The telomeric DNA damage response occurs in the absence of chromatin decompaction.

Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.

Genome-wide analysis of in vivo TRF1 binding to chromatin restricts its location exclusively to telomeric repeats.

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that protect them from degradation, end-to-end fusions, and fragility. In mammals, telomeres are composed of TTAGGG tandem repeats bound by a protein complex called shelterin, which has fundamental roles in the regulation of telomere protection and length. The telomeric repeat binding factor 1 (TERF1 or TRF1) is one of the components of shelterin and has been shown to be essential for telomere protection. Telomeric repeats can also be found throughout the genome, as Internal or Interstitial Telomeric Sequences (ITSs). Some of the components of shelterin have been described to bind to ITSs as well as other extra-telomeric regions, which in the case of RAP1 exert a key role in transcriptional regulation. Here, we set to address whether TRF1 can be found at extra-telomeric sites both under normal conditions and upon induction of telomere shortening. In particular, we performed a ChIP-sequencing technique to map TRF1 binding sites in MEFs wild-type and deficient for the telomerase RNA component (Terc(-/-)), with increasingly short telomeres. Our findings indicate that TRF1 is exclusively located at telomeres both under normal conditions, as well as under extreme telomere shortening. These results indicate that in mice not all members of shelterin have extra-telomeric roles as it was described for RAP1.

ATM regulates proteasome-dependent subnuclear localization of TRF1, which is important for telomere maintenance.

Ataxia telangiectasia mutated (ATM), a PI-3 kinase essential for maintaining genomic stability, has been shown to regulate TRF1, a negative mediator of telomerase-dependent telomere extension. However, little is known about ATM-mediated TRF1 phosphorylation site(s) in vivo. Here, we report that ATM phosphorylates S367 of TRF1 and that this phosphorylation renders TRF1 free of chromatin. We show that phosphorylated (pS367)TRF1 forms distinct non-telomeric subnuclear foci and that these foci occur predominantly in S and G2 phases, implying that their formation is cell cycle regulated. We show that phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. We find that a phosphomimic mutation of S367D abrogates TRF1 binding to telomeric DNA and renders TRF1 susceptible to protein degradation. In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers. Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis. Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

Induction of alternative lengthening of telomeres-associated PML bodies by p53/p21 requires HP1 proteins.

Alternative lengthening of telomeres (ALT) is a recombination-mediated process that maintains telomeres in telomerase-negative cancer cells. In asynchronously dividing ALT-positive cell populations, a small fraction of the cells have ALT-associated promyelocytic leukemia nuclear bodies (APBs), which contain (TTAGGG)n DNA and telomere-binding proteins. We found that restoring p53 function in ALT cells caused p21 up-regulation, growth arrest/senescence, and a large increase in cells containing APBs. Knockdown of p21 significantly reduced p53-mediated induction of APBs. Moreover, we found that heterochromatin protein 1 (HP1) is present in APBs, and knockdown of HP1alpha and/or HP1gamma prevented p53-mediated APB induction, which suggests that HP1-mediated chromatin compaction is required for APB formation. Therefore, although the presence of APBs in a cell line or tumor is an excellent qualitative marker for ALT, the association of APBs with growth arrest/senescence and with "closed" telomeric chromatin, which is likely to repress recombination, suggests there is no simple correlation between ALT activity level and the number of APBs or APB-positive cells.

Combination of mRNA and protein microarray analysis in complex cell profiling.

UNLABELLED: This study combines mRNA and protein analysis using cDNA and antibody microarray techniques, respectively. These create a novel, integrated perspective into cellular molecular profiles. The aims of this study were to establish a reliable way of integrating these two approaches in order to obtain complex molecular profiles of the cell and to find suitable methods to normalize the data obtained using these approaches.

Antibody microarray and cDNA microarray techniques were used to study expression alterations in HL-60 cells that were differentiated into granulocytes using all-trans retinoic acid (ATRA). We selected this model to evaluate this combined profiling technique because the expression levels of most of the mRNA and protein species in these cells are not altered; therefore it is easier to track and define those species that are changed. The proteins whose levels were altered included c-myc, c-jun, Pyk2, FAK, PKC, TRF1, NF-kappaB and certain caspase types. These proteins are involved in apoptosis and hematopoietic differentiation pathways, and some have also been reported to have oncogenic potential. We compared the results obtained using the two methods, verified them by immunoblotting analysis, and devised normalization approaches.

This is one of the first demonstrations that a combination of antibody microarray and cDNA microarray techniques is required for complex molecular profiling of cells based on multiple parameters. This approach allows a more detailed molecular phenotype of the given sample to be obtained. The results obtained using a combination of the two profiling methods are consistent with those from previous studies that used more traditional methods.

A role for monoubiquitinated FANCD2 at telomeres in ALT cells.

Both Fanconi anemia (FA) and telomere dysfunction are associated with chromosome instability and an increased risk of cancer. Because of these similarities, we have investigated whether there is a relationship between the FA protein, FANCD2 and telomeres. We find that FANCD2 nuclear foci colocalize with telomeres and PML bodies in immortalized telomerase-negative cells. These cells maintain telomeres by alternative lengthening of telomeres (ALT). In contrast, FANCD2 does not colocalize with telomeres or PML bodies in cells which express telomerase. Using a siRNA approach we find that FANCA and FANCL, which are components of the FA nuclear core complex, regulate FANCD2 monoubiquitination and the telomeric localization of FANCD2 in ALT cells. Transient depletion of FANCD2, or FANCA, results in a dramatic loss of detectable telomeres in ALT cells but not in telomerase-expressing cells. Furthermore, telomere loss following depletion of these proteins in ALT cells is associated with decreased homologous recombination between telomeres (T-SCE). Thus, the FA pathway has a novel function in ALT telomere maintenance related to DNA repair. ALT telomere maintenance is therefore one mechanism by which monoubiquitinated FANCD2 may promote genetic stability.

