Maintenance Of Telomere Integrity Research Articles

ZNF524 directly interacts with telomeric DNA and supports telomere integrity

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing nonhomologous end joining (NHEJ) through its subunit TRF2. Here, we describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity, and reveal base-specific sequence recognition by cocrystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

Polyadenylated Telomeric Noncoding RNA Functions as a Pivotal Therapeutic Target of Anti-Ageing to Stabilize Telomere Length of Chromosomes Via Collaborating With Zscan4c.

Telomeres are closely associated with the development of cell aging. Shortening or erosion of telomeres will cause cell mortality, suggesting that the maintenance of telomere integrity facilitates cell anti-senescence. However, the mechanism of how to keep the telomere length remains fragmentary. Here, we found that polyadenylated telomeric noncoding RNA (TERRA) can promote the self-renewal when overexpressed in mouse embryonic stem cells (mESCs), implying that TERRA with polyadenylation is critical for mESC maintenance. Further studies revealed that TERRA with a polyadenylated tail plays an important role in the sustenance of telomere length. High-throughput sequencing and quantitative real-time PCR show that zinc finger and SCAN domain containing 4C (Zscan4c) may be a potential target of TERRA. Zscan4c is negatively regulated by TERRA and collaborates with TERRA to stabilize the telomere length of chromosomes in mESCs. Our study not only identifies TERRA as a potential novel factor of telomere length regulation and uncovers the new molecular mechanism of cell anti-aging, but also indicates that Zscan4c could be a key therapeutic target candidate for therapy in dysfunctional chromosome diseases. These data will expand our understanding of the cell fate regulatory network and will be beneficial to drug discovery and theragnostics for antiaging and anticancer therapy in the future.

Histone Modifications and the Maintenance of Telomere Integrity.

Telomeres, the nucleoprotein structures at the ends of eukaryotic chromosomes, play an integral role in protecting linear DNA from degradation. Dysregulation of telomeres can result in genomic instability and has been implicated in increased rates of cellular senescence and many diseases, including cancer. The integrity of telomeres is maintained by a coordinated network of proteins and RNAs, such as the telomerase holoenzyme and protective proteins that prevent the recognition of the telomere ends as a DNA double-strand breaks. The structure of chromatin at telomeres and within adjacent subtelomeres has been implicated in telomere maintenance pathways in model systems and humans. Specific post-translational modifications of histones, including methylation, acetylation, and ubiquitination, have been shown to be necessary for maintaining a chromatin environment that promotes telomere integrity. Here we review the current knowledge regarding the role of histone modifications in maintaining telomeric and subtelomeric chromatin, discuss the implications of histone modification marks as they relate to human disease, and highlight key areas for future research.

Tumor Cell-Accelerated Senescence Is Associated With DNA-PKcs Status and Telomere Dysfunction Induced by Radiation.

Whether telomere structure integrity is related to radiosensitivity is not well investigated thus far. In this study, we investigated the relation between telomere instability and radiation-induced accelerated senescence. Partial knockdown of DNA-dependent catalytic subunit of protein kinase (DNA-PKcs) in human breast cancer cell line MCF-7 was established by small interfering RNA. Radiosensitivity of control and DNA-PKcs knockdown MCF-7 cells was analyzed by clonogenetic assay. Cell growth was measured by real-time cell electronic sensing. Senescence and apoptosis were evaluated by β-galactosidase histochemical staining and fluorescence-activated cell sorting, respectively. DNA damage was determined by long polymerase chain reaction (PCR). Telomere length and integrity were analyzed by real-time PCR and cytogenetic assay, respectively. DNA-PKcs knockdown MCF-7 cells were more sensitive to X-irradiation than control cells. Further investigation revealed that accelerated senescence is more pronounced than apoptosis in cells after radiation, particularly in DNA-PKcs knockdown cells. The cytogenetic assay and kinetics of DNA damage repair revealed that the role of telomere end-capping in DNA-PKcs, rather than DNA damage repair, was more relevant to radiosensitivity. To our knowledge, this is the first study to show that DNA-PKcs plays an important role in radiation-induced accelerated senescence via maintenance of telomere integrity in MCF-7 cells. These results could be useful for future understanding of the radiation-induced genome instability and its consequences.

