scholarly journals Control of Human Telomere Length by TRF1 and TRF2

2000 ◽  
Vol 20 (5) ◽  
pp. 1659-1668 ◽  
Author(s):  
Agata Smogorzewska ◽  
Bas van Steensel ◽  
Alessandro Bianchi ◽  
Stefan Oelmann ◽  
Matthias R. Schaefer ◽  
...  
Keyword(s):  
Telomere Length ◽  
Negative Regulator ◽  
Loop Model ◽  
Telomeric Dna ◽  
Telomere Elongation ◽  
Original Length ◽  
Population Doublings ◽  
Ttaggg Repeat

ABSTRACT 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.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

scholarly journals Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

2006 ◽  
Vol 27 (3) ◽  
pp. 1017-1026 ◽  
Author(s):  
Bryan E. Snow ◽  
Maria Mateyak ◽  
Jana Paderova ◽  
Andrew Wakeham ◽  
Caterina Iorio ◽  
...  
Keyword(s):  
Telomere Length ◽  
Chromosomal Rearrangements ◽  
Dna Helicase ◽  
Negative Regulator ◽  
Wild Type ◽  
Genetic Redundancy ◽  
Telomeric Dna ◽  
Dna Helicases

ABSTRACT Pif1 is a 5′-to-3′ DNA helicase critical to DNA replication and telomere length maintenance in the budding yeast Saccharomyces cerevisiae. ScPif1 is a negative regulator of telomeric repeat synthesis by telomerase, and recombinant ScPif1 promotes the dissociation of the telomerase RNA template from telomeric DNA in vitro. In order to dissect the role of mPif1 in mammals, we cloned and disrupted the mPif1 gene. In wild-type animals, mPif1 expression was detected only in embryonic and hematopoietic lineages. mPif1 − / − mice were viable at expected frequencies, displayed no visible abnormalities, and showed no reproducible alteration in telomere length in two different null backgrounds, even after several generations. Spectral karyotyping of mPif1 − / − fibroblasts and splenocytes revealed no significant change in chromosomal rearrangements. Furthermore, induction of apoptosis or DNA damage revealed no differences in cell viability compared to what was found for wild-type fibroblasts and splenocytes. Despite a novel association of mPif1 with telomerase, mPif1 did not affect the elongation activity of telomerase in vitro. Thus, in contrast to what occurs with ScPif1, murine telomere homeostasis or genetic stability does not depend on mPif1, perhaps due to fundamental differences in the regulation of telomerase and/or telomere length between mice and yeast or due to genetic redundancy with other DNA helicases.

scholarly journals The Yeast Telomere Length Counting Machinery Is Sensitive to Sequences at the Telomere-Nontelomere Junction

1999 ◽  
Vol 19 (1) ◽  
pp. 31-45 ◽  
Author(s):  
Alo Ray ◽  
Kurt W. Runge
Keyword(s):  
Telomere Length ◽  
Feedback Mechanism ◽  
Yeast Cells ◽  
Telomere Elongation ◽  
Negative Feedback Mechanism ◽  
Folded Structure ◽  
Length Regulation ◽  
Threshold Length

ABSTRACT Saccharomyces cerevisiae telomeres consist of a continuous 325 ± 75-bp tract of the heterogeneous repeat TG1-3 which contains irregularly spaced, high-affinity sites for the protein Rap1p. Yeast cells monitor or count the number of telomeric Rap1p molecules in a negative feedback mechanism which modulates telomere length. To investigate the mechanism by which Rap1p molecules are counted, the continuous telomeric TG1-3 sequences were divided into internal TG1-3 sequences and a terminal tract separated by nontelomeric spacers of different lengths. While all of the internal sequences were counted as part of the terminal tract across a 38-bp spacer, a 138-bp disruption completely prevented the internal TG1-3 sequences from being considered part of the telomere and defined the terminal tract as a discrete entity separate from the subtelomeric sequences. We also used regularly spaced arrays of six Rap1p sites internal to the terminal TG1-3 repeats to show that each Rap1p molecule was counted as about 19 bp of TG1-3 in vivo and that cells could count Rap1p molecules with different spacings between tandem sites. As previous in vitro experiments had shown that telomeric Rap1p sites occur about once every 18 bp, all Rap1p molecules at the junction of telomeric and nontelomeric chromatin (the telomere-nontelomere junction) must participate in telomere length measurement. The conserved arrangement of these six Rap1p molecules at the telomere-nontelomere junction in independent transformants also caused the elongated TG1-3 tracts to be maintained at nearly identical lengths, showing that sequences at the telomere-nontelomere junction had an effect on length regulation. These results can be explained by a model in which telomeres beyond a threshold length form a folded structure that links the chromosome terminus to the telomere-nontelomere junction and prevents telomere elongation.

