The lengths of human telomeres, which protect chromosome ends from degradation and end fusions, are crucial determinants of cell lifespan. During embryogenesis and in cancer, the telomerase enzyme counteracts telomeric DNA shortening. As shown in cancer cells, human telomerase binds the shelterin component TPP1 at telomeres during the S phase of the cell cycle, and adds ~60 nucleotides in a single round of extension, after which telomerase is turned off by unknown mechanisms. Here we show that the human CST (CTC1, STN1 and TEN1) complex, previously implicated in telomere protection and DNA metabolism, inhibits telomerase activity through primer sequestration and physical interaction with the protection of telomeres 1 (POT1)–TPP1 telomerase processivity factor. CST competes with POT1–TPP1 for telomeric DNA, and CST–telomeric-DNA binding increases during late S/G2 phase only on telomerase action, coinciding with telomerase shut-off. Depletion of CST allows excessive telomerase activity, promoting telomere elongation. We propose that through binding of the telomerase-extended telomere, CST limits telomerase action at individual telomeres to approximately one binding and extension event per cell cycle. Our findings define the sequence of events that occur to first enable and then terminate telomerase-mediated telomere elongation.

In mammalian cells, the telomeric repeat binding factor (TRF) homology (TRFH) domain-containing telomeric proteins TRF1 and TRF2 associate with a collection of molecules necessary for telomere maintenance and cell-cycle progression. However, the specificity and the mechanisms by which TRF2 communicates with different signaling pathways remain largely unknown. Using oriented peptide libraries, we demonstrate that the TRFH domain of human TRF2 recognizes [Y/F]XL peptides with the consensus motif YYHKYRLSPL. Disrupting the interactions between the TRF2 TRFH domain and its targets resulted in telomeric DNA-damage responses. Furthermore, our genome-wide target analysis revealed phosphatase nuclear targeting subunit (PNUTS) and microcephalin 1 (MCPH1) as previously unreported telomere-associated proteins that directly interact with TRF2 via the [Y/F]XL motif. PNUTS and MCPH1 can regulate telomere length and the telomeric DNA-damage response, respectively. Our findings indicate that an array of TRF2 molecules functions as a protein hub and regulates telomeres by recruiting different signaling molecules via a linear sequence code.

Mutations in CTC1 lead to the telomere syndromes Coats Plus and dyskeratosis congenita (DC), but the molecular mechanisms involved remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex termed CST, which binds ssDNA, promotes telomere DNA synthesis, and inhibits telomerase-mediated telomere elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical interaction with DNA polymerase a-primase (pola-primase), telomeric ssDNA binding in vitro, accumulation in the nucleus, and/or telomere association in vivo. While having diverse molecular defects, CTC1 mutations commonly lead to the accumulation of internal single-stranded gaps of telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease. Strikingly, mutations in CTC1 may also unleash telomerase repression and telomere length control. Hence, the telomere defect initiated by CTC1 mutations is distinct from the telomerase insufficiencies seen in classical forms of telomere syndromes, which cause short telomeres due to reduced maintenance of distal telomeric ends by telomerase. Our analysis provides molecular evidence that CST collaborates with DNA pola-primase to promote faithful telomere DNA replication.

