Patrick Reichenbach

The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5′-UUAGGG-3′ repeats near their 3′-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5′-UUAGGG-3′ repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action

Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of these molecules are present in the clinical setting, it is important to establish the validity of this assertion. To analyze the effects of these compounds, we used a direct assay with telomerase-enriched extracts. The comparison of potent ligands from various chemical families revealed important differences in terms of effects on telomerase initiation and processivity. Although most quadruplex ligands may lock a quadruplex-prone sequence into a quadruplex structure that inhibits the initiation of elongation by telomerase, the analysis of telomerase-elongation steps revealed that only a few molecules interfered with the processivity of telomerase (i.e., inhibit elongation once one or more repeats have been incorporated). The demonstration that these molecules are actually more effective inhibitors of telomeric DNA amplification than extension by telomerase contributes to the already growing suspicion that quadruplex ligands are not simple telomerase inhibitors but, rather, constitute a different class of biologically active molecules. We also demonstrate that the popular telomeric repeat amplification protocol is completely inappropriate for the determination of telomerase inhibition by quadruplex ligands, even when PCR controls are included. As a consequence, the inhibitory effect of many quadruplex ligands has been overestimated.

TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

ABSTRACT Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres—TPP1 and the protein that tethers TPP1 to the complex, TIN2—results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.

Molecular dissection of telomeric repeat-containing RNA biogenesis unveils the presence of distinct and multiple regulatory pathways.

ABSTRACT Telomeres are transcribed into telomeric repeat-containing RNA (TERRA), large, heterogeneous, noncoding transcripts which form part of the telomeric heterochromatin. Despite a large number of functions that have been ascribed to TERRA, little is known about its biogenesis. Here, we present the first comprehensive analysis of the molecular structure of TERRA. We identify biochemically distinct TERRA complexes, and we describe TERRA regulation during the cell cycle. Moreover, we demonstrate that TERRA 5′ ends contain 7-methylguanosine cap structures and that the poly(A) tail, present on a fraction of TERRA transcripts, contributes to their stability. Poly(A)− TERRA, but not poly(A)+ TERRA, is associated with chromatin, possibly reflecting distinct biological roles of TERRA ribonucleoprotein complexes. In support of this idea, poly(A)− and poly(A)+ TERRA molecules end with distinct sequence registers. We also determine that the bulk of 3′-terminal UUAGGG repeats have an average length of 200 bases, indicating that the length heterogeneity of TERRA likely stems from its subtelomeric regions. Finally, we find that TERRA is regulated during the cell cycle, being lowest in late S phase and peaking in early G1. Our analyses offer the basis for investigating multiple regulatory pathways that affect TERRA synthesis, processing, turnover, and function.

Interaction of STAT5 dimers on two low affinity binding sites mediates interleukin 2 (IL-2) stimulation of IL-2 receptor alpha gene transcription.

Stimulation of the interleukin 2 receptor α (IL-2Rα) gene by IL-2 is important for the proliferation of antigen-activated T lymphocytes. IL-2 regulates IL-2Rα transcription via a conserved 51-nucleotide IL-2 responsive enhancer. Mouse enhancer function depends on cooperative activity of three distinct sites. Two of these are weak binding sites for IL-2-activated STAT5 (signal transducer and activator of transcription) proteins, and mutational analysis indicates that binding of STAT5 to both sites is required for IL-2 responsiveness of the enhancer. The STAT5 dimers interact to form a STAT5 tetramer. The efficiency of tetramerization depends on the relative rotational orientation of the two STAT motifs on the DNA helix. STAT5 tetramerization on enhancer mutants correlates well with the IL-2 responsiveness of these mutants. This provides strong evidence that interactions between STAT dimers binding to a pair of weak binding sites play a biological role by controlling the activity of a well characterized, complex cytokine-responsive enhancer.

