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T de Lange

Publications and source records attributed to T de Lange.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mammalian telomeres end in a large duplex loop.

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.

Animals↗
PubMed1999

p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2.

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.

Adenoviridae↗
PubMed1999

Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes.

Tankyrase is a human poly(ADP-ribose) polymerase that was initially identified through its interaction with the telomeric protein TRF1, a negative regulator of telomere length. In vitro poly(ADP-ribosyl)ation by tankyrase inhibits TRF1 binding to telomeric DNA suggesting a role for tankyrase in telomere function. We previously demonstrated that tankyrase co-localizes with TRF1 at the ends of human chromosomes in metaphase. Here we show that tankyrase localizes to additional subcellular sites in a cell cycle dependent manner. In interphase, tankyrase co-localized with TRF1 to telomeres, but in addition was found to reside at nuclear pore complexes, as evidenced by indirect immunofluorescence, subcellular fractionation and immunoelectron microscopy. At mitosis, concomitant with nuclear envelope breakdown and nuclear pore complex disassembly, tankyrase was found to relocate around the pericentriolar matrix of mitotic centrosomes. This complex staining pattern along with the observation that tankyrase did not contain a nuclear localization signal suggested that its telomeric localization might be regulated, perhaps by TRF1. Indeed, localization of exogenously-expressed tankyrase to telomeres was dependent upon co-transfection with TRF1. These data indicate that the subcellular localization of tankyrase can be regulated by both the cell cycle and TRF1.

Cell Cycle↗
PubMed1998

Tankyrase, a poly(ADP-ribose) polymerase at human telomeres.

Tankyrase, a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP), was identified and localized to human telomeres. Tankyrase binds to the telomeric protein TRF1 (telomeric repeat binding factor-1), a negative regulator of telomere length maintenance. Like ankyrins, tankyrase contains 24 ankyrin repeats in a domain responsible for its interaction with TRF1. Recombinant tankyrase was found to have PARP activity in vitro, with both TRF1 and tankyrase functioning as acceptors for adenosine diphosphate (ADP)-ribosylation. ADP-ribosylation of TRF1 diminished its ability to bind to telomeric DNA in vitro, suggesting that telomere function in human cells is regulated by poly(ADP-ribosyl)ation.

Adenosine Diphosphate Ribose↗
PubMed1998

TRF1 promotes parallel pairing of telomeric tracts in vitro.

Human telomeres consist of long arrays of TTAGGG repeats bound to the telomere-specific proteins, TRF1 and TRF2. Here we describe the structure of in vitro complexes formed between telomeric DNA and TRF1 as deduced by electron microscopy. Visualization of TRF1 bound to DNA containing six or 12 tandem TTAGGG repeats revealed a population of DNAs containing a spherical protein complex localized just to the repeats. Mass analysis of the protein complexes suggested binding of TRF1 dimers and tetramers to the TTAGGG repeats. The DNA was not significantly compacted or extended by protein binding. TRF1 formed filamentous structures on longer telomeric repeat arrays (>/=27 repeats) consistent with the presence of an array of bound TRF1 dimers. Unexpectedly, there was a strong propensity for two telomeric tracts to form paired synapses over the TRF1 covered segment. Up to 30% of the TRF1-bound DNAs could be found in a paired configuration with a strong bias for a parallel as contrasted to an antiparallel arrangement. TRF1-induced pairing was confirmed using a ligation assay which detected the formation of DNA multimers dependent on the presence of TRF1 and a 27mer repeat array in the DNA. These findings suggests that this protein may have an architectural role at telomeres. We discuss the possibility that TRF1-dependent changes in the conformation of telomeres are involved in the regulation of telomere length.

DNA↗
PubMed1998

TRF2 protects human telomeres from end-to-end fusions.

The mechanism by which telomeres prevent end-to-end fusion has remained elusive. Here, we show that the human telomeric protein TRF2 plays a key role in the protective activity of telomeres. A dominant negative allele of TRF2 induced end-to-end chromosome fusions detectable in metaphase and anaphase cells. Telomeric DNA persisted at the fusions, demonstrating that TTAGGG repeats per se are not sufficient for telomere integrity. Molecular analysis suggested that the fusions represented ligation of telomeres that have lost their single-stranded G-tails. Therefore, TRF2 may protect chromosome ends by maintaining the correct structure at telomere termini. In addition, expression of mutant forms of TRF2 induced a growth arrest with characteristics of senescence. The results raise the possibility that chromosome end fusions and senescence in primary human cells may be caused by loss by TRF2 from shortened telomeres.

Anaphase↗
PubMed1997

TRF1 is a dimer and bends telomeric DNA.

