore, because several disease states and pathological processes have been linked to telomere shortening, several academic laboratories and companies have explored intervention strategies to slow down the rate of telomere attrition.

Several lifestyle factors can also significantly affect telomere health and the rate of telomere shortening. Among the most studied factors associated with shorter telomeres are psychosocial: depression, anxiety, and childhood adversity (1,2). Other lifestyle factors associated with telomere length include smoking, physical activity, drugs and toxins, and oxidative stress. Indeed, the decades of research implicating oxidative stress in the aging process has recently begun addressing and demonstrating a similarly deleterious role of oxidative stress on telomere length (3-5). As a result, antioxidant intake and subsequent plasma concentrations may be newly emerging biomarkers of telomere status (6-8).

Oxidative stress is defined as an overabundance of reactive oxygen species (ROS). Excessive oxidative stress damages DNA, proteins, and lipids. While ROS are produced under normal conditions, oxidative stress occurs under conditions of poor health. To prevent oxidative stress, the body requires antioxidant nutrients such as glutathione precursors like the amino acid cysteine (found in IsaLean® Shakes) along with specific enzymes (9,10). Among these antioxidant enzymes is catalase, which functions to convert toxic and DNA-damaging hydrogen peroxide (H2O2) into water (11,12).

With this is mind, Isagenix partnered with scientists in the School of Nutrition and Health Promotion at Arizona State University to conduct a clinical evaluation in an independent, randomized, placebo-controlled, double-blinded study to evaluate Product B’s effect on catalase and other enzymes. Third-generation Product B contains a proprietary blend of plant botanicals, antioxidants, and other bioactives that provide significant protection against telomere shortening in cellular systems.

However, obtaining clinical support as a potential natural product against telomere shortening proved difficult due to analytical shortcomings and methodological issues in measuring telomere length. For this reason most studies considering the impact of dietary, environmental, or lifestyle factors on telomeres are frequently observed in studies with thousands of study participants.

In this study researchers had subjects consume either Product B or a placebo for 12 weeks and observed that subjects supplemented with Product B demonstrated a significant elevation of catalase in red blood cells (increase by 30 percent vs. placebo). Considering the role catalase may play in the aging process, this was very exciting news and the relevance of this finding was not lost on the researchers, who commented, “The increase in catalase observed by Product B is an exciting development considering the relationship between the enzyme and increased lifespan in animal studies.”

Now, Isagenix has developed fourth generation Product B® IsaGenesis®, which contains a novel lipid-soluble blend to increase absorption and bioavailability.

Clinical and experimental evidence is slowly emerging supporting the benefits of the nutritional antioxidants, plant botanicals, and other bioactives provided by Ageless Essentials™ Daily Pack, containing Product B IsaGenesis, IsaOmega Supreme®, C-Lyte®, Essentials for Men™ or Essentials for Women™, and Ageless Actives™.

In conjunction with a healthy diet, weight and stress management, quality and sufficient sleep, and regular exercise, Product B IsaGenesis may provide the most optimal protection against age-accelerated telomere shortening and a longer, healthier life.

Puterman E et al. Determinants of telomere attrition over 1 year in healthy older women: stress and health behaviors matter. Mol Psychiatr 2014 Jul 29. doi: 10.1038/mp.2014.70. [Epub ahead of print].

Shalev I et al. Stress and telomere biology: a lifespan perspective. Psychoneuroendocrinology 201338:1835-42.

Correia-Melo C et al. Telomeres, oxidative stress and infl ammatory factors: partners in cellular senescence? Longev Healthspan 20143:1. doi: 10.1186/2046-2395-3-1.

Demissie S et al. Insulin resistance, oxidative stress, hypertension, and leukocyte telomere length in men from the Framingham Heart Study. Aging Cell 20065:325-30.

Salpea KD et al. Association of telomere length with type 2 diabetes, oxidative stress and UCP2 gene variation. Atherosclerosis 2010209:42-50.

Xu Q et al. Multivitamin use and telomere length in women. Am J Clin Nutr 200989:1857-63.

Tiainen AM et al. Leukocyte telomere length and its relation to food and nutrient intake in an elderly population. Eur J Clin Nutr 201266:1290-4.

Paul L. Diet, nutrition and telomere length. J Nutr Biochem 201122:895-901.

Cutler RG. Oxidative stress and aging: catalase is a longevity determinant enzyme. Rejuvenation Res 20058:138-40.

Schriner SE et al. Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 2005308:1909-11.

Woo SR et al. Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres. Biochem Biophys Res Commun 2012417:204-10.