Oxidative stress: Role in maintenance of telomere integrity

Oxidative stress: Role in maintenance of telomere integrity

Forced expression of Nanog or Esrrb preserves the ESC status in the absence of nucleostemin expression.

Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in a plethora of functions including ribosomal biogenesis and maintenance of telomere integrity. In addition to its expression in cancerous cells, the NS gene is expressed in stem cells including embryonic stem cells (ESCs). Previous knockdown and knockout studies have demonstrated that NS is important to preserve the self-renewality and high expression levels of pluripotency marker genes in ESCs. Here, we found that forced expression of Nanog or Esrrb, but not other pluripotency factors, resulted in the dispensability of NS expression in ESCs. However, the detrimental phenotypes of ESCs associated with ablation of NS expression were not mitigated by forced expression of Rad51 or a nucleolar localization-defective NS mutant that counteracts the damage associated with loss of NS expression in other NS-expressing cells such as neural stem/progenitor cells. Thus, our results indicate that NS participates in preservation of the viability and integrity of ESCs, which is distinct from that in other NS-expressing cells. Stem Cells 2015;33:1089–1101

Abstract 2393: The BARD1 BRCT domains are essential for maintenance of telomere integrity

Abstract BARD1 has tumor suppressor functions by binding to BRCA1 and to p53 via RING finger and ankyrin repeats, respectively. The BARD1-BRCA1 dimer has E3 ligase activity, and BARD1-p53 interaction induces apoptosis. The C-terminus of BARD1 is binding to many proteins that are targets of the BRCA1-BARD1 ubiquitin ligase, and expression of N-terminally truncated RING-less BARD1 isoforms was correlated with poor prognosis in various cancers, consistent with their oncogenic functions by antagonistic binding to target proteins. Expression and interaction assays showed that the BARD1 BRCT domains were critically involved in telomere maintenance and interacted with the telomere binding protein TRF2. Overexpression of BARD1 or BRCA1 lead to TRF2 degradation, suggesting that TRF2 presents a novel target of the BARD1-BRCA1 E3 ubiquitination ligase. Repression of BARD1 or BRCA1 by siRNA leads to genetic instability, as shown before, and telomere alterations. We observed distinct telomeric alterations in blood cells of patients with a BARD1 germline mutation that causes a translation stop and translation of a truncated protein lacking the BRCT domains. Cells of carriers of a BRCA1 mutation that disrupts the BARD1-BRCA1 interaction show similar telomere alterations. These in vitro and in vivo observations suggest that BARD1 and BRCA1 play distinct roles in maintaining telomere integrity in a dosage dependent manner. BARD1δ, an isoform lacking RING and ankyrin motifs, but retaining the BRCT domains, is abundantly expressed in all cancers investigated so far. Overexpression of BARD1δ in vitro blocked cell cycle progression and induced chromosomal and telomere abnormalities, suggesting that BARD1δ also affected telomere integrity. We conclude that the correct dose of BARD1 and/ or its BRCT domains is critically important for maintaining telomere integrity. Expression of BARD1δ in cancer, or reduced expression of BARD1 in mutation carriers causes chromosomal instability and promotes carcinogenesis and tumor progression. Citation Format: Irmgard Irminger-Finger, Maxim Pilyugin, Magdalena Ratajska. The BARD1 BRCT domains are essential for maintenance of telomere integrity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2393. doi:10.1158/1538-7445.AM2014-2393

