Regulation of human telomerase activity: repression by normal chromosome 3 abolishes nuclear telomerase reverse transcriptase transcripts but does not affect c-Myc activity.

Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)-PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerase-negative cells, the number of molecules was below the sensitivity of the assay (<0.004 molecules/cell). Intron-containing, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulated. Microcell transfer of a normal chromosome 3 into the human breast carcinoma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromosome 3 hybrids, even in cells permanently expressing a transfected hTERT cDNA. However, chromosome 3 transfer did not affect the expression of green fluorescent protein reporter constructs driven by up to 7.4 kb of noncoding DNA flanking the 5' end of the hTERT gene. Because direct up-regulation of hTERT through c-Myc overexpression had previously been reported, we investigated whether chromosome 3 transfer affected c-Myc activity. An at least 30-fold reduction of immature intron-containing hTERT RNA was observed after the introduction of a normal chromosome 3, but expression levels of c-Myc, Mad1, and other c-Myc target genes were unchanged. Our results suggest that telomerase is regulated primarily at the level of hTERT transcription by complex mechanisms involving regulatory elements distant from the 5' flanking region, and that the putative hTERT repressor on chromosome 3 does not regulate the expression of hTERT through c-Myc or one of its coregulators.

Rearrangements of minisatellites in the human telomerase reverse transcriptase gene are not correlated with its expression in colon carcinomas.

Telomerase activation is crucial in human carcinogenesis. The limiting component of telomerase, the catalytic subunit (hTERT), is undetectable in normal somatic cells but present in most tumor cells, including the earliest stages of colon carcinoma. The mechanisms involved in the differential expression in normal and tumor cells are not understood. In normal cells hTERT expression is shut down by a repressor, and upregulation could be a consequence of cis-acting changes in the hTERT gene, making it resistant to repression. We have identified a polymorphic and a monomorphic minisatellite in the second intron of the hTERT gene, and polymorphic one in intron 6. The polymorphic minisatellite in intron 2 contains binding sites for c-Myc, which has been shown to upregulate hTERT transcription. Screening colon carcinoma DNAs for rearrangements of hTERT minisatellites we detected no changes in 33 samples from tumors, most of which express hTERT. This indicates that size rearrangements of the hTERT minisatellites are not required for telomerase expression in colon carcinomas. Minor changes and one LOH were seen in five tumors.

DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.

STAT transcription factors are expressed in many cell types and bind to similar sequences. However, different STAT gene knock-outs show very distinct phenotypes. To determine whether differences between the binding specificities of STAT proteins account for these effects, we compared the sequences bound by STAT1, STAT5A, STAT5B, and STAT6. One sequence set was selected from random oligonucleotides by recombinant STAT1, STAT5A, or STAT6. For another set including many weak binding sites, we quantified the relative affinities to STAT1, STAT5A, STAT5B, and STAT6. We compared the results to the binding sites in natural STAT target genes identified by others. The experiments confirmed the similar specificity of different STAT proteins. Detailed analysis indicated that STAT5A specificity is more similar to that of STAT6 than that of STAT1, as expected from the evolutionary relationships. The preference of STAT6 for sites in which the half-palindromes (TTC) are separated by four nucleotides (N(4)) was confirmed, but analysis of weak binding sites showed that STAT6 binds fairly well to N(3) sites. As previously reported, STAT1 and STAT5 prefer N(3) sites; however, STAT5A, but not STAT1, weakly binds N(4) sites. None of the STATs bound to half-palindromes. There were no specificity differences between STAT5A and STAT5B.

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.

Constitutive expression of a chimeric receptor that delivers IL-2/IL-15 signals allows antigen-independent proliferation of CD8+CD44high but not other T cells.

We have prepared transgenic mice whose T cells constitutively express a chimeric receptor combining extracellular human IL-4R and intracellular IL-2Rbeta segments. This receptor can transmit IL-2/IL-15-like signals in response to human, but not mouse, IL-4. We used these animals to explore to what extent functional IL-2R/IL-15R expression controls the capacity of T cells to proliferate in response to IL-2/IL-15-like signals. After activation with Con A, naive transgenic CD8+ and CD4+ T cells respond to human IL-4 as well as to IL-2. Without prior activation, they failed to proliferate in response to human IL-4, although human IL-4 did prolong their survival. Thus, IL-2-induced proliferation of activated T cells requires at least one other Ag-induced change apart from the induction of a functional IL-2R. However, a fraction of CD8+CD44high T cells proliferate in human IL-4 without antigenic stimulation or syngeneic feeder cells. In contrast, CD4+CD44high T cells are not constitutively responsive to human IL-4. We conclude that although all transgenic T cells express a functional chimeric receptor, only some CD8+CD44high T cells contain all molecules required for entry into the cell cycle in response to human IL-4 or IL-15.

