p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. (1/246)

Although broken chromosomes can induce apoptosis, natural chromosome ends (telomeres) do not trigger this response. It is shown that this suppression of apoptosis involves the telomeric-repeat binding factor 2 (TRF2). Inhibition of TRF2 resulted in apoptosis in a subset of mammalian cell types. The response was mediated by p53 and the ATM (ataxia telangiectasia mutated) kinase, consistent with activation of a DNA damage checkpoint. Apoptosis was not due to rupture of dicentric chromosomes formed by end-to-end fusion, indicating that telomeres lacking TRF2 directly signal apoptosis, possibly because they resemble damaged DNA. Thus, in some cells, telomere shortening may signal cell death rather than senescence.  (+info)

TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family. (2/246)

The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. Until recently, it was thought that the general transcriptional machinery was largely invariant and relied on a single TBP, whereas a large and diverse collection of activators and repressors were primarily responsible for imparting specificity to transcription initiation. However, it now appears that the "basal" transcriptional machinery also contributes to specificity via tissue-specific versions of TBP-associated factors as well as a tissue-specific TBP-related factor (TRF1) responsible for gene selectivity in Drosophila. Here we report the cloning of a TBP-related factor (TRF2) that is found in humans, Drosophila, Caenorhabditis elegans, and other metazoans. Like TRF1 and TBP, TRF2 binds transcription factor IIA (TFIIA) and TFIIB and appears to be part of a larger protein complex. TRF2's primary amino acid structure suggests divergence in the putative DNA binding domain, and not surprisingly, it fails to bind to DNA containing canonical TATA boxes. Most importantly, TRF2 is associated with loci on Drosophila chromosomes distinct from either TBP or TRF1, so it may have different promoter specificity and regulate a select subset of genes. These findings suggest that metazoans have evolved multiple TBPs to accommodate the vast increase in genes and expression patterns during development and cellular differentiation.  (+info)

Mammalian telomeres end in a large duplex loop. (3/246)

Mammalian telomeres contain a duplex array of telomeric repeats bound to the telomeric repeat-binding factors TRF1 and TRF2. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3' overhang, activation of p53, and end-to-end chromosome fusions. Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3' telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres.  (+info)

Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. (4/246)

Telomerase-negative immortalized human cells maintain their telomeres by a mechanism known as alternative lengthening of telomeres (ALT). We report here that ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB). APBs are large donut-shaped nuclear structures containing PML protein, telomeric DNA, and the telomere binding proteins human telomere repeat binding factors 1 and 2. Immunostaining showed that APBs also contain replication factor A, RAD51, and RAD52, proteins involved in DNA synthesis and recombination. During immortalization, APBs appeared at exactly the same time as activation of ALT. APBs were found in ALT tumors and cell lines but not in mortal cell strains or in telomerase-positive cell lines or tumors.  (+info)

Altered telomere nuclear matrix interactions and nucleosomal periodicity in ataxia telangiectasia cells before and after ionizing radiation treatment. (5/246)

Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G(1), G(2), and metaphase. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. We examined these interactions in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene.  (+info)

Human TATA-binding protein-related factor-2 (hTRF2) stably associates with hTFIIA in HeLa cells. (6/246)

The TATA-binding protein (TBP)-related factor TRF1, has been described in Drosophila and a related protein, TRF2, has been found in a variety of higher eukaryotes. We report that human (h)TRF2 is encoded by two mRNAs with common protein coding but distinct 5' nontranslated regions. One mRNA is expressed ubiquitously (hTRF2-mRNA1), whereas the other (hTRF2-mRNA2) shows a restricted expression pattern and is extremely abundant in testis. In addition, we show that hTRF2 forms a stable stoichiometric complex with hTFIIA, but not with TAFs, in HeLa cells stably transfected with flag-tagged hTRF2. Neither recombinant human (rh)TRF2 nor the native flag.hTRF2-TFIIA complex is able to replace TBP or TFIID in basal or activated transcription from various RNA polymerase II promoters. Instead, rhTRF2, but not the flag.hTRF2-TFIIA complex, moderately inhibits basal or activated transcription in the presence of rhTBP or flag.TFIID. This effect is either completely (TBP-mediated transcription) or partially (TFIID-mediated transcription) counteracted by addition of free TFIIA. Neither rhTRF2 nor flag. hTRF2-TFIIA has any effect on the repression of TFIID-mediated transcription by negative cofactor-2 (NC2) and neither substitutes for TBP in RNA polymerase III-mediated transcription.  (+info)

DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. (7/246)

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK(cs)-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK(cs) gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.  (+info)

Control of human telomere length by TRF1 and TRF2. (8/246)

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.  (+info)