The human telomere binding protein hPot1 binds to the most distal single-stranded extension of telomeric DNA in vitro, and probably in vivo, as well as associating with the double-stranded telomeric DNA binding proteins TRF1 and TRF2 through the bridging proteins PTOP (also known as PIP1 or TINT1) and TIN2. Disrupting either the DNA binding activity of hPot1 or its association with PTOP results in elongated telomeres, suggesting a role for hPot1 in telomere length regulation. However, mutations to POT1 and Cdc13p, the fission and budding yeast genes encoding the structural orthologs of this protein, leads to telomere instability and cell death. Thus, it is possible that the hPot1 protein may also serve to cap and protect telomeres in humans. Indeed, we now find that knocking down the expression of hPot1 in human cells causes apoptosis or senescence, as well as an increase in telomere associations and anaphase bridges, telltale signs of telomere instability. In addition, knockdown cells also displayed chromatin bridges between interphase cells, reminiscent of the cut phenotype that was first described in fission yeast and in which cytokinesis progresses despite a failure of chromatid separation. However, unlike the yeast cut phenotypes, we suggest that the cut-like phenotype observed in hPot1 knockdown cells is a consequence of the fusion of chromosome ends and that this fusion impedes proper chromosomal segregation. We conclude that hPot1 protects chromosome ends from illegitimate recombination, catastrophic chromosome instability, and abnormal chromosome segregation.

Telomerase is the enzyme essential to complete the replication of the terminal DNA of most eukaryotic chromosomes. In humans, this enzyme is composed of the telomerase reverse transcriptase (hTERT) and telomerase RNA (hTR) subunits. hTR has been found in the nucleolus, a site of assembly of ribosomes as well as other ribonucleoproteins (RNPs). We therefore tested whether the hTERT component is also found in the nucleolus, where it could complex with the hTR RNA to form a functional enzyme. We report here that hTERT does indeed localize to the nucleolus, and we mapped the domain responsible for this localization to the hTRbinding region of the protein by deletion analysis. Substitution mutations in two of the three conserved hTRbinding domains in this nucleolar localization domain (NoLD) abolished nucleolar localization. However, another mutation that impeded hTR binding did not alter this subcellular localization. Additionally, wild type hTERT was detected in the nucleolus of cells that failed to express hTR. Taken together, we propose that the nucleolar localization of hTERT involves more than just the association with the hTR subunit. Furthermore, the coincidental targeting of both the hTR and hTERT subunits to the nucleolus supports the premise that the assembly of telomerase occurs in the nucleolus.Telomerase is a reverse transcriptase ribonucleoprotein (RNP) 1 complex composed of a reverse transcriptase catalytic protein subunit (TERT) that copies a template region of an accompanying RNA subunit (TR) onto telomeres as DNA (1). In humans, this enzyme is of great medical importance because of its pivotal role in unlimited cellular proliferation, a hallmark of cancer cells (2). The union of the RNA and protein subunits to form an RNP is essential for telomerase activity (3).Based on a comparison of the amino acid sequence of TERT from organisms of many different kingdoms and on mutational analysis, it has been possible to identify a number of discrete domains in TERT. The central region of the catalytic subunit contains seven motifs found in reverse transcriptases, which define the catalytic core (4 -17). The C terminus of TERT is highly divergent, both at the sequence and the functional level (18,19). The N-terminal region is more conserved, containing domain I and the DAT (dissociates activities of telomerase) domain (18, 20) followed by domains II and III (18,20,21) and the T motif (1, 5, 6), which are essential for telomere elongation. Substitution mutations in domains II and III or the T motif decrease TERT binding to the telomerase RNA in yeast (18), ciliate (22, 23), or human cells (20,24) and correspondingly result in a dysfunctional enzyme. Deletion analysis has also defined the region extending from amino acids 326 to 613, which harbors all three of the aforementioned domains, as the minimum region required for hTR binding (22), although mutations in domain I can also have some effect on hTR binding (24).The site of assembly of the telomerase RNP has not been determined; however, there is growing e...

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of RMS known as alveolar (aRMS) have a 5-year survival rate of <30%. aRMS tissues exhibit a number of genetic changes, including lossof-function of the p53 and Rb tumor suppressor pathways, amplification of MYCN, stabilization of telomeres, and most characteristically, reciprocal translocation of loci involving the PAX and FKHR genes, generating the PAX7-FKHR or PAX3-FKHR fusion proteins. We previously showed that PAX3-FKHR expression in primary human myoblasts, cells that can give rise to RMS, cooperated with loss of p16INK4A to promote extended proliferation. To better understand the genetic events required for aRMS formation, we then stepwise converted these cells to their transformed counterpart. PAX3-FKHR, the catalytic unit of telomerase hTERT, and MycN, in cooperation with down-regulation of p16 INK4A /p14 ARF expression, were necessary and sufficient to convert normal human myoblasts into tumorigenic cells that gave rise to aRMS tumors. However, the order of expression of these transgenes was critical, as only those cells expressing PAX3-FKHR early could form tumors. We therefore suggest that the translocation of PAX3 to FKHR drives proliferation of myoblasts, and a selection for loss of p16 INK4A /p14 ARF . These early steps, coupled with MycN amplification and telomere stabilization, then drive the cells to a fully tumorigenic state. [Cancer Res 2008;68(23):9583-8]

Purpose Rhabdomyosarcoma (RMS) is a malignancy with features of skeletal muscle, and the most common soft-tissue sarcoma of childhood. Survival for high risk groups is ~30% at 5 years and there are no durable therapies tailored to its genetic aberrations. During genetic modeling of the common RMS variants, embryonal (eRMS) and alveolar (aRMS), we noted that the RTK FGFR4 was upregulated as an early event in aRMS. Herein, we evaluated the expression of FGFR4 in eRMS compared to aRMS, and whether FGFR4 had similar or distinct roles in their tumorigenesis. Experimental Design Human RMS cell lines and tumor tissue were analyzed for FGFR4 expression by immunoblot and IHC. Genetic and pharmacologic loss-of-function of FGFR4 using virally-transduced shRNAs and the FGFR small molecule inhibitor PD173074, respectively, were used to study the role of FGFR4 in RMS cell lines in vitro and xenografts in vivo. Expression of the anti-apoptotic protein BCL2L1 was also examined. Results FGFR4 is expressed in both RMS subtypes, but protein expression is higher in aRMS. The signature aRMS gene fusion product, PAX3-FOXO1, induced FGFR4 expression in primary human myoblasts. In eRMS, FGFR4 loss-of-function reduced cell proliferation in vitro and xenograft formation in vivo. In aRMS, it diminished cell survival in vitro. In myoblasts and aRMS, FGFR4 was necessary and sufficient for expression of BCL2L1, while in eRMS, this induction was not observed, suggesting differential FGFR4 signaling. Conclusion These studies define dichotomous roles for FGFR4 in RMS subtypes, and support further study of FGFR4 as a therapeutic target.