Linn S. DNA damage by iron and hydrogen peroxide in vitro and in vivo. Drug Metab Rev 199830:313-26.


Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1

When telomeres are rendered dysfunctional through replicative attrition of the telomeric DNA or by inhibition of shelterin, cells show the hallmarks of ataxia telangiectasia mutated (ATM) kinase signalling. In addition, dysfunctional telomeres might induce an ATM-independent pathway, such as ataxia telangiectasia and Rad3-related (ATR) kinase signalling, as indicated by the phosphorylation of the ATR target CHK1 in senescent cells and the response of ATM-deficient cells to telomere dysfunction. However, because telomere attrition is accompanied by secondary DNA damage, it has remained unclear whether there is an ATM-independent pathway for the detection of damaged telomeres. Here we show that damaged mammalian telomeres can activate both ATM and ATR and address the mechanism by which the shelterin complex represses these two important DNA damage signalling pathways. We analysed the telomere damage response on depletion of either or both of the shelterin proteins telomeric repeat binding factor 2 (TRF2) and protection of telomeres 1 (POT1) from cells lacking ATM and/or ATR kinase signalling. The data indicate that TRF2 and POT1 act independently to repress these two DNA damage response pathways. TRF2 represses ATM, whereas POT1 prevents activation of ATR. Unexpectedly, we found that either ATM or ATR signalling is required for efficient non-homologous end-joining of dysfunctional telomeres. The results reveal how mammalian telomeres use multiple mechanisms to avoid DNA damage surveillance and provide an explanation for the induction of replicative senescence and genome instability by shortened telomeres.


Characteristic structural features

The chromodomain superfamily, which contains the HP1 family, can be subdivided into three major classes on the basis of domain organization [13]. One class, characterized by the presence of a single chromodomain, includes Polycomb and mammalian modifier 3. A second class is identified by paired tandem chromodomains, as found in DNA-binding/helicase proteins, such as yeast CHD1 and mammalian CHD-1 to CHD-4. The third class consists of proteins containing both a chromodomain and the highly related chromoshadow domain, which includes all members of the HP1 family.

The sequence and structure of HP1 proteins can be divided into three regions (Figure 2b). First, the chromodomain is a module at the amino terminus that is responsible for HP1 binding to di- and trimethylated lysine 9 (K9 in the single-letter amino-acid code) of histone H3 these methyl groups are epigenetic marks for gene silencing [15, 16]. Second, the carboxy-terminal chromoshadow domain is involved in homo- and/or heterodimerization and interaction with other proteins. Third, the chromodomain is separated from the chromoshadow domain by a variable linker or hinge region containing a nuclear localization sequence. Each of these three segments will be discussed in detail from a structural perspective.

The chromodomain

The structure of the amino-terminal chromodomain alone has been analyzed by nuclear magnetic resonance spectroscopy [17]. The domain folds into a globular conformation approximately 30 Å in diameter, consisting of an antiparallel three-stranded β sheet packed against an α helix in the carboxy-terminal segment of the domain [17] (Figure 2c). A hydrophobic groove is formed on one side of the β sheet, which is composed of conserved nonpolar residues. Interestingly, comparison of this structure with the databases reveals a similar structure in two archaeal histone-like proteins, Sac7d and Sso7d [17]. This structure in Sac7d binds to the major groove of DNA in a nonspecific manner as a result of the net positive charge on the exterior of the β sheet. Unlike these archaeal DNA-binding proteins, however, in HP1 the β sheet has an overall negative charge, implicating the chromodomain as a protein-interaction motif rather than a DNA-binding motif.

The gene-silencing function of HP1 depends on an interaction between the chromodomain and the methyl K9 histone H3 mark [12, 18]. The hydrophobic pocket of the chromodomain provides the appropriate environment for docking onto this methylated residue. The bound segment of the H3 tail adopts a β-strand conformation, lying coplanar to and antiparallel with two β strands of the chromodomain, which completes a three-stranded β sheet [19, 20]. In addition, the methylammonium group in K9 is effectively caged by three aromatic side chains, whereas the surrounding residues of K9 contact specific sites within the chromodomain. This positioning makes sense of the functional defects and loss of methyl K9 binding upon mutation of key hydrophobic amino acids located in the amino-terminal part of Drosophila HP1 (Tyr24, Val26, Trp45 and Tyr48) [20]. Interestingly, no other combinations of naturally occurring amino acids have been found that interact with the chromodomain, indicating that the methylated histone mark is the sole binding partner for this domain [21].

