POT1 Association with TRF2 Regulates Telomere Length

Abstract

Deleting the OB folds encoding the telomeric single-stranded DNA (ssDNA)-binding activity of the human telomeric protein POT1 induces significant telomere elongation, suggesting that at least one critical aspect of the regulation of telomere length is disrupted by this POT1 ⌬OB mutant protein. POT1 is known to associate with two proteins through the protein interaction domain retained in POT1⌬OB -the telomeric doublestranded DNA-binding protein TRF2 and the telomere-associated protein TPP1. We report that introducing a mutation that reduces association of POT1 with TRF2, but not a mutation that reduces the association with TPP1, abrogates the ability of POT1 ⌬OB to promote telomere elongation. Mechanistically, expression of POT1 ⌬OB reduced the association of TRF2 with POT1, RAP1, and TIN2; however, of these proteins, only ectopic expression of POT1 suppressed the telomere elongation induced by POT1 ⌬OB . Lastly, replacing endogenous POT1 with a full-length POT1 mutant defective in the association with TRF2 induced telomere elongation. Thus, we conclude that the association of POT1 with both ssDNA and TRF2 is critical for telomere length homeostasis.Telomeres are DNA-protein complexes that protect the ends of eukaryotic chromosomes from degradation and detection as sites of DNA damage (reviewed in reference 23). Telomeric DNA is composed of tandem arrays of repetitive double-stranded DNA (dsDNA), wherein the G-rich strand extends beyond the C-rich strand. The resultant 3Ј singlestranded DNA (ssDNA) overhang can invade the dsDNA, forming a lariat structure termed the t-loop (28).The regulation of telomere length affects mammalian biology at both the cellular and organismal levels. In normal human somatic cells, telomeres progressively shorten (32, 33) to a critical length before entering a state of permanent growth arrest termed senescence (1,12,21,34,35). Abnormally short telomeres in humans and mice are associated with various anemias, cirrhosis of the liver, and other disorders due to the premature induction of senescence, particularly in highly proliferative tissues (7,11,61). Conversely, de novo elongation of the telomere by the reverse transcriptase telomerase (27, 54) ensures maintenance of telomere length in the germ line and endows cultured cells with an immortal life span (8,17,18,20,30,36,70,78). Moreover, telomerase activation and subsequent stabilization of telomere length occur in the vast majority of cancer cells (41, 62) and are required for cellular immortalization and the tumorigenic conversion of normal human cells (19,30,31,36,78).The dsDNA portion of telomeres is bound directly by two proteins, TRF1 and TRF2 (9, 23). In turn, TRF1 and TRF2 are bridged by the protein TIN2 (23, 43, 76) and bind other telomere-associated proteins such as tankyrase 1 (23, 63, 64) and RAP1 (48, 56). Telomeric ssDNA is bound directly by the protein POT1 (4, 5, 23, 52). POT1 acts in a heterodimer with the protein TPP1; this heterodimer promotes POT1 binding to telomeric ssDNA at least in vitro (72, 73), protects tel...

Tpz1 controls a telomerase-nonextendible telomeric state and coordinates switching to an extendible state via Ccq1

“…Telomere length phenotypes of POT1/TPP1 knockdown in human cells are controversial (Colgin et al 2003;Veldman et al 2004), and deletion of pot1 + or tpz1 + in fission yeast leads to telomere deprotection. These observations support the proposal that inhibition of telomerase through integration of telomere length information is transduced from the dsDNA-binding TRF1 complex, including TRF2, TIN2, and TPP1, to the telomere terminus through recruiting POT1 to the very end, thereby controlling telomere accessibility to telomerase (Marcand et al 1997;Loayza and de Lange 2003;Barrientos et al 2008;Kendellen et al 2009). Alternatively, one or more shelterin components could also directly act on telomerase to enforce their negative roles.…”

Extra-Telomeric Roles of Telomeric Proteins

“…The actual length of the telomeres varies in various species and also within mammals. TRF1 and TRF2 bind the doublestranded (TTAGGG)n repeats of telomeres while POT1 binds to 3' single-stranded G overhangs (Broccoli et al 1997;Smith et al 1998;Ye and de Lange 2004;de Lange 2005;Xin et al 2007;Kendellen et al 2009;Abreu et al 2010). Rap1 was discovered as a TRF2 interacting factor and named such due to its sequence homology with the yeast Rap1 (Li et al 2000).…”

“…Shelterin is composed of six core members that include TRF1, TRF2 (Telomeric Repeat binding Factor 1 and 2), TIN2 (TRF1-and TRF2-Interacting Nuclear Factor 2), POT1 (Protection Of Telomeres 1), TPP1 (formerly known as TINT1, PTOP or PIP1) and RAP1 (Repressor/Activator Protein 1) (Broccoli et al 1997;Smith et al 1998;Ye and de Lange 2004;de Lange 2005;Xin et al 2007;Kendellen et al 2009;Abreu et al 2010). Three members of this complex, TRF1, TRF2 and POT1, bind directly to telomeric DNA repeats and anchor the rest of the complex along the length of the telomeres.…”

Dissecting the genetic architecture of familial melanoma

“…It has been shown that the ACD-POT1 sub-complex also has a higher binding affinity to ssDNA than POT1 does on its own [143]. Literature shows that if the function of POT1 is inhibited the length of telomeres is increased through telomerase activity, indicating that this subcomplex is able to regulate the elongation of chromosome ends [144][145][146][147][148]. In our study, we show that missense mutations in the OB-domains of POT1 result in an elongation of telomeres in carriers.…”