Abstract 542: Regulation of the telomere healing process by the lncRNA TERRA

Abstract Telomere length is regulated by epigenetic marks that modulate, among other things, the expression of the telomerase catalytic subunit. Telomeres and sub-telomeric regions are enriched in histone marks associated to constitutive heterochromatin. The marks before mentioned are essential for the maintenance of telomere integrity and therefore for genomic stability, but there is another kind of regulation that depends on ncRNAs. This is the case of the lncRNA Telomere repeat-containing RNA (TERRA), an element required for telomeric integrity, considered to hinder chromosomal instability where anaphase bridges, sister chromatid fusions and multi-telomeric regions can take place. This instability leads to chromosomal aberrations that could produce a malignant cell lineage, however, in order for this event to take place, a telomere healing process must occur so that the ribonucleoproteic complex of the telomere can be stabilized once more. For this reason it is important to study the role that the lncRNA TERRA plays in telomere stabilization and how its malfunctioning can bring forth an carcinogenic process. The telomeric regions of interest were analyzed using the data base of the University of California in Santa Cruz (UCSC). We chose the short arm of chromosome 5, where telomerase is encoded, and looked for elements associated to promoter regions such as the presence of the nuclear factor CTCF, Polymerase II, histone marks related with promoters (H3K4me3 and H3K4me1), potential transcription start sites (TSS) and sequences with enhancer potential. We worked with two cell lines of the same cell lineage K562 and SC, in order to have a leukemic and a non-transformed cell model. In order to enrich the amount of TERRA after RNA extraction, we synthesized cDNA using both dT primers and telomere repeat-containing oligonucleotides. The cDNA was used to evaluate the total amount of TERRA in both cell lines using the expression of the ncRNAs U6, HOTAIR and H19 as an amplification reference. We demonstrated that the short arm of chromosome 5 does codify for TERRA, and we verified that only the DNA strand that encodes TERRA was transcribed. Furthermore, we confirmed that the predicted TSS was functional. qPCR showed that there are changes in TERRA expression that take place in accordance with population doubling events. This was observed in the analyzed telomeres of both cell lines used. These results could be related with the natural shortening of telomeres that happens in non-transformed cells, but since such a process is absent in leukemic cells, further investigation is required to answer this query. Citation Format: Diego A. Oliva-Rico, Rodrigo Gonzalez-Barrios, Eunice Fabian-Morales, Luis A. Herrera. Regulation of the telomere healing process by the lncRNA TERRA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 542. doi:10.1158/1538-7445.AM2014-542

Telomeres, a busy platform for cell signaling.

Telomeres are the terminal structures at the ends of linear chromosomes that represent a solution to the end replication problem. Specific binding of the six-protein subunit complex shelterin to telomeric, repetitive TTAGGG DNA sequences contributes to the stable architecture and maintenance of telomeres. Proteins involved in the DNA damage response are also localized at telomeres, and play a role in the surveillance and maintenance of telomere integrity. The enzyme responsible for telomere extension is telomerase, a ribonucleoprotein with reverse transcriptase activity. In the absence of telomerase, telomeres shorten to a length threshold that triggers the DNA damage response and replicative senescence. Here, we will summarize the latest findings concerning vertebrate telomere structure and epigenetics, and we present data regarding the impact of short telomeres upon cell signaling. In particular, in murine embryonic stem cells lacking telomerase, we found that distribution of cytosolic/nuclear β-catenin, a key component of the Wnt signaling pathway, changes when telomeres become critically short. We discuss implications and future perspectives of the effect of epigenetic modifications and/or conformational changes of telomeres on cell metabolism and signaling networks. Such an analysis may unveil potential therapeutic targets for pathologies like cancer, where the integrity of telomeres is altered.

