Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/β-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/β-catenin signalling by serving as a cofactor in a β-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior–posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert−/− mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/ β-catenin signalling pathway.

Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human telomerase consists of at least three principal subunits, including the telomerase reverse transcriptase (TERT), the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein1), uniquely enriched in Cajal bodies, nuclear sites of RNP processing important for telomerase function. TCAB1 associates with active telomerase enzyme, with established telomerase components, and with small Cajal body RNAs involved in modifying splicing RNAs. Depletion of TCAB1 using RNA interference prevents TERC from associating with Cajal bodies, disrupts telomerase-telomere association and abrogates telomere synthesis by telomerase. Thus, TCAB1 controls telomerase trafficking and is required for telomere synthesis in human cancer cells.TERT and TERC comprise the minimal catalytic core of the telomerase enzyme (1), whereas dyskerin is an RNA binding protein that recognizes the H/ACA sequence motif shared by TERC and two groups of non-coding RNAs involved in RNA modification -small Cajal body (sca) RNAs and small nucleolar (sno) RNAs (2,3). Dyskerin functions in part to support telomerase RNP biogenesis and TERC stability (4,5). TERT, TERC and dyskerin are all components of active telomerase (6), and mutations in any of these genes can cause the human stem cell disorder dyskeratosis congenita (7). Other potential components of active telomerase include three evolutionarily conserved dyskerin-associated proteins, NOP10, NHP2 and GAR1 (8-10), and EST1A, a homologue of the yeast telomerase protein Est1p (11,12). However, the size of active human telomerase, estimated in the 0.65 to 2 MDa range (6,13,14), suggests the existence of additional components. We reasoned that other dyskerin-associated proteins may be telomerase components, and we therefore sought to purify dyskerin complexes.To study dyskerin, we expressed tagged dyskerin protein at endogenous levels in the absence of competing endogenous protein (Fig. S1) and isolated dyskerin complexes using a dual affinity chromatography strategy. Purified dyskerin complexes were analyzed by SDS-PAGE and nanoLC-MS/MS for identification of co-purifying proteins (Fig. 1A,B). Dense peptide coverage was obtained for dyskerin and for the dyskerin-associated ATPases pontin and reptin (14). Each of the evolutionarily conserved dyskerin-binding proteins NHP2, NOP10 and GAR1 was detected, as were the dyskerin-associated proteins Nopp140 and NAF1, a nucleoplasmic factor required for assembly of H/ACA RNPs including telomerase (15) (Fig. 1B). In addition, this approach identified the WD40 repeat protein WDR79, a protein that had not been previously implicated in dyskerin or telomerase function.We further characterized WDR79, hereafter referred to as TCAB1 (Fig. 1B, S2). Endogenous TCAB1 was specifically bound to Flag-dyskerin immunoprecipitated from Flagdyskerin +shRNA HeLa cells, as were endogenous ponti...

Telomerase is a multisubunit ribonucleoprotein (RNP) complex that adds telomere repeats to the ends of chromosomes. Three essential telomerase components have been identified thus far: the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC), and the TERC-binding protein dyskerin. Few other proteins are known to be required for human telomerase function, limiting our understanding of both telomerase regulation and mechanisms of telomerase action. Here, we identify the ATPases pontin and reptin as telomerase components through affinity purification of TERT from human cells. Pontin interacts directly with both TERT and dyskerin, and the amount of TERT bound to pontin and reptin peaks in S phase, evidence for cell-cycle-dependent regulation of TERT. Depletion of pontin and reptin markedly impairs telomerase RNP accumulation, indicating an essential role in telomerase assembly. These findings reveal an unanticipated requirement for additional enzymes in telomerase biogenesis and suggest alternative approaches for inhibiting telomerase in cancer.

Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES) products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf), L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs) in the available databases. Moreover, this analysis was able to confirm the presence of 274 “hypothetical” proteins inferred from gene prediction algorithms applied to the B. malayi (Bm) genome. Not surprisingly, the majority (160/274) of these “hypothetical” proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase), MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females) compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host–parasite interaction.