Telomere shortening is proportional to the size of the G-rich telomeric 3'-overhang.

Most normal diploid human cells do not express telomerase activity and are unable to maintain telomere length with ongoing cell divisions. We show that the length of the single-stranded G-rich telomeric 3'-overhang is proportional to the rate of shortening in four human cell types that exhibit different rates of telomere shortening in culture. These results provide direct evidence that the size of the G-rich overhang is not fixed but subject to regulation. The potential ability to manipulate this rate has profound implications both for slowing the rate of replicative aging in normal cells and for accelerating the rate of telomere loss in cancer cells in combination with anti-telomerase therapies.

Normal human telomeres are not late replicating.

Telomeres in yeast are late replicating. Genes placed next to telomeres in yeast can be repressed (telomere positional effects), leading to the hypothesis that telomeres may be heterochromatic and may control the expression of subtelomeric genes. In addition, yeast telomeres are processed to have a transient long overhang at the end of S phase. The applicability of the yeast data to human biology was examined by determining the timing of telomere replication and processing in normal human diploid fibroblasts. Telomeres were purified from synchronized cells that had been labeled with 5-bromodeoxyuridine (BrdU) at hourly intervals, and the fraction of labeled telomeres was analyzed by retrieval with anti-BrdU antibodies. We determined that normal human telomeres replicate throughout S phase rather than being very late replicating. Furthermore, the overall timing of replication was unaffected by telomere length in young versus old cells or cells whose telomeres had been elongated following transfection with the catalytic subunit of telomerase. Finally, the asymmetry in the length of the G-rich overhang in daughter telomeres produced by leading versus lagging strand synthesis was shown to be established within 1 h of telomere replication, indicating there is no significant delay between synthesis and the processing events that contribute to the establishment of asymmetric overhangs. Therefore, the timings of replication and processing of human telomeres are very different from those of yeast.

Two inactive fragments of the integral RNA cooperate to assemble active telomerase with the human protein catalytic subunit (hTERT) in vitro.

We have mapped the 5' and 3' boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3' end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.

Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels.

The human telomerase RNA component (hTR) is present in normal somatic cells at lower levels than in cancer-derived cell lines. To understand the mechanisms regulating hTR levels in different cell types, we have compared the steady-state hTR levels in three groups of cells: (i) normal telomerase-negative human diploid cells; (ii) normal cells transfected with the human telomerase catalytic subunit, hTERT; and (iii) cells immortalized in vitro and cancer cells expressing their own endogenous hTERT. To account for the differences in steady-state hTR levels observed in these cell types, we compared the transcription rate and half-life of hTR in a subset of these cells. The half-life of hTR in telomerase-negative cells is about 5 days and is increased 1.6-fold in the presence of hTERT. The transcription rate of hTR is essentially unchanged in cells expressing exogenous hTERT, and the increased steady-state hTR level appears to be due to the increased half-life. However, the transcription rate of hTR is greatly increased in cells expressing endogenous hTERT, suggesting some overlap in transcriptional regulatory control. We conclude that the higher hTR level in cells expressing an endogenous telomerase can be a result of both increased transcription and a longer half-life and that the longer half-life might be partially a result of protection or stabilization by the telomerase catalytic subunit. The 4-week half-life of hTR in H1299 tumor cells is the longest half-life yet reported for any RNA.

Functional requirement of p23 and Hsp90 in telomerase complexes.

Most normal human diploid cells have no detectable telomerase; however, expression of the catalytic subunit of telomerase is sufficient to induce telomerase activity and, in many cases, will bypass normal senescence. We and others have previously demonstrated in vitro assembly of active telomerase by combining the purified RNA component with the reverse transcriptase catalytic component synthesized in rabbit reticulocyte extract. Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase. To our knowledge, p23 and Hsp90 are the first telomerase-associated proteins demonstrated to contribute to telomerase activity.

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Normal human chromosomes have long G-rich telomeric overhangs at one end.

Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200 +/- 75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3' overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.

Regulation of HIV-1 gene expression by NF-IL6.

Previous studies have shown that in human T-cells (Jurkat) and hepatoma cells (HepG2), exogenous NF-IL6 can activate HIV-1 gene expression even in the absence of its consensus binding elements in the viral long terminal repeat (LTR). To identify the LTR elements that mediate this response, we have analysed constructs containing mutated and deleted LTR sequences. We have also examined heterologous plasmids to evaluate a potential requirement for the natural LTR sequences in producing HIV-1 gene activation by NF-IL6. As observed for Jurkat and HepG2 cells, we find that in the absence of NF-IL6 binding elements, NF-IL6 can elicit LTR-mediated gene expression in cotransient expression assays performed in monocytic (U937) cells. However, we detect distinct modes of regulation depending on cell type. In U937 cells, the basal LTR sequences retain a significant fraction (42%) of NF-IL6 responsiveness in the absence of upstream regulatory elements in the LTR while these elements are required for maximal response. In HepG2 cells, NF-IL6 elicits a relatively low level of gene activation from the basal LTR elements; response to NF-IL6 is restored with either the Sp1 binding sequences or the other upstream regulatory elements in the LTR. In addition, even though NF-IL6 induces a relatively low gene activity from the basal LTR sequences analysed in HepG2 cells, study of a heterologous construct indicates that these sequences are required for the responsiveness of Sp1 elements to NF-IL6 in this cellular background.

NF-IL6-mediated transcriptional activation of the long terminal repeat of the human immunodeficiency virus type 1.

An upstream control region in the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) includes a potential negative regulatory element (NRE1). Cotransfecting multimers of a sequence spanning this element with an LTR-CAT construct produced an increase in chloramphenicol acetyltransferase (CAT) activity in Jurkat and HepG2 cells, providing further evidence and support for the existence of an NRE. In screening experiments aimed at identifying those factors that regulate HIV-1 transcription through interactions with the NRE1 region, we isolated a cDNA for NF-IL6. Previous studies have shown that NF-IL6 is a key nuclear factor that activates gene expression in response to interleukin 6. By methylation interference analysis, we have localized the NF-IL6 binding site within the NRE1 region and found that it overlaps an E box that has previously been implicated as the binding element for a negative regulator of HIV-1 expression. Through a database search, we identified an additional consensus binding sequence for NF-IL6 in the LTR of many HIV-1 variants and found that over this sequence, purified NF-IL6 can produce an extended footprint that overlaps one of the binding sites for NF-kappa B. A product of the nf-il6 gene activated transcription from several LTR-CAT constructs in transient transfection assays. Thus, NF-IL6 could play a central role in the control of HIV-1 gene expression and this protein might be a key mediator in signaling pathways where HIV-1 is activated by interleukin 6.

Evidence for leucine zipper motif in lactose repressor protein.

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.