Interleukin-2 (IL-2) regulates the accessibility of the IL-2-responsive enhancer in the IL-2 receptor alpha gene to transcription factors.

Interleukin-2 (IL-2) responsiveness of T lymphocytes is controlled through transcription of the IL-2 receptor (IL-2R) alpha subunit by antigen and by IL-2 itself. IL-2 induces IL-2Ralpha transcription via an IL-2-responsive enhancer (IL-2rE), whose activity depends on the cooperative binding of IL-2-induced STAT5 to two sites and of constitutively active Elf-1 to a third one. Here we describe the changes in IL-2rE chromatin that occur in normal T lymphocytes upon activation of IL-2Ralpha expression. In cells induced to transiently express IL-2Ralpha with concanavalin A (which mimics antigen), none of the IL-2rE sites is occupied despite the presence of Elf-1 and STAT1, which bind to the IL-2rE in vitro. The two STAT binding sites are occupied rapidly upon IL-2 stimulation, concomitantly with STAT5 activation. Occupation of the Elf-1 binding site is delayed, although Elf-1 concentration and binding activity are not modified by IL-2. Digestion of T-cell chromatin with DNase I and micrococcal nuclease shows that IL-2 induces the appearance of nuclease-hypersensitive sites flanking the IL-2rE. Thus IL-2, in addition to activating STAT5, appears to regulate IL-2Ralpha transcription by making IL-2Ralpha chromatin accessible to transcription factors.

Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor alpha gene.

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2R alpha gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4- CD8- cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4- CD8- thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

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 alpha (IL-2Ralpha) gene by IL-2 is important for the proliferation of antigen-activated T lymphocytes. IL-2 regulates IL-2Ralpha 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.

A conserved IL-2 responsive enhancer in the IL-2R alpha gene.

IL-2 stimulates expression of the alpha subunit of the high affinity IL-2 receptor (IL-2R alpha) in antigen-activated T lymphocytes, by increasing IL-2R alpha gene transcription. This response is mediated by a 52 nt IL-2 responsive enhancer (IL-2rE) that is conserved between mouse and man. The mouse enhancer is 1.3 kb upstream of the transcription start site and co-localizes with an inducible DNasel hypersensitive site, whereas the human homologue maps to -4 kb. The human IL-2rE is functional in rodent cells. Both enhancers contain two potential STAT binding sites and an Ets consensus motif. One of the STAT motifs overlaps with a binding site for GATA factors. Functional analysis of the mouse and human enhancers indicates that IL-2-activated STAT5 and the constitutively active Ets protein Elf-1 play a predominant role in controlling IL-2rE activity.

Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene.

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

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 alpha (IL-2R alpha) gene transcription in CD4-CD8- murine T lymphocyte precursors. Here we map the cis-acting elements that mediate interleukin responsiveness of the mouse IL-2R alpha gene using a thymic lymphoma-derived hybridoma (PC60). The transcriptional response of the IL-2R alpha gene to stimulation by IL-1 + IL-2 is biphasic. IL-1 induces a rapid, protein synthesis-independent appearance of IL-2R alpha mRNA that is blocked by inhibitors of NF-kappa B 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-2R alpha transcripts. Transient transfection experiments show that several elements in the promoter-proximal region of the IL-2R alpha gene contribute to IL-1 responsiveness, most importantly an NF-kappa B 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-2R alpha expression has been induced. IL-2 responsiveness requires three distinct elements within the enhancer. Two of these are potential binding sites for STAT proteins.

Interleukins (IL)-1 and IL-2 control IL-2 receptor alpha and beta expression in immature thymocytes.

Functional high-affinity interleukin-2 receptors (IL-2R) contain three transmembrane proteins, IL-2R alpha, beta and gamma. We have investigated the expression of IL-2R alpha and beta genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL-2R alpha on their surface. Stimulation of IL-2R alpha+ and IL-2R alpha- immature thymocytes with phorbol 12-myristate 13-acetate and calcium ionophore induces synthesis of IL-2R alpha and IL-2R beta mRNA. Most of this response depends on autocrine stimulation by IL-2. IL-1 synergizes with IL-2 to induce a 120-fold increase in IL-2R alpha mRNA and a 14-fold increase in IL-2R beta mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL-2 plays a major role in the regulation of IL-2R expression in normal immature thymocyte. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.