Ectopic Human Telomerase Catalytic Subunit Expression Maintains Telomere Length But Is Not Sufficient for CD8+ T Lymphocyte Immortalization

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8+ T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8+ lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

Mouse interleukin-2 receptor alpha gene expression. Interleukin-1 and interleukin-2 control transcription via distinct cis-acting elements

We have shown that interleukin-1 (IL-1) and IL-2 control IL-2 receptor α (IL-2Rα) gene transcription in CD4CD8 murine T lymphocyte precursors. Here we map the cis-acting elements that mediate interleukin responsiveness of the mouse IL-2Rα gene using a thymic lymphoma-derived hybridoma (PC60). The transcriptional response of the IL-2Rα gene to stimulation by IL-1 + IL-2 is biphasic. IL-1 induces a rapid, protein synthesis-independent appearance of IL-2Rα mRNA that is blocked by inhibitors of NF-κB activation. It also primes cells to become IL-2 responsive and thereby prepares the second phase, in which IL-2 induces a 100-fold further increase in IL-2Rα transcripts. Transient transfection experiments show that several elements in the promoter-proximal region of the IL-2Rα gene contribute to IL-1 responsiveness, most importantly an NF-κB site conserved in the human and mouse gene. IL-2 responsiveness, on the other hand, depends on a 78-nucleotide segment 1.3 kilobases upstream of the major transcription start site. This segment functions as an IL-2-inducible enhancer and lies within a region that becomes DNase I hypersensitive in normal T cells in which IL-2Rα expression has been induced. IL-2 responsiveness requires three distinct elements within the enhancer. Two of these are potential binding sites for STAT proteins.

Protein–RNA and protein–protein interactions mediate association of human EST1A/SMG6 with telomerase

The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme telomerase. In an attempt to better understand hEST1A function, we have started to dissect the molecular interactions between hEST1A and telomerase. Here, we demonstrate that the interaction between hEST1A and telomerase is mediated by protein–RNA and protein–protein contacts. We identify a domain within hEST1A that binds the telomerase RNA moiety hTR while full-length hEST1A establishes in addition RNase-resistant and hTR-independent protein–protein contacts with the human telomerase reverse transcriptase polypeptide (TERT). Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity. Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall Kd = 25 nM) but low specificity. We propose that hEST1A assembles specifically with telomerase in the context of the hTR–hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific protein–protein contacts with hTERT.

Human CD8+ T cells expressing HLA‐DR and CD28 show telomerase activity and are distinct from cytolytic effector T cells

Cycling lymphocytes may express the enzyme telomerase which is involved in maintenance of telomere length and cell proliferation potential. In CD8+ T cells freshly isolated from peripheral blood, we found that in vivo cycling cells expressed HLA‐DR. Furthermore, CD28‐positive cells are known to have longer telomeres than CD28‐negative T cells. Therefore we used HLA‐DR‐ and CD28‐specific antibodies to sort CD8+ T cells and measure telomerase activity ex vivo. Relatively high levels of telomerase activity were found in HLA‐DR / CD28 double‐positive cells. In contrast, HLA‐DR‐negative and CD28‐negative cells had almost no telomerase activity. In summary, HLA‐DR expression correlates with proliferation, and CD28 expression with proliferative potential. We have previously identified that ex vivo cytolytic CD8+ T cells are CD56 (NCAM) positive. Here we show that HLA‐DR+ cells were rarely CD56+ and vice versa. This demonstrates that telomerase‐expressing and cytolytic CD8+ T cells can be separated on the basis of the cell surface markers HLA‐DR and CD56. Thus, activated CD8+ T cells specialize and exert distinct functions correlating with surface molecule expression.

Low- to high-throughput analysis of telomerase modulators with Telospot

We designed a method termed Telospot to discover and characterize telomerase modulators as anticancer drugs or chemical biology tools. Telospot is based on a highly efficient human telomerase expression system and the detection of telomerase DNA reaction products in macroarray format. Telospot offers a highly scalable, cost- and time-effective alternative to presently available telomerase assays, which are limited by the requirement for PCR, telomerase purification or technologies not amenable to high throughput.