Loss of hPot1 Function Leads to Telomere Instability and a cut-like Phenotype

Veldman, Timothy; Etheridge, Katherine T.; Counter, Christopher M.

doi:10.1016/j.cub.2004.12.031

Cited by 132 publications

(142 citation statements)

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“…5 B-D and SI Appendix, Fig. S5 A and B), consistent with previous hPOT1 knockdown studies that have shown that it causes apoptosis in immortalized cancer cell lines (45,50) and senescence in primary cells (45,50,51). We further observed that the phenotypes resulting from hPOT1 knockdown were rapidly lost.…”

Section: Discussionsupporting

confidence: 90%

“…We stably transduced HMECs expressing the aforementioned hTERT mutants at 4 mo after senescence of control cells with an hPOT1 or control shRNA vector (45). By using quantitative real-time RT-PCR (qPCR), we observed 60% to 80% knockdown of endogenous hPOT1 mRNA expression in HMECs expressing WT hTERT, hTERT IA− , or hTERT N-DAT 116 (Fig.…”

Section: Cellular Lifespan Extension By Htert Is Independent Of Telomerementioning

confidence: 98%

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Separation of telomerase functions by reverse genetics

Mukherjee

Firpo

Wang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The canonical function of the human telomerase protein (hTERT) is to synthesize telomeric DNA, but it has other biological activities, including enhancing cell proliferation, decreasing apoptosis, regulating DNA damage responses, and increasing cellular proliferative lifespan. The mechanistic relationships among these activities are not understood. We previously demonstrated that ectopic hTERT expression in primary human mammary epithelial cells diminishes their requirement for exogenous mitogens, thus giving them a proliferative advantage in a mitogen-depleted environment. Here, we show that this phenotype is caused by a combination of increased cell division and decreased apoptosis. In addition, we use a panel of hTERT mutants to demonstrate that this enhanced cell proliferation can be uncoupled not only from telomere elongation, but also from other telomerase activities, including cellular lifespan extension and regulation of DNA damage responses. We also find that the proliferative function of hTERT, which requires hTERT catalytic activity, is not caused by increased Wnt signaling, but is accompanied by alterations in key cell cycle regulators and is linked to an hTERT-catalyzed decrease in the levels of the RNA component of mitochondrial RNA processing endoribonuclease. Thus, enhanced cell proliferation is an independent function of hTERT that could provide a new target for the development of anti-telomerase cancer therapeutic agents.

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Section: Discussionsupporting

confidence: 90%

Section: Cellular Lifespan Extension By Htert Is Independent Of Telomerementioning

confidence: 98%

Separation of telomerase functions by reverse genetics

Mukherjee

Firpo

Wang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Since this residue is critically important for telomere extension by telomerase, POT1 functions to negatively regulate telomere length by competing with telomerase for access to the telomeric substrate (Loayza and de Lange, 2003;Kelleher et al, 2005;Lei et al, 2005). In addition, depletion of POT1 levels by RNAi (Veldman et al, 2004;Yang et al, 2005) or conditional deletion of the Pot1a gene in mice (Hockemeyer et al, 2006;Wu et al, 2006) results in increased chromosomal aberrations, which suggests that POT1 plays an important role in protecting telomeric ends. Recently, POT1 was found to physically interact with WRN and stimulate its helicase activity (Opresko et al, 2005).…”