scholarly journals Telomere-Associated Protein TIN2 Is Essential for Early Embryonic Development through a Telomerase-Independent Pathway

2004 ◽  
Vol 24 (15) ◽  
pp. 6631-6634 ◽  
Author(s):  
Y. Jeffrey Chiang ◽  
Sahn-Ho Kim ◽  
Lino Tessarollo ◽  
Judith Campisi ◽  
Richard J. Hodes
Keyword(s):  
Embryonic Development ◽  
Telomere Length ◽  
Embryonic Lethality ◽  
Negative Regulator ◽  
Telomeric Dna ◽  
Interacting Protein ◽  
Telomere Elongation ◽  
Dna Repeat ◽  
Early Embryonic Lethality ◽  
Length Regulation

ABSTRACT TIN2 is a negative regulator of telomere elongation that interacts with telomeric DNA repeat binding factor 1 (TRF1) and affects telomere length by a telomerase-dependent mechanism. Here we show that inactivation of the mouse TRF1-interacting protein 2 (TIN2) gene results in early embryonic lethality. We further observed that the embryonic lethality of TIN2 mutant mice was not affected by inactivation of the telomerase reverse transcriptase gene, indicating that embryonic lethality is not the result of telomerase-dependent changes in telomere length or function. Our findings suggest that TIN2 has a role independent of telomere length regulation that is essential for embryonic development and cell viability.

scholarly journals Rescue of an hTERT Mutant Defective in Telomere Elongation by Fusion with hPot1

2004 ◽  
Vol 24 (8) ◽  
pp. 3552-3561 ◽  
Author(s):  
Blaine N. Armbruster ◽  
Corinne M. Linardic ◽  
Tim Veldman ◽  
Niharika P. Bansal ◽  
Diane L. Downie ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Telomeric Dna ◽  
Telomere Elongation ◽  
Telomere Binding Protein ◽  
Catalytically Active ◽  
Bona Fide ◽  
Per Se ◽  
Telomeric Protein

ABSTRACT The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.

scholarly journals Telomerase RNA Template Mutations Reveal Sequence-Specific Requirements for the Activation and Repression of Telomerase Action at Telomeres

2000 ◽  
Vol 20 (8) ◽  
pp. 2941-2948 ◽  
Author(s):  
John C. Prescott ◽  
Elizabeth H. Blackburn
Keyword(s):  
Enzymatic Activity ◽  
Activity Levels ◽  
Telomeric Dna ◽  
Telomere Shortening ◽  
Telomere Elongation ◽  
Telomere Binding Protein ◽  
Telomere Function ◽  
Double Base ◽  
Telomere Lengthening