Methylation occurs on other lysines within histone H3, as well as the other histones. In fact, methylation on K27 of H3 occurs within a highly similar amino-acid sequence context as K9 - ARKS. This mark on K27 serves as a binding site for the Polycomb chromodomain [22]. The discrimination between these two highly related repressive marks has been examined [23]. The chromodomains of HP1 and Polycomb are structured similarly, but their peptide-binding grooves show distinct features that provide this discrimination. The main differences lie in the extent of protein-peptide interactions - Polycomb interacts with a larger number of the peptide residues surrounding the methyl lysine - and in context recognition, as HP1 finely discriminates the peptide residues in the immediate vicinity. Therefore, although the posttranslational mark, the surrounding histone sequence and the overall chromodomain structure are strikingly similar between them, the mode in which Polycomb and HP1 bind histone H3 and make essential interacting contacts are different.

The chromoshadow domain

The overall structure of the chromoshadow domain is very similar to that of the chromodomain, with a globular conformation of approximately the same size [24] (Figure 2c). Like the chromodomain, the chromoshadow domain is composed of three β strands to complete an antiparallel sheet. Unlike the chromodomain, which has a subsequent single α helix that folds against the sheet, the chromodomain has two carboxy-terminal α helices.

Although the chromodomain remains monomeric in solution, the chromoshadow domain readily dimerizes under the same conditions [25]. The dimer interface involves a symmetrical interaction on helix α2, which lies at an angle of 35° to helix α2 of the other HP1 molecule [24]. Conserved residues that are unique to the chromoshadow domain are located at the dimer interface. As a result, this dimer structure creates a nonpolar groove that can accommodate HP1-interacting proteins containing the consensus sequence PXVXL [24] (see below).

The linker region

The two highly conserved chromo- and chromoshadow domains are separated by a less conserved linker or hinge region. This region contains the most variable amino-acid sequence between HP1 proteins, between proteins both from the same species and from different species. The structure of the linker region has been proposed to be flexible and exposed to the surface [26]. The variable nature of this region has been resulted in some difficulty in capturing its three-dimensional structure with a variety of methods.

The linker is highly amenable to posttranslational modifications, especially phosphorylation [27–30]. In addition, modifications within this region have been shown to affect localization, interactions and function. The linker could therefore be a central control region in the regulation of HP1 proteins.


We studied telomere length in 32 CML patients who discontinued imatinib after achieving complete molecular remission and 32 age-sex-matched controls. The relative telomere length (RTL) was determined by q-PCR as the telomere to single copy gene (36B4) ratio normalized to a reference sample (K-562 DNA). Age-corrected RTL (acRTL) was also obtained. The 36-month probability of treatment-free remission (TFR) was 59.4 %. TFR patients showed shorter acRTL compared to relapsed (mean ± SD =𠂐.01 ±𠂐.14 vs 0.20 ±𠂐.21 p =𠂐.01). TFR was significantly higher in CML patients with acRTL 𢙀.09 (78.9 vs 30.8 %, p =𠂐.002). CML stem cells harboring longer telomeres possibly maintain a proliferative potential after treatment discontinuation.

Electronic supplementary material

The online version of this article (doi:10.1186/s13045-016-0293-y) contains supplementary material, which is available to authorized users.

Telomeres are specialized nucleoprotein structures composed of long arrays of TTAGGG repeats localized at the ends of human chromosomes able to maintain genome stability and integrity and to protect the cell from progressive DNA shortening during repeated division [1]. Telomere biology has been more extensively studied in chronic myeloid leukemia (CML) than in any other blood cancer. Shorter telomeres have been associated with CML, disease progression, poor prognosis, higher Hasford score, and acquisition of cytogenetic aberrations [2𠄴]. As yet, no studies have considered the possible association between telomere length and treatment-free remission (TFR) after discontinuation of therapy with tyrosine kinase inhibitors (TKIs).

Thirty-two chronic-phase CML patients discontinued TKI treatment after achieving complete molecular remission (CMR) for at least 18 months. All patients received imatinib therapy for more than 24 months. Two patients underwent second-line treatment with nilotinib because of molecular relapse. The median follow-up after discontinuation was 30 months (range 18�). A complete molecular response was defined as undetectable breakpoint cluster region-Abelson (BCR/ABL1) by real-time quantitative polymerase chain reaction (qRT-PCR) with a sensitivity of the assay corresponding to molecular response (MR)4 and MR4.5. Peripheral blood samples from 32 age- and sex-matched healthy individuals were used for control purposes. The relative telomere length (RTL) was determined by q-PCR according to the technique described by Cawthon in 2002 [5] (Additional file 1). Age-corrected RTL (acRTL) represented the difference in telomere length between patients and age- and sex-matched controls.

