Arabidopsis POT1 associates with the telomerase RNP and is required for telomere maintenance

POT1 is a single-copy gene in yeast and humans that encodes a single-strand telomere binding protein required for chromosome end protection and telomere length regulation. In contrast, Arabidopsis harbors multiple, divergent POT-like genes that bear signature N-terminal OB-fold motifs, but otherwise share limited sequence similarity. Here, we report that plants null for AtPOT1 show no telomere deprotection phenotype, but rather exhibit progressive loss of telomeric DNA. Genetic analysis indicates that AtPOT1 acts in the same pathway as telomerase. In vitro levels of telomerase activity in pot1 mutants are significantly reduced and are more variable than wild-type. Consistent with this observation, AtPOT1 physically associates with active telomerase particles. Although low levels of AtPOT1 can be detected at telomeres in unsynchronized cells and in cells arrested in G2, AtPOT1 binding is significantly enhanced during S-phase, when telomerase is thought to act at telomeres. Our findings indicate that AtPOT1 is a novel accessory factor for telomerase required for positive telomere length regulation, and they underscore the coordinate and extraordinarily rapid evolution of telomere proteins and the telomerase enzyme.     

SGS1 is required for telomere elongation in the absence of telomerase

In S. cerevisiae, mutations in genes that encode telomerase components, such as the genes EST1, EST2, EST3, and TLC1, result in the loss of telomerase activity in vivo. Two telomerase-independent mechanisms can overcome the resulting senescence. Type I survival is characterized by amplification of the subtelomeric Y' elements with a short telomere repeat tract at the terminus. Type II survivors arise through the abrupt addition of long tracts of telomere repeats. Both mechanisms are dependent on RAD52 and on either RAD50 or RAD51. We show here that the telomere elongation pathway in yeast (type II) is dependent on SGS1, the yeast homolog of the gene products of Werner's (WRN) and Bloom's (BLM) syndromes. Survival in the absence of SGS1 and EST2 is dependent upon RAD52 and RAD51 but not RAD50. We propose that the RecQ family helicases are required for processing a DNA structure specific to eroding telomeres.     

An Arabidopsis splicing RNP variant STEP1 regulates telomere length homeostasis by restricting access of nuclease and telomerase

Telomere is an essential DNA-protein complex composed of repetitive DNA and binding proteins to protect the chromosomal ends in eukaryotes. Telomere length is regulated by a specialized RNA-dependent DNA polymerase, telomerase and associated proteins. We show here a potential role of STEP1 that was previously isolated by affinity chromatography in controlling telomere length. While STEP1 requires both RNA-binding domains for telomere binding and subsequent DNA protection, it requires only one RBD to interact with telomerase. The differential telomerase inhibitory activity depending on STEP1 concentrations may suggest that STEP1 contributes to controlling telomere length homeostasis, likely by limiting the accessibility of nuclease or telomerase to telomeric DNA.     

依赖端粒酶的端粒维持机制

端粒是真核生物染色体的末端重要结构复合物,对维持染色体稳定性起着重要作用。端粒酶的主要功能是复制端粒末端DNA,维持端粒长度。端粒酶活性调节与肿瘤发生和细胞衰老有着密切关系。本简要综述近年来依赖端粒酶的端粒维持机理的研究进展。     

ATM is required for telomere maintenance and chromosome stability during Drosophila development

ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.     

AtLAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence

The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T‐DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch‐angle phenotype and the gene consequently named AtLAZY1. Time‐resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch‐angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch‐angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery.     

Arabidopsis MSI1 is required for epigenetic maintenance of reproductive development

WD40 repeat proteins similar to yeast MSI1 are conserved in animals and plants, in which they participate in complexes involved in chromatin metabolism. Although MSI1-like proteins are well characterised biochemically, their function in the development of multicellular eukaryotes is not well understood. We constructed Arabidopsis plants in which the AtMSI1 protein level was altered. Strong ectopic expression of AtMSI1 produced no visible altered phenotype, but reduction of AtMSI1 dramatically affected development. The primary shoot apical meristem was unable to develop organs after the transition to flowering. Flowers that developed on floral shoots from axillary meristems experienced a progressive loss of floral morphology, including a reduction in size of the petals and stamens and the development of carpel-like sepals. Ovule development was disrupted in all flowers, resulting in complete female sterility. Molecular analysis of the mutant plants revealed that AtMSI1 is required to maintain the correct temporal and organ-specific expression of homeotic genes, including AGAMOUS and APETALA2. In contrast, FAS1 and FAS2, which together with AtMSI1 form the chromatin assembly complex CAF-1, are not required for repression of these genes. Therefore, AtMSI1 has specific functions in addition to CAF-1-mediated chromatin assembly. Efficient formation of heterochromatin, but not methylation of centromeric DNA repeats, depends on AtMSI1 presence demonstrating a key role of AtMSI1 in maintenance of chromatin structure.     

A common variant in the telomerase RNA component is associated with short telomere length

Background

Telomeres shorten as cells divide. This shortening is compensated by the enzyme telomerase. We evaluated the effect of common variants in the telomerase RNA component (TERC) gene on telomere length (TL) in the population-based Health Aging and Body Composition (Health ABC) Study and in two replication samples (the TwinsUK Study and the Amish Family Osteoporosis Study, AFOS).

