Quantitative telomeric chromatin isolation protocol for human cells.

The ends of eukaryotic chromosomes, known as telomeres, consist of repetitive DNA sequences, multiple proteins and noncoding RNAs. Telomeres are dynamic structures that play crucial roles as guardians of genome stability and tumor suppressors. Defects in telomere length or protein composition can accelerate aging and are seen in telomere syndromes, which affect various proliferative tissues such as the bone marrow or the lungs. One of the biggest challenges in the telomere field is to identify the molecular changes at telomeres that occur during normal development, in cancer and in telomere syndromes. To tackle this problem, our laboratory has established a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analyzed by mass spectrometry. QTIP involves stable isotope labeling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states.

The human CST complex is a terminator of telomerase activity.

The lengths of human telomeres, which protect chromosome ends from degradation and end fusions, are crucial determinants of cell lifespan. During embryogenesis and in cancer, the telomerase enzyme counteracts telomeric DNA shortening. As shown in cancer cells, human telomerase binds the shelterin component TPP1 at telomeres during the S phase of the cell cycle, and adds ~60 nucleotides in a single round of extension, after which telomerase is turned off by unknown mechanisms. Here we show that the human CST (CTC1, STN1 and TEN1) complex, previously implicated in telomere protection and DNA metabolism, inhibits telomerase activity through primer sequestration and physical interaction with the protection of telomeres 1 (POT1)­TPP1 telomerase processivity factor. CST competes with POT1­TPP1 for telomeric DNA, and CST­telomeric-DNA binding increases during late S/G2 phase only on telomerase action, coinciding with telomerase shut-off. Depletion of CST allows excessive telomerase activity, promoting telomere elongation. We propose that through binding of the telomerase-extended telomere, CST limits telomerase action at individual telomeres to approximately one binding and extension event per cell cycle. Our findings define the sequence of events that occur to first enable and then terminate telomerase-mediated telomere elongation.

The Rat1p 5' to 3' exonuclease degrades telomeric repeat-containing RNA and promotes telomere elongation in Saccharomyces cerevisiae.

Vertebrate telomeres are transcribed into telomeric repeat-containing RNA (TERRA) that associates with telomeres and may be important for telomere function. Here, we demonstrate that telomeres are also transcribed in Saccharomyces cerevisiae by RNA polymerase II (RNAPII). Yeast TERRA is polyadenylated and stabilized by Pap1p and regulated by the 5' to 3' exonuclease, Rat1p. rat1-1 mutant cells accumulate TERRA and harbor short telomeres because of defects in telomerase-mediated telomere elongation. Overexpression of RNaseH overcomes telomere elongation defects in rat1-1 cells, indicating that RNA/DNA hybrids inhibit telomerase function at chromosome ends in these mutants. Thus, telomeric transcription combined with Rat1p-dependent TERRA degradation is important for regulating telomerase in yeast. Telomere transcription is conserved in different kingdoms of the eukaryotic domain.

Human UPF1 interacts with TPP1 and telomerase and sustains telomere leading-strand replication.

Eukaryotic up-frameshift 1 (UPF1) is a nucleic acid-dependent ATPase and 5'-to-3' helicase, best characterized for its roles in cytoplasmic RNA quality control. We previously demonstrated that human UPF1 binds to telomeres in vivo and its depletion leads to telomere instability. Here, we show that UPF1 is present at telomeres at least during S and G2/M phases and that UPF1 association with telomeres is stimulated by the phosphoinositide 3-kinase (PI3K)-related protein kinase ataxia telangiectasia mutated and Rad3-related (ATR) and by telomere elongation. UPF1 physically interacts with the telomeric factor TPP1 and with telomerase. Akin to UPF1 binding to telomeres, this latter interaction is mediated by ATR. Moreover, the ATPase activity of UPF1 is required to prevent the telomeric defects observed upon UPF1 depletion, and these defects stem predominantly from inefficient telomere leading-strand replication. Our results portray a scenario where UPF1 orchestrates crucial aspects of telomere biology, including telomere replication and telomere length homeostasis.