Section: Pot1mentioning

confidence: 99%

WRN at telomeres: implications for aging and cancer

Multani

Chang

2007

Journal of Cell Science

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Werner Syndrome (WS) is a fascinating autosomal recessive disorder affecting ~10 per million individuals (Epstein et al., 1966). Patients appear normal until the second decade of life, when they develop pathologies that phenocopy many aspects of normal human aging, including alopecia, ischemic heart disease, osteoporosis, bilateral ocular cataracts, type II diabetes mellitus, and hypogonadism (Thannhauser, 1945). WS patients also experience an increased risk of rare non-epithelial cancers, especially mesenchymal neoplasms such as sarcomas (Goto et al., 1996). Death usually occurs in the fourth decade from cardiovascular compromise or cancer. Fibroblasts isolated from WS patients characteristically senesce prematurely in culture (Faragher et al., 1993) and display increased chromosomal aberrations (Salk et al., 1981;Melcher et al., 2000). Since WS is caused by mutation of a single gene, WRN, these observations suggest that the WRN gene product functions to maintain genome stability.Cloning of WRN ten years ago revealed that it encodes a protein containing a highly conserved 3Ј to 5Ј DNA helicase domain of the RecQ family (reviewed by Martin and Oshima, 2000). RecQ helicase family members are involved in diverse biochemical processes, including DNA recombination, replication and repair, and WRN has been implicated in all of these (reviewed by Hickson, 2003). WRN also possesses several other conserved domains, including a 3Ј to 5Ј exonuclease domain, a nuclear localization sequence, and a multifunctional DNA/protein-binding domain (DPBD) that interacts with both DNA and proteins (reviewed by Orren, 2006). Through these domains WRN interacts with many factors that participate in diverse aspects of DNA metabolism beyond the scope of this commentary. Here, we focus on recent progress that indicates WRN might have a role at telomeres. Discussion of other aspects of WRN biology can be found elsewhere (Hickson, 2003; Comai and Li, 2004;Bohr, 2005;Orren, 2006). Telomere structure and maintenanceTelomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes and play crucial roles in maintaining genomic stability by providing both end-protection and a mechanism for generating chromosomal ends (LeBel and Wellinger, 2006). In mammals, telomeres consist of TTAGGG repetitive sequences that terminate in a 3Ј single-stranded Grich overhang. Telomeres can fold into a structure termed the t-loop, in which the 3Ј single-stranded overhang invades a duplex region of the telomere to sequester the overhang, forming a single-stranded displacement (D) loop (Griffith et al., 1999;Murti and Prescott, 1999). This telomeric conformation probably protects natural DNA ends from being recognized as double-strand breaks (DSBs) that would otherwise activate DNA damage checkpoint responses or participate in aberrant recombination events (reviewed by d'Adda di Fagagna et al., 2004). However, an extended telomeric conformation must also exist to facilitate replication of telomeres during S phase. Telomeres are maintained by the e...

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“…POT1b deficiency results in the excessive resection of the 5Ј end, leading to an increase of the 3Ј-overhang sequences as well as progressive telomere shortening (10,11). Like POT1a, human POT1 is required to hide telomeres from DNA damage surveillance, and it can complement the loss of POT1a at mouse telomeres if coexpressed with human TPP1 (hTPP1) (10,13,16,25). It has been speculated that POT1b harbors functions not conserved in its human homologue, as human POT1 does not complement POT1b in terms of the 3Ј-overhang regulation of mouse telomeres.…”

mentioning

confidence: 99%

Functional Dissection of Human and Mouse POT1 Proteins

Palm

Hockemeyer

Kibe

et al. 2009

Molecular and Cellular Biology

119

144

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The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.Mammalian chromosome ends associate with shelterin, the protein complex dedicated to telomere protection and homeostasis (reviewed in references 5 and 20). The DNA of mammalian telomeres consists of long arrays of doublestranded (ds) TTAGGG repeats ending in a single-stranded (ss) protrusion of the 3Ј end, referred to as the 3Ј overhang. The 3Ј-overhang strand can invade the duplex part of telomeres, forming a lariat structure called the t-loop (8). Three shelterin components interact with telomeric DNA. POT1 recognizes ss TTAGGG repeats and therefore can associate with the telomeric 3Ј overhang as well as with the displaced strand at the base of the t-loop. TRF1 and TRF2 cover the duplex part of telomeric DNA, and they recruit the other shelterin components Rap1, TIN2, and TPP1 to telomeres through protein-protein interactions. POT1 cannot localize to telomeres alone but instead requires heterodimerization with TPP1; this interaction is crucial for the association of POT1 with telomeres, as TPP1 tethers POT1 to TRF1 and TRF2 by a TPP1-TIN2 bridge and improves the DNA binding affinity of POT1 (12,19,26).Shelterin masks telomeres from the cellular machinery that recognizes and repairs DNA lesions. If shelterin function is perturbed, DNA damage response factors accumulate at telomeres, leading to the activation of the ATM and ATR kinase signaling pathways (2, 10, 15, 29). Distinct components of shelterin independently repress ATM and ATR signaling: TRF2 is required to prevent ATM activation at telomeres, while POT1 represses the activation of ATR (16). Dysfunctional telomeres trigger various DNA repair reactions and can be processed like dsDNA breaks through nonhomologous end joining or through homology-direc...

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Loss of hPot1 Function Leads to Telomere Instability and a cut-like Phenotype

Cited by 132 publications

References 28 publications

Separation of telomerase functions by reverse genetics

Separation of telomerase functions by reverse genetics

WRN at telomeres: implications for aging and cancer

Functional Dissection of Human and Mouse POT1 Proteins

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