Telomeric 3' overhangs in chronic HBV-related hepatitis and hepatocellular carcinoma.

Telomeric 3' overhang is a key component of telomere structure, but little is known about its role in hepatocarcinogenesis. We examined the 3' overhang and telomere length, mRNA levels of hTERT, POT1, TRF1 and cytokeratin 19 (CK19) in 41 hepatitis B virus (HBV)-related hepatocellular carcinomas (HCCs) and adjacent non-HCCs of B viral chronic hepatitis/cirrhosis. 3' overhang length was positively correlated with telomere length (p < 0.001). In non-HCCs, the 3' overhang shortened with increasing age (p = 0.043). Twenty-six HCCs had shorter and 15 HCCs had longer 3' overhangs than the adjacent non-HCCs. The mRNA levels of hTERT, POT1 and TRF1 were upregulated in HCCs than in non-HCCs. HCCs with lengthened 3' overhangs expressed higher hTERT mRNA levels than those with shortened 3' overhangs, when compared to 3' overhangs in non-HCCs (p = 0.044). POT1 and TRF1 showed no significant difference according to the 3' overhangs. HCCs with long 3' overhangs had higher mitosis (p = 0.046) and more frequent multipolar mitosis compared to those with short 3' overhangs (p = 0.034). HCCs with high cytokeratin 19 mRNA levels, a marker for hepatic progenitor cells, had longer 3' overhangs than HCCs with low cytokeratin 19 mRNA levels (p= 0.019). In conclusion, the 3' overhang erosion might be closely related to the number of cell divisions in telomerase-negative hepatocytes of chronic hepatitis/cirrhosis. In telomerase-positive HCCs, an altered 3' overhang are involved in HBV-related hepatocarcinogenesis and hTERT might be involved in regulation of 3' overhang.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging.

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

Quantum dot-based western blot technology for ultrasensitive detection of tracer proteins.

The present study describes two quantum dot-based Western blotting protocols for ultrasensitive detection of "tracer" proteins in cell lysates.

The Werner syndrome helicase and exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 and TRF2.

Werner syndrome (WS) is characterized by features of premature aging and is caused by loss of the RecQ helicase protein WRN. WS fibroblasts display defects associated with telomere dysfunction, including accelerated telomere erosion and premature senescence. In yeast, RecQ helicases act in an alternative pathway for telomere lengthening (ALT) via homologous recombination. We found that WRN associates with telomeres when dissociation of telomeric D loops is likely during replication and recombination. In human ALT cells, WRN associates directly with telomeric DNA. The majority of TRF1/PCNA colocalizing foci contained WRN in live S phase ALT cells but not in telomerase-positive HeLa cells. Biochemically, the WRN helicase and 3' to 5' exonuclease act simultaneously and cooperate to release the 3' invading tail from a telomeric D loop in vitro. The telomere binding proteins TRF1 and TRF2 limit digestion by WRN. We propose roles for WRN in dissociating telomeric structures in telomerase-deficient cells.

Gene expression of telomerase related proteins in human normal oral and ectocervical epithelial cells.

We analyzed telomerase activities and gene expressions of telomerase components: hTERT, hTR, hTEP1, telomeric repeat binding factors: TRF1, TRF2, and c-myc, Max and Mad in human normal oral and ectocervical epithelial keratinocytes, comparing with those of squamous carcinoma cells and HPV16- or SV40-immortalized cells. Significant telomerase activity and hTERT expression were detected in primary keratinocytes. However, both were dramatically down-regulated during serial passages. The down-regulation of hTERT mRNA was associated with augmented expression of TRF1. Expression of c-myc was slightly decreased, whereas Mad was expressed in parallel with that of hTERT during passages. We also detected an alternate splicing of hTERT transcript in two of four cancer cells and normal aged epithelial cells. These results suggest that the senescence of normal oral and ectocervical keratinocytes is accompanied with up-regulation of TRF1 and down-regulation of telomerase activity due to transcriptional suppression of active form of hTERT in vitro.

[Preparation and characterization of monoclonal antibody against human telomeric repeat binding factor 1].

OBJECTIVE: To prepare a monoclonal antibody against human telomeric repeat binding factor 1 (TRF1) protein and explore its biological characteristics. METHODS: BALB/c mice were immunized with GST-TRF1(33-277) fusion protein for the preparation of monoclonal antibody by hybridoma technique. The obtained antibody was used for clinical assay by Western-blot and immunohistochemical staining. RESULTS: One strain of hybridoma was obtained. It was confirmed by Western-blot that the antibody specifically recognized the 60 kD TRF1 protein. Immunohistochemical staining of the antibody showed that TRF1 protein located in the cytoplasm of epithelial cells and bone marrow cells. CONCLUSION: A TRF1 monoclonal antibody, with high specificity was developed. It is useful for detection of TRF1 protein in tissue specimens.