The characteristics of 32 chronic phase CML patients are shown in Table  1 . Thirteen patients (41 %) showed loss of CMR. All relapsed patients regained CMR after restarting treatment with TKIs. The 36-month cumulative probability of TFR was 59.4 %. RTL was assessed at a mean of 26 months from discontinuation (range 20�). RTL was assessed at a mean of 26 months from discontinuation (range 18�). The majority of relapses occurred within 9 months of therapy interruption (mean 8.7 months, range 2�). In these patients, RTL was assessed after relapse. Overall, median RTL was slightly shorter in patients than in controls (0.97 vs 1.05). The median value of acRTL in the CML cohort was 0.09 (range 𢄠.26, +0.86). The Mann-Whitney U test showed shorter acRTL in TFR patients compared to patients with molecular relapse (mean ± SD =𠂐.01 ±𠂐.14 vs 0.20 ±𠂐.21 p =𠂐.01) (Additional file 2). Although the male gender was more frequent in TFR patients, we did not find any significant difference in telomere length between male and female. Patients were stratified according to the median value of acRTL 𢙀.09. TFR was significantly higher in CML patients with acRTL 𢙀.09 in comparison to those with longer telomeres (78.9 vs 30.8 %, p =𠂐.002) (Fig.  1 ).

Table 1

Characteristics of 32 CML patients according to treatment-free remission (TFR) or molecular relapse after imatinib discontinuation

Patients in TFR no. 19 (59 %) Relapsed patients no. 13 (41 %) p
Age at diagnosis (mean, range)74 (47�) 56 (37�) 0.004
Leukocytes at diagnosis 휐 3 /uL (mean, range)50.47 (8.15�) 69 (19.8�) ns
Platelets at diagnosis 휐 3 /uL (mean, range)472 (178�) 357 (171�) ns
Months to CMR (median, range)28 (3�) 30 (6�) ns
Months of TKIs (median, range)86 (24�) 84 (45�) ns
Months of TKIs 㹠 (no., %)1368.41292.3ns
Male gender (no., %)1684.2861.5ns
Sokal risk (no., %)
 Low631.6861.5ns
 Intermediate1052.6323.1ns
 High315.8215.4ns
First-line TKI treatment (no., %)1157.91184.6ns
Previous IFN treatment (no., %)842.1215.4ns
Imatinib first-line TKI treatment (no., %)1910013100ns
Nilotinib second-line TKI treatment (no., %)15.317.7ns
Age-corrected relative telomere length (mean ± SD)0.01 ±𠂐.14 0.20 ±𠂐.21 0.01

pres present, CMR complete molecular response, TKIs tyrosine kinase inhibitors, IFN interferon, ns not significant, SD standard deviation


To investigate whether RG7834 treatment phenocopies PAPD5 knockdown, we depleted DKC1 in HeLa cells (supplemental Figure 1A-B) and treated them with either DMSO (control) or 5 μM of RG7834 for 2 days. Similar to previous results, 13 knockdown of DKC1 led to a significant reduction in TERC levels (Figure 1A-B). Importantly, treatment with RG7834 led to a significant increase in TERC levels in DKC1 knockdown cells, indicating that RG7834 is able to increase TERC levels in DKC1-deficient cells. Because the depletion of PARN also leads to TERC degradation mediated by 3′ end adenylation by PAPD5, 13 we silenced PARN in HeLa cells and treated them with DMSO or 5 μM of RG7834 for 2 days. We found that PARN knockdown leads to a significant reduction in TERC levels, which is completely rescued by RG7834 treatment. Thus, similar to the genetic silencing of PAPD5, treatment with RG7834 increased TERC levels in both PARN- and DKC1-deficient cells, without affecting levels of either DKC1 or PARN in individual knockdown cells. Finally, RG7834 treatment did not affect TERC levels in HeLa cells that were not subject to silencing of either DKC1 or PARN (supplemental Figure 1C).