Abstract 2123: ATM mediated homeostasis of ‘shelterin’ at telomere is compromised by G-quadruplex leading to DNA damage

Abstract Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27 inhibits growth of malignant cells. Telomere integrity is maintained by the ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. ATM has been shown to play a role in the maintenance of telomere integrity. U937, histiocytic lymphoma cells were sensitive to Pu-27 as evident by MTT and colony formation assay and frequently showed chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal breaks (27%). RT-PCR array of Pu-27 treated U937 cells revealed downregulation of ‘shelterin’ proteins TRF2, TRF1, POT1 and TIN2; induction of DNA damage response sensor γ-H2AX; downregulation of upstream kinase ATM and other downstream DNA damage mediator RAD17, RAD50, BRCA1, 53BP1, CHK1, CHK2. To understand the specific role of ‘shelterin’ protein complex, we stably transfected U937 cells with a vector expressing a dominant negative mutant of a shelterin protein TRF2 (dn-U937). These dn-UP37 cells became relatively resistant when treated with Pu-27 and showed no changes of basal level expression of ‘shelterin’ protein complex but induction of γ-H2AX and DNA repair inducing molecules BRCA1 and CHK1. This suggests that intact TRF2 is required for Pu-27 mediated U937 cellular sensitivity and it may acted by altering the telomere-‘shelterin’ complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. To elucidate the specific role of ATM, we have shown by MTT and colony formation assay that ATM-deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM-proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and no alteration of basal level of ‘shelterin’ protein complex in Pu-27 treated ATM deficient cells (as determined by RT-PCR array). A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these data suggest that downregulation of ATM destabilizes telomere-shelterin homeostasis at telomere ends in response to Pu-27 of U937 cells. Continuous exposure of G-quadruplex results in refractory DNA damage repair leading to uncompensated genomic stability and cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2123. doi:1538-7445.AM2012-2123

Phasor Diagram Analysis of Telomeric G-Quadruplex Stability and Heterogeneity

Telomeres function to protect the end of linear chromosomes from damage and degradation. Human telomeres consists of several kilobases of the tandem repeat d(TTAGGG), which terminate in a 3′-single stranded overhang capable of forming G-Quadruplex structures. Telomeric G-Quadruplexes have potential roles in the maintenance of telomere integrity and the control of telomerase activity, an enzyme responsible for elongation of the telomeric overhang that is over expressed in a wide variety of cancers. Investigation of intramolecular G-Quadruplexes corresponding to the human telomere is complicated by the formation of multiple rapidly interconverting conformations. The multiple telomere G-Quadruplex conformations are identified by unique folding geometries containing distinct connecting loop regions.Time-resolved fluorescent measurements have been conducted to characterize the stability and heterogeneity of the telomeric G-Quadruplex forming sequence d(AGGG(TTAGGG)3). The fluorescent adenine analog, 2-aminopurine, served as an internal fluorescent probe of G-Quadruplex conformation and was used to specifically monitor loop regions. 2-aminopurine demonstrates a complex fluorescent decay upon incorporation into nucleic acid polymers that can be simplified by transformation of time-resolved data to an (S,G) phasor diagram. Phasor diagrams were used to monitor G-Quadruplex formation upon addition of NaCl or KCl and elucidated a unique KCl-dependent transition. The digestion of G-Quadruplex structure by a single-strand specific nuclease and acrylamide quenching are also evaluated through phasor diagrams. This work demonstrates the utility of phasor diagrams for the study of nucleic acid systems and the ability to sensitively monitor complex fluorescent decays that may complicate analysis through nonlinear regression techniques. Supported by awards RO1CA35635 and RO1GM077422 from the NCI and NIGMS.

Abstract 3886: The G-quadruplex induced DNA damage at telomeres is regulated by ATM