A three-state model for the regulation of telomerase by TERRA and hnRNPA1.

Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeat-containing RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5'-UUAGGG-3' repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms. Also, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has been implicated in telomere length control. In vivo, TERRA is partially associated with hnRNPA1, and hnRNPA1 is also detected at telomeres. We demonstrate that on binding of TERRA, hnRNPA1 can alleviate the TERRA-mediated inhibition of telomerase. However, when in excess over TERRA, hnRNPA1 becomes itself an inhibitor of telomere extension, on binding of the telomeric DNA substrate. Yet, hnRNPA1 has no notable direct effects on the telomerase catalysis. Our in vitro results suggest that TERRA-mediated telomerase inhibition may be prevented by hnRNPA1 in vivo. Telomere extension by telomerase may require balanced levels of TERRA and hnRNPA1 at telomeres. Thus, TERRA and hnRNPA1 can function as a bimolecular regulator to turn telomerase and the telomere on and off.

Subtelomeric repetitive elements determine TERRA regulation by Rap1/Rif and Rap1/Sir complexes in yeast.

Telomeric repeat-containing RNA (TERRA) has been implicated in the control of heterochromatin and telomerase. We demonstrate that yeast TERRA is regulated by telomere-binding proteins in a chromosome-end-specific manner that is dependent on subtelomeric repetitive DNA elements. At telomeres that contain only X-elements, the Rap1 carboxy-terminal domain recruits the Sir2/3/4 and Rif1/2 complexes to repress transcription in addition to promoting Rat1-nuclease-dependent TERRA degradation. At telomeres that contain Y' elements, however, Rap1 represses TERRA through recruitment of Rif1 and Rif2. Our work emphasizes the importance of subtelomeric DNA in the control of telomeric protein composition and telomere transcription.

The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase.

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5'-UUAGGG-3' repeats near their 3'-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5'-UUAGGG-3' repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

Protein RNA and protein protein interactions mediate association of human EST1A/SMG6 with telomerase.

The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme telomerase. In an attempt to better understand hEST1A function, we have started to dissect the molecular interactions between hEST1A and telomerase. Here, we demonstrate that the interaction between hEST1A and telomerase is mediated by protein-RNA and protein-protein contacts. We identify a domain within hEST1A that binds the telomerase RNA moiety hTR while full-length hEST1A establishes in addition RNase-resistant and hTR-independent protein-protein contacts with the human telomerase reverse transcriptase polypeptide (TERT). Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity. Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall K(d) = 25 nM) but low specificity. We propose that hEST1A assembles specifically with telomerase in the context of the hTR-hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific protein-protein contacts with hTERT.

Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG3(T2AG3)3 and (T2AG3)4 human telomere sequences in Na+ and K+ solutions.

The quadruplex structures of the human telomere sequences AG3(T2AG3)3 I and (T2AG3)4 II were investigated in the presence of Na+ and K+ ions, through the cross-linking of adenines and guanines by the cis- and trans-[Pt(NH3)2(H2O)2](NO3)2 complexes 1 and 2. The bases involved in chelation of the cis- and trans-Pt(NH3)2 moieties were identified by chemical and 3'-exonuclease digestions of the products isolated after denaturing gel electrophoresis. These are the four adenines of each sequence and four out of the 12 guanines. Two largely different structures have been reported for I: A from NMR data in Na+ solution and B from X-ray data of a K+-containing crystal. Structure A alone agrees with our conclusions about the formation of the A1-G10, A13-G22, A1-A13 platinum chelates at the top of the quadruplex and A7-A19, G4-A19 and A7-G20 at the bottom, whether the Na+ or K+ ion is present. At variance with a recent proposal that structures A and B could be the major species in Na+ and K+ solutions, respectively, our results suggest that structure A exists predominantly in the presence of both ions. They also suggest that covalent platinum cross-linking of a human telomere sequence could be used to inhibit telomerase.