RG7834 treatment rescues TERC levels and localization in DKC1- and PARN-depleted cells. (A) Representative northern blots for TERC levels in HeLa cells under indicated conditions. Numbers below panels: average ± standard deviation for 3 biological replicates. (B) Quantification of TERC by quantitative reverse transcription polymerase chain reaction in HeLa cells under the indicated conditions (n = 3 biological replicates). Values are expressed in relation to scrambled control. (C) Representative images for 4′,6-diamidino-2-phenylindole (DAPI) (nucleus), coilin (cajal body), TERC, and merge of individual channels obtained from HeLa cells transfected with indicated siRNAs and treated with RG7834. White arrows indicate TERC localization within the cell. Scale bar, 5 μm. Numbers in image panels: Quantification of fraction of cells with TERC colocalized to cajal bodies from at least 30 independent cells and 3 replicates. Cells with at least one TERC focus colocalized with coilin were counted. (D) Telomerase activity by telomere repeat amplification in HeLa cells transfected with indicated siRNAs and treated with 5 μM of RG7834 or DMSO. Range of protein concentrations represent fourfold serial dilutions. *P < .05 **P < .01. LC, loading control.

RG7834 treatment rescues TERC levels and localization in DKC1- and PARN-depleted cells. (A) Representative northern blots for TERC levels in HeLa cells under indicated conditions. Numbers below panels: average ± standard deviation for 3 biological replicates. (B) Quantification of TERC by quantitative reverse transcription polymerase chain reaction in HeLa cells under the indicated conditions (n = 3 biological replicates). Values are expressed in relation to scrambled control. (C) Representative images for 4′,6-diamidino-2-phenylindole (DAPI) (nucleus), coilin (cajal body), TERC, and merge of individual channels obtained from HeLa cells transfected with indicated siRNAs and treated with RG7834. White arrows indicate TERC localization within the cell. Scale bar, 5 μm. Numbers in image panels: Quantification of fraction of cells with TERC colocalized to cajal bodies from at least 30 independent cells and 3 replicates. Cells with at least one TERC focus colocalized with coilin were counted. (D) Telomerase activity by telomere repeat amplification in HeLa cells transfected with indicated siRNAs and treated with 5 μM of RG7834 or DMSO. Range of protein concentrations represent fourfold serial dilutions. *P < .05 **P < .01. LC, loading control.

We have previously shown that in DKC1- or PARN-depleted cells, TERC accumulates in cytoplasmic puncta called “cyTER bodies,” 13 instead of its normal localization to cajal bodies in the nucleus. 22,23 To investigate whether RG7834 treatment rescued TERC localization to cajal bodies in the nucleus, we treated DKC1- or PARN-depleted cells with RG7834 for 2 days. We found that in WT control cells, TERC localized to cajal bodies, suggesting normal accumulation and trafficking of the telomerase RNA component (Figure 1C). However, in control DKC1 knockdown cells, only ∼15% of cells showed TERC localization to cajal bodies, with a significant number of cells exhibiting TERC accumulation in cyTER bodies. Upon treatment with RG7834, ∼38% of cells had TERC localized to cajal bodies, which is consistent with the 1.5× increase in TERC levels in these cells. RG7834 treatment also rescued TERC localization to cajal bodies in PARN-depleted cells. In PARN knockdown cells, ∼34% of cells had TERC in cajal bodies, with TERC also mostly being localized to cyTER bodies in these cells. However, upon RG7834 treatment, ∼85% of cells had TERC localized to cajal bodies. Finally, treatment with RG7834 was also able to increase telomerase activity in DKC1- and PARN-depleted cells (detected by using 2-step telomere repeat amplification assays) (Figure 1D supplemental Figure 1D), which is consistent with the increase in TERC levels (Figure 1A-B). No toxicity was observed in RG7834-treated cells for the duration of these experiments. Taken together, these results suggest that the chemical inhibition of RG7834 is effective at increasing TERC levels, correcting TERC localization to cajal bodies, and increasing telomerase activity in DKC1- or PARN-depleted cells, indicating restored telomerase function in these conditions.

We next investigated whether RG7834 exhibits a similar effect on TERC levels and function in a physiologically relevant model of DC. We have previously generated (through CRISPR/Cas9 gene editing) DKC1_A353V mutant hESCs, which exhibit reduced TERC levels and telomerase activity, progressive telomere shortening, and impaired definitive hematopoietic specification. 21 Treatment of DKC1_A353V hESCs with RG7834 caused a >15-fold reduction in the 3′-end oligoadenylation of TERC, indicating efficient PAPD5 inhibition in these cells (Figure 2A). Inhibition of 3′ end oligoadenylation led to a significant increase in TERC levels in DKC1_A353V hESCs (4 days of treatment) (supplemental Figure 2A), which was sustained up to 30 days upon continuous treatment with different concentrations of RG7834 (Figure 2B). Although the levels of the telomerase reverse transcriptase (TERT) remained unchanged during this period, the increased TERC levels found in DKC1_A353V hESCs treated with RG7834 caused a noticeable rise in telomerase activity in DKC1_A353V hESCS treated with different concentrations of the PAPD5/7 inhibitor (Figure 2C quantification in supplemental Figure 2B).