Abstract Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. Our group has shown that quadruplex-forming oligonucleotides selectively inhibit proliferation of transformed cells. One of these oligonucleotides, AS1411, is currently in a Phase IIb clinical trial. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27, also inhibits growth of malignant cells. We have hypothesized that one potential mechanism of this effect is competition for telomere-binding proteins. If this is correct, we would expect this effect to be mediated by the DNA repair response. Telomere integrity is maintained by the shelterin protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. Like many other DNA damage response proteins, ATM has been shown to play a role in the maintenance of telomere integrity. We have shown that ATM deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and downregulation of shelterin proteins TRF2 and TRF1 in Pu-27 treated ATM deficient cells (as determined by western blotting). U937 leukemia cells, which were sensitive to Pu-27, became relatively resistant when they were stably transfected with a vector expressing a dominant negative mutant of the shelterin protein TRF2 (dn-TRF2). This suggests that Pu-27 may act by altering the telomere-shelterin complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, this data suggests that ATM plays a pivotal role in Pu-27 induced DNA damage at telomere ends, which occurs in response to a Pu-27 induced alteration in telomere-shelterin homeostasis. It is likely that this response is induced in a p53 independent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2011-3886

Unraveling the roles of WRN and DNA-PKcs at telomeres

In eukaryotic cells, the ends of linear chromosomes are maintained by nucleoprotein complexes called telomeres, which are composed of long stretches of repetitive DNA sequences bound by specific telomere binding proteins. The repetitive DNA sequence (repeats of TTAGGG in humans) is composed of double stranded DNA and, at its extreme end, a single-stranded G-rich 3' overhang, referred to as the G-tail. The telomere folds back on itself to form a lariat-shaped loop called the T (for telomere) loop, and the G-tail invades the double stranded DNA of the T-loop to form a D (for displacement) loop. This entire structure is stabilized by interaction with proteins of the shelterin complex, namely TRF1, TRF2, TIN2, RAP1, TPP1 and POT1 [1,2]. The T-loop structure is thought to protect the ends of linear chromosomes from being recognized by the cell as DNA double strand breaks (DSBs) [3]. Paradoxically, numerous proteins known to be involved in the detection and repair of DSBs are also found at telomeres. One such protein is the DNA-dependent protein kinase catalytic subunit, DNA-PKcs, which plays a critical role in the repair of DSBs by non-homologous end-joining in mammalian cells [4,5]. Another is the Werner Syndrome protein, WRN, a multifunctional protein with 3'-5' DNA helicase and 3'-5' exonuclease activities [6]. Both DNA-PKcs and WRN have functions not only in DNA repair and maintenance of genomic stability, but also in the maintenance of telomere integrity. Loss of DNA-PKcs results in telomere uncapping and chromosomal fusions [7], whereas loss of WRN results in telomere shortening and is associated with a premature aging phenotype [6]. One of the current challenges in telomere biology is to understand how these proteins and protein-DNA complexes regulate telomere length, maintain telomere integrity, and prevent the initiation of a DNA damage response and cellular senescence. In this issue of Aging, Kusumoto-Matsuo and colleagues [8] report that DNA-PKcs stimulates the helicase activity of WRN towards model D loop structures in vitro. DNA-PKcs did not affect the exonuclease activity of WRN or the unwinding activity of the related RecQ helicase, BLM, suggesting that this effect is specific for the WRN helicase activity. Significantly, WRN and DNA-PKcs were shown to cooperate to protect the 3' single stranded ends of telomeres in vivo by preventing G-tail shortening. These findings have important implications for how DNA-PKcs and WRN cooperate to resolve these complex telomeric DNA structures. Moreover, since shortening of the G-overhang contributes to onset of cellular senescence [9], these findings suggest that the WRN/DNA-PKcs interaction might contribute to preventing cellular senescence. This study is the first to report a functional interaction between WRN and DNA-PKcs at telomeres and provides a stepping-stone to further elucidate the critical roles that these proteins play in telomere integrity and cellular senescence. It also raises some interesting questions. For example, it will be interesting to determine whether other proteins present at telomeres such as TRF2, which has recently been shown to regulate the activity of WRN [10], affects the regulation of WRN by DNA-PKcs. Equally interesting will be to determine the molecular mechanism by which DNA-PKcs stimulates the helicase activity of WRN and whether the DNA-PKcs binding partner, Ku, plays a role in this process. It will also be interesting to determine whether WRN-mediated unwinding promotes telomerase accessibility to the telomere for example during S-phase. Interestingly, the authors have also shown that DNA-PKcs stimulates the activity of WRN towards D-loops composed of non-telomeric DNA sequences, suggesting that these findings may extend to D-loop structures formed during other nuclear processes such as homologous recombination. In conclusion, these findings bring us one step closer to unraveling the complex interplay between DNA damage response proteins and telomeres and to understanding how these complex and dynamic structures contribute to the maintenance of genomic stability and the prevention of premature aging.