Methodology

Five variants were identified in the TERC region by sequence analysis and only one SNP was common (rs2293607, G/A). The frequency of the G allele was 0.26 and 0.07 in white and black, respectively. Testing for association between TL and rs2293607 was performed using linear regression models or variance component analysis conditioning on relatedness among subjects.

Results

The adjusted mean TL was significantly shorter in 665 white carriers of the G allele compared to 887 non-carriers from the Health ABC Study (4.69±0.05 kbp vs. 4.86±0.04 kbp, measured by quantitative PCR, p = 0.005). This association was replicated in another white sample from the TwinsUK Study (6.90±0.03 kbp in 301 carriers compared to 7.06±0.03 kbp in 395 non-carriers, measured by Southern blots, p = 0.009). A similar pattern of association was observed in whites from the family-based AFOS and blacks from the Health ABC cohort, although not statistically significant, possibly due to the lower allele frequency in these populations. Combined analysis using 2,953 white subjects from 3 studies showed a significant association between TL and rs2293607 (β = −0.19±0.04 kbp, p = 0.001).

Conclusion

Our study shows a significant association between a common variant in TERC and TL in humans, suggesting that TERC may play a role in telomere homeostasis.     

The breast cancer susceptibility gene BRCA2 is required for the maintenance of telomere homeostasis

Inactivating mutations in the breast cancer susceptibility gene BRCA2 cause gross chromosomal rearrangements. Chromosome structural instability in the absence of BRCA2 is thought to result from defective homology-directed DNA repair. Here, we show that BRCA2 links the fidelity of telomere maintenance with genetic integrity. Absence of BRCA2 resulted in signs of dysfunctional telomeres, such as telomere shortening, erosions, and end fusions in proliferating mouse fibroblasts. BRCA2 localized to the telomeres in S phase in an ATR-dependent manner, and its absence resulted in the accumulation of common fragile sites, particularly at the G-rich lagging strand, and increased the telomere sister chromatid exchange in unchallenged cells. The incidence of common fragile sites and telomere sister chromatid exchange increased markedly after treatment with replication inhibitors. Congruently, telomere-induced foci were frequently observed in the absence of Brca2, denoting activation of the DNA damage response and abnormal chromosome end joining. These telomere end fusions constituted a significant portion of chromosome aberrations in Brca2-deficient cells. Our results suggest that BRCA2 is required for telomere homeostasis and may be particularly important for the replication of G-rich telomeric lagging strands.     

Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP

Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.     

Compromised telomere maintenance in hypomethylated Arabidopsis thaliana plants

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are important for the maintenance of genomic stability. Telomeres were considered as typical heterochromatic regions, but in light of recent results, this view should be reconsidered. Asymmetrically located cytosines in plant telomeric DNA repeats may be substrates for a DNA methyltransferase enzyme and indeed, it was shown that these repeats are methylated. Here, we analyse the methylation of telomeric cytosines and the length of telomeres in Arabidopsis thaliana methylation mutants (met 1-3 and ddm 1-8), and in their wild-type siblings that were germinated in the presence of hypomethylation drugs. Our results show that cytosine methylation in telomeric repeats depends on the activity of MET1 and DDM1 enzymes. Significantly shortened telomeres occur in later generations of methylation mutants as well as in plants germinated in the presence of hypomethylation drugs, and this phenotype is stably transmitted to the next plant generation. A possible role of compromised in vivo telomerase action in the observed telomere shortening is hypothesized based on telomere analysis of hypomethylated telomerase knockout plants. Results are discussed in connection with previous data in this field obtained using different model systems.     

Replication proteins influence the maintenance of telomere length and telomerase protein stability

We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres. The telomere elongation was telomerase dependent in the replication mutants analyzed. Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Polalpha, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a polalpha mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Polalpha was severely compromised compared to those in the wild-type polalpha cells. Interestingly, ectopic expression of wild-type polalpha in this polalpha mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability.     

Fission yeast Rhp51 is required for the maintenance of telomere structure in the absence of the Ku heterodimer

The Schizosaccharomyces pombe Ku70–Ku80 heterodimer is required for telomere length regulation. Lack of pku70⁺ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80⁺ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70⁺ increased the single-stranded overhang in both G₂ and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.     

Functional interaction between DNA-PKcs and telomerase in telomere length maintenance

DNA-PKcs is the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex that functions in the non-homologous end-joining of double-strand breaks, and it has been shown previously to have a role in telomere capping. In particular, DNA-PKcs deficiency leads to chromosome fusions involving telomeres produced by leading-strand synthesis. Here, by generating mice doubly deficient in DNA-PKcs and telomerase (Terc(-/-)/DNA-PKcs(-/-)), we demonstrate that DNA-PKcs also has a fundamental role in telomere length maintenance. In particular, Terc(-/-)/DNA-PKcs(-/-) mice displayed an accelerated rate of telomere shortening when compared with Terc(-/-) controls, suggesting a functional interaction between both activities in maintaining telomere length. In addition, we also provide direct demonstration that DNA-PKcs is essential for both end-to-end fusions and apoptosis triggered by critically short telomeres. Our data predict that, in telomerase-deficient cells, i.e. human somatic cells, DNA-PKcs abrogation may lead to a faster rate of telomere degradation and cell cycle arrest in the absence of increased apoptosis and/or fusion of telomere-exhausted chromosomes. These results suggest a critical role of DNA-PKcs in both cancer and aging.