Markus Nabholz

Regulation of the human telomerase reverse transcriptase gene

Most somatic human cells lack telomerase activity because they do not express the telomerase reverse transcriptase (hTERT) gene. Conversely, most cancer cells express hTERT and are telomerase positive. For most tumors it is not clear whether hTERT expression is due to their origin from telomerase positive stem cells or to reactivation of the gene during tumorigenesis. Telomerase negative cells lack detectable cytoplasmic and nuclear hTERT transcripts; in telomerase positive cells 0.2 to 6 mRNA molecules/cell can be detected. This suggests that expression is regulated by changes in the rate of hTERT gene transcription. In tumor cell lines hTERT expression behaves like a recessive trait, indicating that lack of expression in normal cells is due to one or several repressors. Studies with monochromosomal hybrids indicate that several chromosomes may code for such repressors. A number of transcription factors, tumor suppressors, cell cycle inhibitors, cell fate determining molecules, hormone receptors and viral proteins have been implicated in the control of hTERT expression; but these studies have not yet provided a clear explanation for the tumor specific expression of the hTERT gene, and the cis-acting elements which are the targets of repression in normal cells still have to be identified.

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.

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 α chain of the interleukin-2 receptor (IL-2Rα) 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-2Rα 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-2Rα gene expression. These results show that regulation of the endogenous IL-2Rα 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-2Rα 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.

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

ABSTRACT Interleukin-2 (IL-2) responsiveness of T lymphocytes is controlled through transcription of the IL-2 receptor (IL-2R) α subunit by antigen and by IL-2 itself. IL-2 induces IL-2Rα 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-2Rα expression. In cells induced to transiently express IL-2Rα 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-2Rα transcription by making IL-2Rα chromatin accessible to transcription factors.

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-2Rβ 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.

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.

A Conserved IL-2 Responsive Enhancer in the IL-2Rα Gene

Abstract IL-2 stimulates expression of the a subunit of the high affinity IL-2 receptor (IL-2Rα) in antigen-activated T lymphocytes, by increasing IL-2Rα 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.

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.