ABSTRACT Telomeric DNA is maintained within a length range characteristic of an organism or cell type. Significant deviations outside this range are associated with altered telomere function. The yeast telomere-binding protein Rap1p negatively regulates telomere length. Telomere elongation is responsive to both the number of Rap1p molecules bound to a telomere and the Rap1p-centered DNA-protein complex at the extreme telomeric end. Previously, we showed that a specific trinucleotide substitution in the Saccharomyces cerevisiae telomerase gene (TLC1) RNA template abolished the enzymatic activity of telomerase, causing the same cell senescence and telomere shortening phenotypes as a complete tlc1 deletion. Here we analyze effects of six single- and double-base changes within these same three positions. All six mutant telomerases had in vitro enzymatic activity levels similar to the wild-type levels. The base changes predicted from the mutations all disrupted Rap1p binding in vitro to the corresponding duplex DNAs. However, they caused two classes of effects on telomere homeostasis: (i) rapid, RAD52-independent telomere lengthening and poor length regulation, whose severity correlated with the decrease in in vitro Rap1p binding affinity (this is consistent with loss of negative regulation of telomerase action at these telomeres; and (ii) telomere shortening that, depending on the template mutation, either established a new short telomere set length with normal cell growth or was progressive and led to cellular senescence. Hence, disrupting Rap1p binding at the telomeric terminus is not sufficient to deregulate telomere elongation. This provides further evidence that both positive and negativecis-acting regulators of telomerase act at telomeres.

scholarly journals Cdc13 N-Terminal Dimerization, DNA Binding, and Telomere Length Regulation

2010 ◽  
Vol 30 (22) ◽  
pp. 5325-5334 ◽  
Author(s):  
Meghan T. Mitchell ◽  
Jasmine S. Smith ◽  
Mark Mason ◽  
Sandy Harper ◽  
David W. Speicher ◽  
...  
Keyword(s):  
Dna Binding ◽  
Telomere Length ◽  
Functional Characterization ◽  
Point Mutations ◽  
Telomeric Dna ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Length Regulation ◽  
Telomere Length Regulation

ABSTRACT The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

scholarly journals Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

1989 ◽  
Vol 9 (11) ◽  
pp. 5073-5080 ◽  
Author(s):  
M Kozak
Keyword(s):  
Context Effects ◽  
Consensus Sequence ◽  
Negative Regulator ◽  
Late Event ◽  
Free Translation ◽  
Initiator Codon ◽  
Translation Systems ◽  
Short Versions

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.

scholarly journals Negative-feedback regulation of FGF signalling by DUSP6/MKP-3 is driven by ERK1/2 and mediated by Ets factor binding to a conserved site within the DUSP6/MKP-3 gene promoter

2008 ◽  
Vol 412 (2) ◽  
pp. 287-298 ◽  
Author(s):  
Maria Ekerot ◽  
Marios P. Stavridis ◽  
Laurent Delavaine ◽  
Michael P. Mitchell ◽  
Christopher Staples ◽  
...  
Keyword(s):  
Binding Site ◽  
Negative Feedback ◽  
Fluorescent Protein ◽  
Gene Promoter ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Negative Feedback Regulation ◽  
Fgf Signalling

DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling.

scholarly journals Genome-wide CRISPR-Cas9 screen identified KLF11 as a druggable suppressor for sarcoma cancer stem cells

2021 ◽  
Vol 7 (5) ◽  
pp. eabe3445
Author(s):  
Yicun Wang ◽  
Jinhui Wu ◽  
Hui Chen ◽  
Yang Yang ◽  
Chengwu Xiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Negative Feedback ◽  
Direct Interaction ◽  
Therapy Resistance ◽  
Negative Regulator ◽  
Chemotherapy Response ◽  
Genome Wide

Cancer stem cells (CSCs) are involved in tumorigenesis, recurrence, and therapy resistance. To identify critical regulators of sarcoma CSCs, we performed a reporter-based genome-wide CRISPR-Cas9 screen and uncovered Kruppel-like factor 11 (KLF11) as top candidate. In vitro and in vivo functional annotation defined a negative role of KLF11 in CSCs. Mechanistically, KLF11 and YAP/TEAD bound to adjacent DNA sites along with direct interaction. KLF11 recruited SIN3A/HDAC to suppress the transcriptional output of YAP/TEAD, which, in turn, promoted KLF11 transcription, forming a negative feedback loop. However, in CSCs, this negative feedback was lost because of epigenetic silence of KLF11, causing sustained YAP activation. Low KLF11 was associated with poor prognosis and chemotherapy response in patients with sarcoma. Pharmacological activation of KLF11 by thiazolidinedione effectively restored chemotherapy response. Collectively, our study identifies KLF11 as a negative regulator in sarcoma CSCs and potential therapeutic target.

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