Androgen receptor (AR) is a hormone-regulated transcription factor that mediates a wide array of biological processes including sexual differentiation, spermatogenesis, and prostate cancer progression. The transcriptional activity of AR and other members of the nuclear receptor superfamily are modulated by coregulatory proteins. In this study, we have investigated the regulation of AR transcriptional activity by the silencing mediator for retinoid and thyroid hormone receptors (SMRT). We found that AR possesses an intrinsic transcriptional repression activity, and AR interacts directly with SMRT. One interacting surface on AR is mapped to the ligand-binding domain, and the presence of a DNA binding/hinge region enhances this interaction. The binding surface on SMRT is mapped to the C-terminal ID2 region, and mutation in the ID2 corepressor motif inhibits the interaction. Overexpression of SMRT inhibits dihydrotestosterone-dependent transactivation by AR and further suppresses the antiandrogen flutamide-mediated inhibition of AR activity. We provide evidence to suggest that the mechanisms of SMRT-mediated inhibition of AR activity involves inhibition of AR N/C interaction and competition with the p160 coactivator. Our data establish a significant role of SMRT in modulating AR transcriptional activity. Androgen receptor (AR)1 is a member of the nuclear receptor (NR) superfamily that mediates the biological effects of the male sex hormone androgens in a wide range of physiological processes, including sexual differentiation and maturation, spermatogenesis, and gonadotropin regulation (1). AR is also involved in the development and progression of prostate cancer, which is one of the most frequently diagnosed cancers in males. Indeed, somatic mutations of the AR gene have been found in prostate tumors, which may contribute to androgenindependent growth of the cancer cell (2). Treatment with antiandrogens can partially or completely arrest prostate cancer cell proliferation. Similar to other steroid receptors the unliganded AR is held in an inactive conformation by association with heat shock proteins in the cytoplasm. The heat shock protein complex assists in maintaining an AR conformation that is active to ligand binding. Upon ligand binding, the heat shock protein complex dissociates from the receptor (3), allowing AR to homodimerize and translocate to the nucleus, where it recognizes and binds specific promoter elements to activate gene expression (4).Like other members of the NR superfamily, AR contains an N-terminal AF-1 transactivation domain (or A/B domain), a centrally located DNA-binding domain (DBD or C domain), a hinge region (or D domain), and a ligand-binding domain (LBD or E domain). The E domain is also involved in receptor dimerization and contains ligand-dependent AF-2 function. The A/B domain is the most variable among NRs, and in the case of AR, it contains two separate transactivation domains that are required for full ligand-inducible transcription activity (5, 6). The A/B region also contains a...

Extrachromosomal telomere repeat (ECTR) DNA is unique to cancer cells that maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, but the role of ECTRs in ALT development remains elusive. We found that induction of ECTRs in normal human fibroblasts activated the cGAS-STING-TBK1-IRF3 signaling axis to trigger IFNβ production and a type I interferon response, resulting in cell-proliferation defects. In contrast, ALT cancer cells are commonly defective in sensing cytosolic DNA. We found that STING expression was inhibited in ALT cancer cell lines and transformed ALT cells. Notably, the ALT suppressors histone H3.3 and the ATRX-Daxx histone chaperone complex were also required to activate the DNA-sensing pathway. Collectively, our data suggest that the loss of the cGAS-STING pathway may be required to evade ECTR-induced anti-proliferation effects and permit ALT development, and this requirement may be exploited for treatments specific to cancers utilizing the ALT pathway.

Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ϳ12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric pro- During mammalian DNA replication, linear chromosomal ends will gradually erode because of the inability of the DNA replication machinery to replicate the extreme 5Ј terminus of a linear DNA sequence (1, 2). This inherent "end replication problem" is circumvented through specialized chromosomal end structures (telomeres) and the action of the RNA-containing DNA polymerase -telomerase (3-9). Telomere homeostasis is essential for genome stability, cell survival, and growth.Telomeres and telomerase help to ensure genome integrity in eukaryotes by enabling complete replication of the ends of linear DNA molecules, and preventing chromosomal rearrangements or fusion. For dividing cells such as stem cells and the majority of cancer cells, the telomerase is an essential positive regulator of their telomere length and ultimately determines the proliferative potential of these cells.Mammalian telomeres consist of a series of (TTAGGG)n sequence repeats and terminate in 3Ј single-stranded DNA overhangs that are extendable by the telomerase (10). Exposed linear chromosome ends or naturally occurring doublestranded breaks pose additional risks including activation of DNA damage responses. The ends of telomeres in mammalian cells appear to fold back in a T-loop structure, with the 3Ј G-rich single-stranded overhang invading into the doublestranded telomere regions to form the D-loop (11). The structure of the telomeres, coupled with the coordinated action of a collection of proteins that protect the ends of chromosomes (12-15), contributes to the maintenance of telomere integrity, genome stability, and proper cell cycle progression.In mammals, the most widely studied telomere-associated proteins include the double-stranded DNA binding proteins TRF1 and TRF2 (16,17), the single-stranded telomeric DNA binding protein POT1 (18), and three associated factors (RAP1, TIN2, and TPP1) (19 -23). Work from our lab and others suggest that TPP1, along with POT1, TIN2, TRF1, TRF2, and RAP1, form a higher order complex (the telosome/ shelterin) at the telomeres (24 -27). Information regarding the state of the telomere ends can be transmitted from TRF1 and TRF2 to POT1, through TPP1 and the other subunits (28). Furthermore, TRF1 and TRF2 function as bona fide protein hubs and interact with a diverse array of factors/complexes that are involved in cell cycle, DNA repair, and recombinat...