Peroxiredoxin 1 Protects Telomeres from Oxidative Damage and Preserves Telomeric DNA for Extension by Telomerase.

Oxidative damage of telomeres can promote cancer, cardiac failure, and muscular dystrophy. Specific mechanisms protecting telomeres from oxidative damage have not been described. We analyzed telomeric chromatin composition during the cell cycle and show that the antioxidant enzyme peroxiredoxin 1 (PRDX1) is enriched at telomeres during S phase. Deletion of the PRDX1 gene leads to damage of telomeric DNA upon oxidative stress, revealing a protective function of PRDX1 against oxidative damage at telomeres. We also show that the oxidized nucleotide 8-oxo-2'deoxyguanosine-5'-triphosphate (8oxodGTP) causes premature chain termination when incorporated by telomerase and that some DNA substrates terminating in 8oxoG prevent extension by telomerase. Thus, PRDX1 safeguards telomeres from oxygen radicals to counteract telomere damage and preserve telomeric DNA for elongation by telomerase.

Cross-links of quadruplex structures from human telomeric DNA by dinuclear platinum complexes show the flexibility of both structures.

The folding of AG(3)(T(2)AG(3))(3) was investigated in the presence of Na(+) or K(+) ions, by using the dinuclear platinum complexes [{trans-PtCl(NH(3))(2)}(2)H(2)N(CH(2))(n)NH(2)]Cl(2) (n = 2 or 6). AG(3)(T(2)AG(3))(3) has been previously found to adopt two different quadruplex structures: the antiparallel one in a solution containing Na(+) and the parallel one in a K(+)-containing crystal. The two structures are strikingly distinct and are not expected to form the same platinum cross-links. Therefore, characterization of the cross-links formed with platinum complexes in solution allowed the predominant conformation(s) to be identified. The bases coordinating the platinum atoms were identified by chemical and 3'-exonuclease digestions. The observed cross-links showed that the parallel structure exists in solution whatever the cation and confirmed the existence of the antiparallel structure in the presence of both cations as previously reported from cross-linking experiments of AG(3)(T(2)AG(3))(3) by mononuclear platinum complexes. Furthermore, the major platinum cross-links were unexpectedly formed between two guanines belonging to the same G-quartet. Their formation was rationalized using molecular dynamics simulations in implicit solvent of the two quadruplex structures. It was shown that they were flexible, allowing some guanines to leave reversibly the top G-quartet and thus rendering their N(7) atom accessible to platinum complexes. Our results also suggest that the human telomere sequence could be a target for such platinum complexes.

Unprecedented selective ipso-nitration of calixarenes monitored by the O-substituents.

The electrophilic ipso-reactions of a tBu-calix[6]arene that presents alternate O-methyl and O-2-methylen-N-methyl-imidazolyl groups (1) at the small rim have been studied. Whereas 1 underwent per-sulfonation in sulfuric acid, it selectively reacted with nitric acid to yield a tris-nitro derivative. The ipso-nitration occurred regioselectively on the calixarene anisol units. The reaction has been studied with various tBu-calixarenes (2-11) presenting alternate anisol and phenol ether units. The regioselectivity of the process appeared to be correlated to the presence of a protonable site on the O-substituent. It is proposed that the corresponding protonated heteroatom (N for the amines, O for the amides and the carboxylic acid), situated in the gamma or epsilon position of the phenoxy moieties, deactivates the corresponding aromatic ring by removing electron density through intramolecular hydrogen bonding. The high control operated by the O-substituents at the small rim even allowed the selective ipso-nitration of partially detertiobutylated calixarene 1(H3). Hence, these findings open new routes to a wide range of nonsymmetrically substituted calixarenes at the large rim.