Tethering Telomeric Double- and Single-stranded DNA-binding Proteins Inhibits Telomere Elongation

Abstract

Mammalian telomeres are composed of G-rich repetitive double-stranded (ds) DNA with a 3 single-stranded (ss) overhang and associated proteins that together maintain chromosome end stability. Complete replication of telomeric DNA requires de novo elongation of the ssDNA by the enzyme telomerase, with telomeric proteins playing a key role in regulating telomerase-mediated telomere replication. In regards to the protein component of mammalian telomeres, TRF1 and TRF2 bind to the dsDNA of telomeres, whereas POT1 binds to the ssDNA portion. These three proteins are linked through either direct interactions or by the proteins TIN2 and TPP1. To determine the biological consequence of connecting telomeric dsDNA to ssDNA through a multiprotein assembly, we compared the effect of expressing TRF1 and POT1 in trans versus in cis in the form of a fusion of these two proteins, on telomere length in telomerase-positive cells. When expressed in trans these two proteins induced extensive telomere elongation. Fusing TRF1 to POT1 abrogated this effect, inducing mild telomere shortening, and generated looped DNA structures, as assessed by electron microscopy, consistent with the protein forming a complex with dsDNA and ssDNA. We speculate that such a protein bridge between dsDNA and ssDNA may inhibit telomerase access, promoting telomere shortening.Telomeres are DNA-protein structures that cap and protect the ends of eukaryotic chromosomes from illegitimate recombination and degradation. In humans, the DNA portion of this structure is composed of the G-rich sequence TTAGGG repeated in tandem hundreds of times (1). This G-strand extends beyond the complementary C-strand to form a ssDNA overhang. Electron microscopy revealed that this ssDNA extension appeared to invade the dsDNA, 2 forming a D-loop, with the intervening dsDNA looping out in what is termed a T-loop. This structure has been speculated to impart some of the replicative and protective functions of telomeres (2).In terms of the protein components of telomeres, telomeric DNA-binding proteins fall into two classes, ssDNA-or dsDNAbinding proteins. In humans, the primary dsDNA telomerebinding proteins are TRF1 (3) and TRF2 (4), whereas the principle ssDNA-binding protein is POT1 (5). Disruption of TRF2 by expression of a dominant-negative version of the protein (6, 7) and disruption of POT1 expression by RNAi or genetic knock out (8 -11) can lead to various degrees of chromosome instability and/or cell arrest or death, whereas knock out of TRF1 is embryonic lethal (12). On the other hand, overexpressing these proteins alters telomere length (13-16). As access of telomerase, the enzyme that elongates the G-strand overhang of telomeres, to telomere ends is mediated by proteins in lower eukaryotes as a means of regulating telomere length; human telomere-binding proteins may also serve in this capacity (17). Thus, telomere-binding proteins can function in telomere stability and/or telomerase-mediated replication of telomeres.Accumulating evidence argues that the ssDNA-and dsDNAte...

TINF2 mutations result in very short telomeres: analysis of a large cohort of patients with dyskeratosis congenita and related bone marrow failure syndromes

“…This idea is strengthened by recent work involving fusing TRF1 to POT1. In this study telomere lengths were shortened, whereas when POT1 and TFR1 are expressed, normally telomere lengths were lengthened, 26 suggesting a link between shelterin interactions and telomere length. What is striking, however, is the similarity in the phenotypes produced by defects in telomere elongation and protection mechanisms both in terms of telomere lengths and clinical phenotypes, suggesting that however telomeres are inappropriately shortened, the clinical outcome is the same.…”

POT1 Association with TRF2 Regulates Telomere Length

“…The idea of POT1-TRF2 regulating telomere length is also supported by the finding that fusing POT1 to the TRF2 paralog, TRF1, both abolishes the telomere elongation observed when POT1 and TRF1 are expressed in trans and induces the in vitro formation of t-loops (25), structures found on telomeres (28) that could inhibit access of telomerase to telomere ends (6,23). Additionally, Pot1 can be recruited to telomeres via an association with telomeric dsDNA-binding protein Taz1 in fission yeast.…”

“…Telomere lengths were assessed using 32 P-labeled (CCCTAA) 3 Southern hybridization of HinFI-and RsaI-digested genomic DNA as previously described (2,25). Mean telomere lengths were determined using the Telometric program (26).…”

RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes

“…Nevertheless, the differential gene expression signature generated in this study yielded genes which can influence apoptosis and might contribute to the mitogenic phenotype of MLL-AF9 positive monoblasts in a less ideal environment possibly present in the in vivo disease state. These genes encode for example POT1 which promotes telomere elongation [35], CALR which promotes DNA synthesis and inhibits growth arrest and senescence (via p21) [36] and HIPK2 which is involved in induction of cell death and differentiation [37]. …”