TRF1 is a mammalian telomeric protein that binds to the duplex array of TTAGGG repeats at chromosome ends. TRF1 has homology to the DNA-binding domain of the Myb family of transcription factors but, unlike most Myb-related proteins, TRF1 carries one rather than multiple Myb-type DNA-binding motifs. Here we show that TRF1 binds DNA as a dimer using a large conserved domain near the N-terminus of the protein for TRF1-TRF1 interactions. Dimerization was observed both in a complex with DNA and in the yeast two-hybrid assay. TRF1 dimers were found to require both Myb repeats for the formation of a stable complex with DNA, indicating a parallel between the DNA-binding mode of TRF1 and other Myb-related proteins. TRF1 was found to have a number of biochemical similarities to Rap1p, a distantly related DNA-binding protein that functions at telomeres in yeast. Rap1p and TRF1 both require two Myb motifs for DNA binding and both factors bind along their cognate telomeric sequences without showing strong cooperative interactions between adjacent proteins. Furthermore, TRF1 was found to bend its telomeric site to an angle of -120 degrees. Since Rap1p similarly distorts telomeric DNA, we propose that DNA bending is important for the function of telomeres in yeast and mammals.

Amino Acid Sequence↗
PubMed1997

Control of telomere length by the human telomeric protein TRF1.

Human telomeres, the nucleoprotein complexes at chromosome ends, consist of tandem arrays of TTAGGG repeats bound to specific proteins. In normal human cells, telomeres shorten with successive cell divisions, probably due to the terminal sequence loss that accompanies DNA replication. In tumours and immortalized cells, this decline is halted through the activation of telomerase, a reverse transcriptase that extends the telomeric TTAGGG-repeat arrays. Telomere length is stable in several immortal human-cell lines, suggesting that a regulatory mechanism exists for limiting telomere elongation by telomerase. Here we show that the human telomeric-repeat binding factor TRF1 (ref. 8) is involved in this regulation. Long-term overexpression of TRF1 in the telomerase-positive tumour-cell line HT1080 resulted in a gradual and progressive telomere shortening. Conversely, telomere elongation was induced by expression of a dominant-negative TRF1 mutant that inhibited binding of endogenous TRF1 to telomeres. Our results identify TRF1 as a suppressor of telomere elongation and indicate that TRF1 is involved in the negative feedback mechanism that stabilizes telomere length. As TRF1 does not detectably affect the expression of telomerase, we propose that the binding of TRF1 controls telomere length in cis by inhibiting the action of telomerase at the ends of individual telomeres.

DNA-Binding Proteins↗
PubMed1997

TRF1, a mammalian telomeric protein.

Telomerase adds TTAGGG repeats onto mammalian chromosome ends, replenishing the terminal sequence loss incurred during DNA replication. This maintenance of telomeric DNA preserves binding sites for telomeric proteins, which form a protective nucleoprotein complex at chromosome ends. The recent isolation of TRF1, the mammalian telomeric-repeat binding factor, should now allow the structure and function of the telomeric complex to be examined in detail.

Amino Acid Sequence↗
PubMed1997

Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2.

Human telomeres are composed of long arrays of TTAGGG repeats that form a nucleoprotein complex required for the protection and replication of chromosome ends. One component of human telomeres is the TTAGGG repeat binding factor 1 (TRF1), a ubiquitously expressed protein, related to the protooncogene Myb, that is present at telomeres throughout the cell cycle. Recent evidence has implicated TRF1 in the control of telomere length. TRF1 is proposed to be an inhibitor of telomerase, acting in cis to limit the elongation of individual chromosome ends. Here we report the cloning of TRF2, a distant homologue of TRF1 that carries a very similar Myb-related DNA-binding motif. Like TRF1, TRF2 was ubiquitously expressed, bound specifically to duplex TTAGGG repeats in vitro and localized to all human telomeres in metaphase chromosomes. TRF2 was shown to have an architecture similar to that of TRF1 in that it carries a C-terminal Myb motif and a large TRF1-related dimerization domain near its N terminus. However, the dimerization domains of TRF1 and TRF2 did not interact, suggesting that these proteins exist predominantly as homodimers. While having similar telomere binding activity and domain organization, TRF2 differed from TRF1 in that its N terminus was basic rather than acidic, and TRF2 was much more conserved than TRF1. The results indicate that the TTAGGG repeat arrays at the ends of human and mouse chromosomes bind to two related proteins. Because TRF1 and TRF2 showed significant differences, we suggest that these factors have distinct functions at telomeres.

Amino Acid Sequence↗
PubMed1997

Comparison of the human and mouse genes encoding the telomeric protein, TRF1: chromosomal localization, expression and conserved protein domains.