RG7834 treatment rescues telomerase activity, increases telomere length, and improves hematopoietic specification in DKC1_A353V mutant hESCs. (A) Quantification of oligo(A) reads at the 3′ end (UGC) of TERC in indicated conditions (average ± standard deviation from 2 independent replicates). TERC reads with more than 2 As at the 3′ end are considered oligoadenylated. (B) Quantification of TERC and TERT by quantitative reverse transcription polymerase chain reaction in DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834 for 30 days (n = 3 biological replicates). (C) Telomerase activity by telomere repeat amplification in DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834. Range of protein concentrations represent fourfold serial dilutions. (D) Telomere length analysis by telomere restriction fragment analysis of DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834 for 90 days. (E) Representative flow cytometric analysis of CD34 and CD43 expression on day 8 of definitive hematopoietic differentiation, following CHIR99021 and SB-431542 treatment in DKC1_A353V cells treated with DMSO or different concentrations of RG7834. (F) Quantification of CD34 + CD43 – population obtained from day 8 differentiation cultures treated with CHIR99021 and SB-431542, as in panel E. (G) CFC potential of definitive hematopoietic progenitors in WT and DKC1_A353V cells treated with DMSO or 1μM of RG7834 (n = 3 biological replicates). Statistical significance was determined by using one- or two-way analysis of variance following a Bonferroni multiple comparison posttest. *P < .05 **P < .01 ***P < .001. BFU-E, burst forming unit-erythroid CFC, colony forming cell MTL, mean telomere length n.s., not significant.

RG7834 treatment rescues telomerase activity, increases telomere length, and improves hematopoietic specification in DKC1_A353V mutant hESCs. (A) Quantification of oligo(A) reads at the 3′ end (UGC) of TERC in indicated conditions (average ± standard deviation from 2 independent replicates). TERC reads with more than 2 As at the 3′ end are considered oligoadenylated. (B) Quantification of TERC and TERT by quantitative reverse transcription polymerase chain reaction in DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834 for 30 days (n = 3 biological replicates). (C) Telomerase activity by telomere repeat amplification in DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834. Range of protein concentrations represent fourfold serial dilutions. (D) Telomere length analysis by telomere restriction fragment analysis of DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834 for 90 days. (E) Representative flow cytometric analysis of CD34 and CD43 expression on day 8 of definitive hematopoietic differentiation, following CHIR99021 and SB-431542 treatment in DKC1_A353V cells treated with DMSO or different concentrations of RG7834. (F) Quantification of CD34 + CD43 – population obtained from day 8 differentiation cultures treated with CHIR99021 and SB-431542, as in panel E. (G) CFC potential of definitive hematopoietic progenitors in WT and DKC1_A353V cells treated with DMSO or 1μM of RG7834 (n = 3 biological replicates). Statistical significance was determined by using one- or two-way analysis of variance following a Bonferroni multiple comparison posttest. *P < .05 **P < .01 ***P < .001. BFU-E, burst forming unit-erythroid CFC, colony forming cell MTL, mean telomere length n.s., not significant.

The sustained treatment (up to 3 months) with RG7834 also led to improved telomere maintenance, analyzed by telomere restriction fragment analysis, in DKC1_A353V hESCs (Figure 2D). Together, these data suggest that RG7834 treatment rescued TERC levels, prevented its 3′ end oligoadenylation, increased telomerase activity, and improved telomere homeostasis in DKC1_A353V hESCs.

Finally, we investigated if treatment with RG7834 was sufficient to reduce DNA damage signaling arising from eroded telomeres, a hallmark of DC. We observed that γH2AX levels were reduced in DKC1_A353V cells treated with different concentrations of RG7834 compared with DMSO-treated cells (supplemental Figure 3A). Importantly, we detected no toxicity associated with RG7834 treatment at the concentrations indicated, during the entire duration of the experiments performed (supplemental Figure 3B-C). In addition, whole-genome RNA-sequencing analysis showed that treatment with 1 μM of RG7834 did not lead to significant changes in gene expression in DKC1_A353V mutant hESCs compared with DMSO-treated cells (supplemental Figure 3D), suggesting that RG7834 treatment affects specific RNAs in hESCs and rules out toxicity associated with genome-wide gene expression perturbations. Combined, these data indicate that similar to the genetic silencing of PAPD5, treatment with RG7834 is able to rescue the major biochemical phenotypes observed in DC models.

