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.

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres.

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.

Proteolytic cleavage of ALF into alpha- and beta-subunits that form homologous and heterologous complexes with somatic TFIIA and TRF2 in male germ cells.

Male germ cells specifically express paralogues of components of the general transcription apparatus including ALF a paralogue of TFIIAalpha/beta. We show that endogenous ALF is proteolytically cleaved to give alpha- and beta-subunits and we map the proteolytic cleavage site by mass spectrometry. Immunoprecipitations show that ALFalpha- and beta-subunits form a series of homologous and heterologous complexes with somatic TFIIA which is coexpressed in male germ cells. In addition, we show that ALF is coexpressed in late pachytene spermatocytes and in haploid round spermatids with transcription factor TRF2, and that these proteins form stable complexes in testis extracts. Our observations highlight how cleavage of ALF and coexpression with TFIIA and TRF2 increases the combinatorial possibilities for gene regulation at different developmental stages of spermatogenesis.

hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere.

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

[Quantitative detection of telomere binding factor 2 gene expression in non-Hodgkin lymphoma with a real-time reverse transcription-polymerase chain reaction assay].

OBJECTIVE: To detect the expression levels of telomere binding factor 2 (TRF2) mRNA in tumor tissue of non-Hodgkin lymphoma (NHL) patients using quantitative real-time RT-PCR. METHODS: The target gene mRNA was amplified with RT-PCR, then was sequentially electrophoresed and purified as standards, and the standard curves of gene expression were established. The expression levels of TRF2 mRNA of lymphoid tissue from NHL and reactive lymphoadenopathy were detected with real-time RT-PCR. RESULTS: The correlation coefficient was 0.996 between the amount of template cDNA and the intensity of fluorescence signal when gene expression standard curves were established. The correlation coefficient of template cDNA amount and grey density of bands derived from gel electrophoresis of real-time RT-PCR final products was 0.779 (P<0.05). Of all NHL patients, expression levels of TRF2 mRNA of follicular lymphoma, mantle cell lymphoma and diffuse large B cell lymphoma were(22.943 +/-9.424) amol, (23.181 +/-5.983) amol and (18.339 +/-7.910) amol, respectively, which had no significant difference compared with reactive lymphoadenopathy [(21.796 +/-4.800) amol, P>0.05]. The expression level of TRF2 mRNA of Burkitt lymphoma was (33.170 +/-12.841) amol, which was significantly higher than that of reactive lymphoadenopathy and other types of NHL (P<0.05). CONCLUSION: Alcohol drinking isn't one of the risk factors of colorectal cancer among Jiashan County population.

TRF2 is in neuroglial cytoplasm and induces neurite-like processes.

TRF2 is a ubiquitous protein that protects telomeres in the nucleus. We found that TRF2 was present at the peripheral nerve axons and the brain neuroglial cell processes extensively. It was in the cytoplasmic membrane as well as nuclear fractions, but not in the soluble cytoplasmic fraction of SH-SY5Y neuroblastoma cells. TRF2 was up-regulated in P19 embryonal carcinoma cells at the early stage of induced neural differentiation with retinoic acid treatment. Upon transfection, TRF2-expressing COS cells often produced neurite-like long cytoplasmic processes. TRF2 is a component of neuroglial cells and appears to be involved in the cytoplasmic process formation that is necessary for neural differentiation.

Cell cycle-dependent 3D distribution of telomeres and telomere repeat-binding factor 2 (TRF2) in HaCaT and HaCaT-myc cells.

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.

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.