Mammalian chromosome ends contain long arrays of TTAGGG repeats that are complexed to a telomere specific protein, the TTAGGG repeat binding factor, TRF1. Here we describe the characterization of genes encoding the human and mouse TRF1 proteins, hTRF1 and mTRF1. The mTRF1 cDNA was isolated based on sequence similarity to the hTRF1 cDNA and the mTRF1 mRNA was shown to be ubiquitously expressed as a single 1.9 kb polyadenylated transcript in mouse somatic tissues. High levels of a 2.1 kb transcript were found in testes. In vitro translation of the mTRF1 cDNA resulted in a 56 kDa protein that binds to TTAGGG repeat arrays. mTRF1 displayed the same sequence specificity as hTRF1, preferring arrays of TTAGGG repeats as a binding substrate over TTAGGC and TTGGGG repeats. Expression of an epitope-tagged version of mTRF1 showed that the protein is located at the ends of murine metaphase chromosomes. In agreement, conceptual translation indicated that mTRF1 and hTRF1 are similarly-sized proteins with nearly identical C-terminal Myb-related DNA binding motifs. In addition, comparison of the predicted mTRF1 and hTRF1 amino acid sequences showed that the acidic nature of the N-terminus of TRF1 is conserved and revealed a highly conserved novel domain of approximately 200 amino acids in the middle of the proteins. However, other regions of the proteins are poorly conserved (<35% identity) and the overall level of identity of the mTRF1 and hTRF1 amino acid sequences is only 67%. The TRF1 genes are not syntenic; the hTRF1 gene localized to human chromosome 8 band q13 while the mTRF1 gene localized to mouse chromosome 17 band E3. The data indicate that the genes for mammalian telomeric proteins evolve rapidly.

Amino Acid Sequence↗
PubMed1996

Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex.

Mammalian telomeres are composed of long arrays of TTAGGG repeats complexed with the TTAGGG repeat binding factor, TRF. Biochemical and ultrastructural data presented here show that the telomeric DNA and TRF colocalize in individual, condensed structures in the nuclear matrix. Telomeric TTAGGG repeats were found to carry an array of nuclear matrix attachment sites occurring at a frequency of at least one per kb. The nuclear matrix association of the telomeric arrays extended over large domains of up to 20-30 kb, encompassing the entire length of most mammalian telomeres. TRF protein and telomeric DNA cofractionated in nuclear matrix preparations and colocalized in discrete, condensed sites throughout the nuclear volume. FISH analysis indicated that TRF is an integral component of the telomeric complex and that the presence of TRF on telomeric DNA correlates with the compact configuration of telomeres and their association with the nuclear matrix. Biochemical fractionation of TRF and telomeric DNA did not reveal an interaction with the nuclear lamina. Furthermore, ultrastructural analysis indicated that the mammalian telomeric complex occupied sites throughout the nuclear volume, arguing against a role for the nuclear envelope in telomere function during interphase. These results are consistent with the view that mammalian telomeres form nuclear matrix-associated, TRF-containing higher order complexes at dispersed sites throughout the nuclear volume.

Adenocarcinoma↗
PubMed1996

Telomerase activation in mouse mammary tumors: lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation.

Activation of telomerase in human cancers is thought to be necessary to overcome the progressive loss of telomeric DNA that accompanies proliferation of normal somatic cells. According to this model, telomerase provides a growth advantage to cells in which extensive terminal sequence loss threatens viability. To test these ideas, we have examined telomere dynamics and telomerase activation during mammary tumorigenesis in mice carrying a mouse mammary tumor virus long terminal repeat-driven Wnt-1 transgene. We also analyzed Wnt-1-induced mammary tumors in mice lacking p53 function. Normal mammary glands, hyperplastic mammary glands, and mammary carcinomas all had the long telomeres (20 to 50 kb) typical of Mus musculus and did not show telomere shortening during tumor development. Nevertheless, telomerase activity and the RNA component of the enzyme were consistently upregulated in Wnt-1-induced mammary tumors compared with normal and hyperplastic tissues. The upregulation of telomerase activity and RNA also occurred during tumorigenesis in p53-deficient mice. The expression of telomerase RNA correlated strongly with histone H4 mRNA in all normal tissues and tumors, indicating that the RNA component of telomerase is regulated with cell proliferation. Telomerase activity in the tumors was elevated to a greater extent than telomerase RNA, implying that the enzymatic activity of telomerase is regulated at additional levels. Our data suggest that the mechanism of telomerase activation in mouse mammary tumors is not linked to global loss of telomere function but involves multiple regulatory events including upregulation of telomerase RNA in proliferating cells.

Animals↗