439 POSTER The cytotoxic activity of the telomere binding agent KML001 in non-small cell lung cancer cells is dependent on telomere length and p53 status and is enhanced by cisplatin

Telomeres are unique DNA tandem repeats that form the ends of eukaryotic chromosomes to protect the chromosomes from degradation and illegitimate recombination. In yeast, loss of telomere may be compensated for through the acquisition of new telomere by RAD52-mediated or RAD52-independent recombinational repair. In this report, the effects of cis-dichlorodiammine-platinum (II) (cisplatin) on telomere length and the role of nucleotide excision repair in telomere maintenance were examined in the yeast Saccharomyces cerevisiae. We showed that the SSL2 (RAD25) DNA repair yeast mutant exhibited a gradual shortening of the telomere in the presence of cisplatin. Further telomere shortening was prevented upon the withdrawal of cisplatin. Complementation of the mutant with the wild-type SSL2 (RAD25) gene abolished the cisplatin-induced telomere degradation. These results suggest that telomeres are susceptible to cisplatin-induced intrastrand crosslinks and that Ssl2 (Rad25) or the nucleotide excision repair pathway may play a critical role in the repair and the maintenance of telomere integrity.

Telomeric Repeat–Containing RNA and RNA Surveillance Factors at Mammalian Chromosome Ends

Telomeres, the DNA-protein complexes located at the end of linear eukaryotic chromosomes, are essential for chromosome stability. Until now, telomeres have been considered to be transcriptionally silent. We demonstrate that mammalian telomeres are transcribed into telomeric repeat-containing RNA (TERRA). TERRA molecules are heterogeneous in length, are transcribed from several subtelomeric loci toward chromosome ends, and localize to telomeres. We also show that suppressors with morphogenetic defects in genitalia (SMG) proteins, which are effectors of nonsense-mediated messenger RNA decay, are enriched at telomeres in vivo, negatively regulate TERRA association with chromatin, and protect chromosome ends from telomere loss. Thus, telomeres are actively transcribed into TERRA, and SMG factors represent a molecular link between TERRA regulation and the maintenance of telomere integrity.

Differential regulation of Ku gene expression in etiolated mung bean hypocotyls by auxins

Plant Ku genes were identified very recently in Arabidopsis thaliana, and their roles in repair of double-stranded break DNA and maintenance of telomere integrity were scrutinized. In this study, the cDNAs encoding Ku70 ( VrKu70) and Ku80 ( VrKu80) were isolated from mung bean ( Vigna radiata L.) hypocotyls. Both genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid implicating that exogenous auxins triggered the effects following their uptake by the cells. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca 2+ chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways.

Structural Basis for Binding of Porphyrin to Human Telomeres,

Maintenance of telomere integrity is a hallmark of human cancer, and the single-stranded 3' ends of telomeric DNA are targets for small-molecule anticancer therapies. We report here the crystal structure of a bimolecular human telomeric quadruplex, of the sequence d(TAGGGTTAGGG), in a complex with the quadruplex-binding ligand 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) to a resolution of 2.09 A. The DNA quadruplex topology is parallel-stranded with external double-chain-reversal propeller loops, consistent with previous structural determinations. The porphyrin molecules bind by stacking onto the TTA nucleotides, either as part of the external loop structure or at the 5' region of the stacked quadruplex. This involves stacked on hydrogen-bonded base pairs, formed from those nucleotides not involved in the formation of G-tetrads, and there are thus no direct ligand interactions with G-tetrads. This is in accord with the relative nonselectivity by TMPyP4 for quadruplex DNAs compared to duplex DNA. Porphyrin binding is achieved by remodeling of loops compared to the ligand-free structures. Implications for the design of quadruplex-binding ligands are discussed, together with a model for the formation of anaphase bridges, which are observed following cellular treatment with TMPyP4.