Characterization of a new tissue-specific transcription factor binding to the simian virus 40 enhancer TC-II (NF-kappa B) element.

We have biochemically and functionally characterized a new transcription factor, NP-TCII, which is present in nuclei from unstimulated T and B lymphocytes but is not found in nonhematopoietic cells. This factor has a DNA-binding specificity similar to that of NF-kappa B but is unrelated to this or other Rel proteins by functional and biochemical criteria. It can also be distinguished from other previously described lymphocyte-specific DNA-binding proteins.

Minimal growth requirements of mature T lymphocytes: interleukin (IL)-1 and IL-6 increase growth rate but not plating efficiency of CD4 cells stimulated with anti-CD3 and IL-2.

We show that interleukin (IL)-2 is necessary and sufficient for the proliferation of both CD4 and CD8 subsets of peripheral murine T cells activated by plastic-bound anti-CD3 monoclonal antibodies (mAb). The frequency of proliferating cells (f) was 0.32 for CD4 cells and 0.63 for CD8 cells. These frequencies were not increased by the addition of IL-1 or IL-6, alone or in combination. These cytokines were unable to induce responsiveness to IL-2 in T cells confirming that they cannot substitute for the signal delivered via the TcR/CD3 complex. On the other hand, IL-1 and IL-6 increase the growth rate of CD4 cells. The addition of IL-6 significantly lowered the mean doubling time (dt) of CD4 cells (dt: 26 h vs. 38 h in the presence of IL-2 alone, p less than 0.01), while the addition of IL-1, ineffective by itself, combined with IL-6 further increased the growth rate of CD4 cells (dt: 23 h, p less than 0.001). The growth rate of CD8 cells stimulated with anti-CD3 and IL-2, was markedly faster than that of CD4 cells (dt: 18 h vs. 38 h, p less than 0.001) and was not significantly influenced by addition of IL-1 and/or IL-6.

A PCR-based assay for reporter gene expression.

Transient transfection is a widely used tool for the identification of cis-acting regulatory elements. These elements are detected by their effect on the expression of a reporter gene, which is quantified by measuring the reporter gene product in the form of mRNA, protein (hGH), or enzymes (CAT, luciferase). Measurements of mRNA levels have several advantages over enzyme or protein assays. However, mRNA quantification by RNase protection or S1 mapping has considerably lower signal-to-background ratio than protein assays and is therefore less sensitive. In this paper we report the development of a system that takes advantage of the polymerase chain reaction (PCR) to quantify rabbit beta-globin reporter gene expression. Cells are co-transfected with constructs whose activity is to be tested and a reference plasmid with a small deletion in the second exon of the beta-globin gene. We show that the ratio of the two amplified cDNA signals is a highly reliable measure of test gene expression. The sensitivity of this assay is at least 1000-fold higher than RNase protection.

Cell specificity of granzyme gene expression.

Granzymes are serine proteases present in secretory granules of cytolytic T lymphocyte lines. We have studied the expression of the granzyme family (granzyme A, B, C, D, E, F, and G) in different lymphoid cell populations and cell lines as well as in nonlymphoid cells and tissues. Our data show that with few exceptions expression of granzyme genes is restricted to T cells and their thymic precursors. In mature T cells granzymes are expressed only upon activation. The same is true for thymocytes, with the exception of grazyme A that is expressed also in non-stimulated cells. In T cells and thymocytes the distribution of mRNAs coding for different granzymes depends on the subpopulation tested and the activation protocol. Highly cytolytic PEL express granzymes A and B but none of the other granzymes.

Control of IL-2 receptor-alpha expression by IL-1, tumor necrosis factor, and IL-2. Complex regulation via elements in the 5' flanking region.

We have analyzed the mechanisms by which IL-1, IL-2, and TNF regulate expression of IL-2R alpha chain in a rodent T cell line. All three cytokines induce detectable IL-2R alpha mRNA by themselves, but there is strong synergy between IL-1 or TNF, on the one hand, and IL-2, on the other. The earliest phase of induction by IL-1 is independent of protein synthesis. IL-1, but not TNF, also stimulates transient secretion of IL-2. This leads to an autocrine stimulation of a further increase in IL-2R alpha mRNA levels. When IL-2 secretion has dropped off, continued IL-2R alpha expression requires both IL-2 and IL-1. Most or all of this regulation is due to changes in the rate of transcription of the IL-2R alpha gene. The response to IL-1 and IL-2 depends on a segment in the IL-2R alpha 5' flanking region, upstream of all cis-acting regulatory elements previously identified in the human gene.