The six human telomeric proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 can form a complex called the telosome/shelterin, which is required for telomere protection and length control. TPP1 has been shown to regulate both POT1 telomere localization and telosome assembly through its binding to TIN2. It remains to be determined where such interactions take place and whether cellular compartmentalization of telomeric proteins is important for telomere maintenance. We systematically investigated here the cellular localization and interactions of human telomeric proteins. Interestingly, we found TIN2, TPP1, and POT1 to localize and interact with each other in both the cytoplasm and the nucleus. Unexpectedly, TPP1 contains a functional nuclear export signal that directly controls the amount of TPP1 and POT1 in the nucleus. Furthermore, binding of TIN2 to TPP1 promotes the nuclear localization of TPP1 and POT1. We also found that disrupting TPP1 nuclear export could result in telomeric DNA damage response and telomere length disregulation. Our findings highlight how the coordinated interactions between TIN2, TPP1, and POT1 in the cytoplasm regulate the assembly and function of the telosome in the nucleus and indicate for the first time the importance of nuclear export and spatial control of telomeric proteins in telomere maintenance.Telomere maintenance by telomerase and telomeric proteins is essential for normal cell growth, and its disregulation may have direct consequences in aging and cancer (2,10,11,22,28,29,44). Recent biochemical and genetic experiments have led to a much clearer understanding of how telomere ends are protected in mammalian cells. To date, six major telomeric proteins-namely, TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 (previously PTOP, PIP1, or TINT1)-have been identified in human cells and shown to participate in telomere regulation (2, 9, 38). These proteins perform related but distinct functions at the telomeres. Understanding the function and molecular interaction of these telomeric proteins is fundamental to telomere biology.Telomeres in mammalian cells consist of long telomeric double-stranded DNA (dsDNA) repeats and short single-stranded DNA (ssDNA) overhangs (12, 13). TRF1 and TRF2 directly associate with the repeats through their myb domains (4). TRF1 is a negative regulator of telomere length since overexpression of TRF1 results in gradual shortening of telomeres (41). Overexpression of dominant-negative mutants or deletion of TRF2 results in DNA damage responses at the telomeres and chromosomal end-to-end fusions, underlining its essential role in telomere end protection (5, 42). TRF2 also works closely with its associated protein RAP1 (24, 31). In TRF2 KO mouse embryonic fibroblast cells, protein levels of RAP1, but not TRF1, were diminished, suggesting that TRF2 and RAP1 act together (5).The human telomere ssDNA overhangs are protected by POT1 and TPP1. POT1 and TPP1 are OB-fold-containing proteins that are structurally homologous to ciliate telomere end binding proteins 43,46). Original...

The cyclic GMP‐AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway mediates anti‐microbial innate immunity by inducing the production of type I interferons (IFNs) and inflammatory cytokines upon recognition of microbial DNA. Recent studies reveal that self‐DNA from tumors and by‐products of genomic instability also activates the cGAS–STING pathway and either promotes or inhibits tumor development. This has led to the development of cancer therapeutics using STING agonists alone and in combination with conventional cancer treatment or immune checkpoint targeting. On the other hand, for cancers lacking the cGAS–STING pathway and thus a regular innate immunity response, oncolytic virus therapy has been shown to have therapeutic potential. We here review and discuss the dichotomous roles of the cGAS–STING pathway in cancer development and therapeutic approaches.