Finally, because bone marrow failure is the leading cause of death in DC, we wanted to analyze the consequences of RG7834 treatment in DKC1_A353V cells during definitive hematopoietic differentiation. There are no animal models that faithfully recapitulate the hematopoietic defects observed in patients harboring pathogenic mutations in DKC1 or PARN we therefore followed established protocols of hESC differentiation into hematopoietic lineages. These protocols recapitulate, in vitro, the major aspects of blood development in vivo, 24 a strategy that we and others have shown to accurately model key aspects of DC. 18,21,25,26 A schematic of our protocol is depicted in supplemental Figure 4. Our data show that although CD34 + CD43 – early hematopoietic progenitors (day 8 of differentiation) (Figure 2E-F) were similar in all samples, definitive hematopoietic colony potential analysis (day 28 of differentiation) revealed that treatment with different concentrations of RG7834 significantly increased the hematopoietic potential of DKC1_A353V cells (Figure 2G). These observations provide compelling evidence that chemical inhibition of PAPD5, in addition to rescuing telomerase function, is sufficient to increase definitive, multilineage, hematopoietic potential in DKC1_A353V mutants.

Our data provide functional evidence that a small molecule inhibitor of PAPD5/7 significantly increases TERC levels, localization, and function in DKC1- and PARN-deficient cells. We show that RG7834 reduces the 3′-end oligoadenylation of TERC, increases TERC levels and telomerase activity, and elongates telomeres in PARN- or DKC1-deficient cells. RG7834 treatment was sufficient to restore the in vitro definitive hematopoietic development of DKC1_A353V hESCs, similarly to what we have recently observed with the genetic silencing of PAPD5. 18 Although these experiments represent the first nongenetic rescue of hematopoietic development from hESCs in DKC1 mutant cells, it has recently been shown that PAPD5 inhibitors are able to promote telomere restoration in different patient-derived DC samples in vitro and in vivo, through xenotransplantation assays 27 these results provide further support for the future use of this technology in the clinic. In addition, the studies presented here indicate that small increases in TERC levels (less than twofold) are sufficient to increase telomerase activity and improve hematopoietic output in DKC1 mutant cells. Future experiments performed in cells harboring mutations in other genes that impair TERC levels/function (including TERC itself, NHP2, NOP10, and ZCCHC8) are necessary for determining the scope and range of effectiveness of PAPD5 inhibition for DC treatment. In addition, the efficiency of PAPD5 inhibition to ameliorate other phenotypes that are commonly associated with telomere shortening, such as pulmonary fibrosis and liver disease, must be assessed, as these conditions cause substantial morbidity and mortality and represent an important unmet need for these patients. Nonetheless, the experiments presented here indicate that the chemical inhibition of PAPD5 by RG7834 or other specific small molecule inhibitors can be a promising therapeutic approach for the treatment of DC or other telomere biology syndromes caused by mutations that reduce TERC levels.


Hallmarks of telomeres in ageing research †

Telomeres are repetitive DNA sequences at the ends of linear chromosomes. Telomerase, a cellular reverse transcriptase, helps maintain telomere length in human stem cells, reproductive cells and cancer cells by adding TTAGGG repeats onto the telomeres. However, most normal human cells do not express telomerase and thus each time a cell divides some telomeric sequences are lost. When telomeres in a subset of cells become short (unprotected), cells enter an irreversible growth arrest state called replicative senescence. Cells in senescence produce a different constellation of proteins compared to normal quiescent cells. This may lead to a change in the homeostatic environment in a tissue-specific manner. In most instances cells become senescent before they can become cancerous thus, the initial growth arrest induced by short telomeres may be thought of as a potent anti-cancer protection mechanism. When cells can be adequately cultured until they reach telomere-based replicative senescence, introduction of the telomerase catalytic protein component (hTERT) into telomerase-silent cells is sufficient to restore telomerase activity and extend cellular lifespan. Cells with introduced telomerase are not cancer cells, since they have not accumulated the other changes needed to become cancerous. This indicates that telomerase-induced telomere length manipulations may have utility for tissue engineering and for dissecting the molecular mechanisms underlying genetic diseases, including cancer. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Progressive telomere shortening characterizes familial breast cancer patients

Telomeres, the complex structures that protect the end of chromosomes, of peripheral blood cells are significantly shorter in patients with familial breast cancer than in the general population. Results of the study carried out by the Human Genetics Group of the Spanish National Cancer Research Centre (CNIO), led by Javier Benitez, to be published in open-access journal PLoS Genetics on July 28th, reflect that familial, but not sporadic, breast cancer cases are characterized by shorter telomeres. Importantly, they also provide evidence for telomere shortening as a mechanism of genetic anticipation, the successively earlier onset of cancer down generations.