Karyotypic characteristics of human uterine leiomyoma and myometrial cell lines following telomerase induction

Uterine leiomyomas are the most frequently diagnosed tumors of the female genital tract and the primary cause of hysterectomy in premenopausal women in the United States. In vitro model systems for studying these tumors are limited, due to poor culture growth. For the present study, myometrial (UtSMC) and uterine leiomyoma (UtLM) cell lines and their human telomerase reverse transcriptase (hTERT) immortalized counterparts were evaluated by GTL-banding and spectral karyotyping. UtSMC, at passage 9 showed the normal female karyotype, 46,XX. UtSMC-hTERT at passage 13 (population doubling [PD] 22 post immortalization) had two cell clones: 46,XX,der(13)t(10;13)(q11.2;p13)]/46,XX. UtLM, at passage 14, and the immortalized counterpart UtLM-hTERT (passage 13, PD 20 post immortalization), had the reciprocal translocation t(12;14)(q14;q23) in all cells and monosomy X in a fraction of cells. UtLM also displayed genomic instability with 15% of cells showing structural abnormalities involving chromosome arms 1p and 5q. UtLM-hTERT was karyotypically more stable than the parental line, thereby reflecting the inhibition of the accumulation of cytogenetic abnormalities by the maintenance of telomere integrity. This phenomenon was not observed in the UtSMC cells.

Telomeres, telomerase, and apoptosis.

Telomeres are specialized high-order chromatin structures that cap the ends of eukaryotic chromosomes. In vertebrates, telomeric DNA is composed of repetitions of the TTAGGG hexanucleotide, is bound to a set of specific proteins, and is elongated by the reverse transcriptase enzyme telomerase. Telomerase activity is promptly detected in cells with an indefinite replicative potential, such as cancer cells, while is almost undetectable in normal cells, which are characterized by a limited life span. Mounting evidence indicates that the maintenance of telomere integrity and telomerase protect cells from apoptosis. Disruption of the telomere capping function and (or) telomerase inhibition elicit an apoptotic response in cancer cells, while restoration of telomerase activity in somatic cells confers resistance to apoptosis. The possible mechanisms linking telomeres, telomerase and apoptosis are discussed in this review, together with the impact of this field in anticancer research.

Frequent occurrence of UVB-induced sister chromatid exchanges in telomere regions and its implication to telomere maintenance

Sister chromatid exchanges (SCEs) are symmetrical exchanges between newly replicated chromatids and their sisters. While homologous recombination may be one of the principal mechanisms responsible for SCEs, the full details of their molecular basis and biological significance remain to be elucidated. Following exposure to ultraviolet light B (UVB), mitomycin C (MMC) and cisplatin, we analyzed the location of SCEs on metaphase chromosomes in Chinese hamster CHO cells. The frequency of SCEs increased over the spontaneous level in proportion to the agent’s dose. UVB-induced SCEs occurred frequently in telomere regions, as cisplatin-induced SCEs did, differing from MMC-induced ones. The remarkable difference of intrachromosomal distribution among the three mutagens may be attributed to the specificity of induced DNA lesions and structures of different chromosome regions. Telomeric DNA at the end of chromosomes is composed of multiple copies of a repeated motif, 5′-TTAGGG-3′ in mammalian cells. Telomeric repeats may be potential targets for UVB and cisplatin, which mainly form pyrimidine dimers and intrastrand d(GpG) cross-links, respectively, resulting in SCE formation. UVB irradiation shortened telomeres and augmented the telomerase activity. The possible implications of the frequent occurrence of SCEs in telomere regions are discussed in connection with the maintenance of telomere integrity.