Mutations in two DNA repair genes, BRCA1 and BRCA2, characterize some, but not all, instances of hereditary breast cancer. Non-BRCA1/2 breast cancer families are heterogeneous, suggesting the existence of other genes conferring susceptibility. The group has investigated the role of telomere length in hereditary breast cancer based on previous information suggesting, first, that short telomeres and subsequent genomic instability contribute to malignant transformation second, that genetic anticipation occurs in breast cancer families and, third, that telomere shortening is associated with anticipation in other genetic diseases.

By analyzing telomere length differences between mothers and daughters from breast cancer families, the authors demonstrated that genetic anticipation is associated with a decrease in telomere length in affected daughters relative to their mothers.

The results allowed the authors not only to conclude that women carrying BRCA1/2 mutation have chromosomes with short telomeres, but also to describe for the first time that genetic anticipation in breast cancer could be explained by telomere shortening. In addition, the study expands the field of research concerning genetic predisposition to breast cancer to include genes involved in telomere maintenance. The significance of generational changes in telomere length has interesting potential clinical applications in the management of familial breast cancer, and could be extended to other hereditary cancer syndromes.

FINANCIAL DISCLOSURE: This work was supported by Asociación Española Contra el Cancer (AECC) and Spanish Fondo de Investigaciones Sanitarias (grant numbers FISPI081298 and FIS-PI081120). The CIBER de Enfermedades Raras is an initiative of the ISCIII. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

COMPETING INTERESTS: The authors have declared that no competing interests exist.

CITATION: Martinez-Delgado B, Yanowsky K, Inglada-Perez L, Domingo S, Urioste M, et al. (2011) Genetic Anticipation Is Associated with Telomere Shortening in Hereditary Breast Cancer. PLoS Genet 7(7): e1002182. doi:10.1371/journal.pgen.1002182

Contact:
Dr. Beatriz Martinez-Delgado and Dr. Javier Benitez
Spanish National Cancer Research Centre (CNIO)
Human Genetics
Melchor Fernandez Almagro 3
Madrid 28029
SPAIN
[email protected]
[email protected]

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Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe

This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres.


Anxiety, exhaustion, and telomeres

It seems like just about everyone these days is stressed out and working too hard. We know it&rsquos not good for us, but how bad is it really?

It might be worse than we&rsquod like to think. Today, PLoS ONE published two separate studies investigating how anxiety and work-related exhaustion are correlated to the length of our telomeres, the protective caps on our chromosomes that help make sure our dividing cells have all the genetic material they need for long, healthy lives.

The short answer: both higher stress and severe exhaustion were found to be correlated with decreased telomere length, with potential implications for aging and long-term health. It&rsquos important to note that the results are only correlative, and don&rsquot show any causation, but they add to a suggestive and growing body of literature in this area.

Telomeres are the portions at the ends of our chromosomes, the packaged-up version of all of our DNA, which are critical for proper cell functioning. The telomeres themselves don&rsquot contain any crucial information, but they protect the important parts of the chromosome from deterioration by slowly sacrificing themselves, a bit at a time, during each cell division. It&rsquos thought that some of the issues associated with aging and some cancers may be caused by telomere shortening, which can lead to dysfunctional cells with incomplete chromosomes that die or go rogue.

The connection between telomere length and health remains somewhat tenuous, but they have become quite a hot spot for research, including the two articles published today. In one of the studies, &ldquoHigh Phobic Anxiety is Related to Lower Leukocyte Telomere Length in Women,&rdquo led by Olivia Okereke of Harvard University, the researchers compared telomere length with anxiety levels for 5,243 women between the ages of 42 and 69, and found that higher anxiety was generally associated with shorter telomere length. The other report, &ldquoWork-related Exhaustion and Telomere Length: A Population-based Study,&rdquo led by Kirsi Ahola of the Finnish Institute of Occupational Health, found a similar correlation between work-related exhaustion, which they interpreted as an indicator of prolonged work stress, and telomere length in 2,911 men and women aged 30 to 64, with severe exhaustion associated with markedly shorter telomeres.

Again, it&rsquos important to remember that correlation does not equal causation, but perhaps these results at least offer a potential suggestion that we should all find a little more time to sit back and relax, something we should probably be doing regardless.



Comments:

  1. Khristian

    Quite right! The idea is great, I support it.

  2. Nikolmaran

    He has specially signed up to the forum to say thank you for the support.

  3. Douzahn

    Congratulations, great idea and on time



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