Silencing and trans-activation of the mouse IL-2 gene in Xenopus oocytes by proteins from resting and mitogen-induced primary T-lymphocytes.

Mouzaki, Αthanasia; Weil, Roger; Muster, Lisbeth; Rungger, Duri

doi:10.1002/j.1460-2075.1991.tb07660.x

Cited by 29 publications

(31 citation statements)

References 27 publications

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“…If indicated, oocytes were heat treated for 60 min at 37ЊC. Extracts were prepared as described elsewhere (18) following a further overnight incubation of the oocytes at 20ЊC.…”

Section: Methodsmentioning

confidence: 99%

Multiple Layers of Regulation of Human Heat Shock Transcription Factor 1

Zuo

Rungger

Voellmy

1995

Molecular and Cellular Biology

227

231

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Upon heat stress, monomeric human heat shock transcription factor 1 (hHSF1) is converted to a trimer, acquires DNA-binding ability, is transported to the nucleus, and becomes transcriptionally competent. It was not known previously whether these regulatory changes are caused by a single activation event or whether they occur independently from one another, providing a multilayered control that may prevent inadvertant activation of hHSF1. Comparison of wild-type and mutant hHSF1 expressed in Xenopus oocytes and human HeLa cells suggested that retention of hHSF1 in the monomeric form depends on hydrophobic repeats (LZ1 to LZ3) and a carboxy-terminal sequence element in hHSF1 as well as on the presence of a titratable factor in the cell. Oligomerization of hHSF1 appears to induce DNA-binding activity as well as to uncover an amino-terminally located nuclear localization signal. A mechanism distinct from that controlling oligomerization regulates the transcriptional competence of hHSF1. Components of this mechanism were mapped to a region, including LZ2 and nearby sequences downstream from LZ2, that is clearly separated from the carboxy-terminally located transcription activation domain(s). We propose the existence of a fold-back structure that masks the transcription activation domain in the unstressed cell but is opened up by modification of hHSF1 and/or binding of a factor facilitating hHSF1 unfolding in the stressed cell. Activation of hHSF1 appears to involve at least two independently regulated structural transitions.The transcriptional enhancement of heat shock protein (hsp) genes by heat shock or other conditions that are stressful to cells is dependent on the presence of so-called heat shock element (HSE) sequences in their promoter regions (3,17,22) that consist of arrays of alternatively oriented NGAAN modules (2, 37). HSEs are binding sites for heat shock transcription factor (HSF) (21,23,36), which is inactive in unstressed cells and active in stressed cells (16,36,38). Organisms differ in their number of distinct HSF species. Whereas Saccharomyces cerevisiae and fruit flies express a single HSF species, birds, mammals, and plants express multiple HSF species (reviewed in reference 31). Furthermore, the strategies employed to regulate HSF activity differ drastically in S. cerevisiae and higher eukaryotes (30). While yeast HSF binds DNA constitutively, heat-activable HSF in higher eukaryotes is incapable of DNA binding in the absence of heat stress. Thus, in higher eukaryotes, HSF activity may be regulated mainly or exclusively at the level of DNA-binding ability. However, a number of situations involving mammalian cells, in which HSF DNAbinding ability was induced but a concomitant increase in hsp gene expression did not occur, have been described (6,14,15,24), suggesting that activation of HSF is a multistage process and that induction of DNA-binding ability may be only an early event in a complex activation process.It has been shown previously that stress activation of hsp genes in mammalian cells is ...

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“…If indicated, oocytes were heat treated for 60 min at 37ЊC. Extracts were prepared as described elsewhere (18) following a further overnight incubation of the oocytes at 20ЊC.…”

Section: Methodsmentioning

confidence: 99%

Multiple Layers of Regulation of Human Heat Shock Transcription Factor 1

Zuo

Rungger

Voellmy

1995

Molecular and Cellular Biology

227

231

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“…Transcription assays with proteins isolated from freshly recovered mouse or humanT cells revealed that, in resting cells, a silencer binds to the purine-rich (Pu) elements present twice in the promoter (at positions -130 and -276 bp) and blocks transcription. Upon cell activation, a positive transcription factor (TF) binds to these elements and derepresses the gene [18,191. These Pubinding TF were found in mouse or humanT cells only, and experiments with purified human T cell subsets revealed that these factors exist only in CD4+ cells [18,191.…”

Section: Introductionmentioning

confidence: 99%

Occurrence of a silencer of the interleukin‐2 gene in naive but not in memory resting T helper lymphocytes

et al. 1993

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In the immune system the first activation of a naive T cell by antigen is a key step in the shaping of the peripheral T cell specificity repertoire and maintenance of self-tolerance. In the present study, analysis of the interleukin-2 (IL-2) gene activation shows that naive human helper T cells (cord blood CD4+ T cells, adult CD4+CD45RO- T cells) regulate IL-2 transcription by a mechanism involving both a silencer and an activator acting on the purine-rich IL-2 promoter elements (NF-AT binding sites). By contrast, memory cells, either in vitro activated helper T cells reverting to a resting state, or CD4+ T (memory) clones, or CD4+CD45RO+ T cells isolated ex vivo, no longer have a silencer. Their IL-2 transcription seems to be controlled solely by the transition from inactive to active functional state of a positive transcription factor binding to these promoter elements as well as its cytoplasmic or nuclear location: in resting memory T cells the activator is located in the cytoplasm and is inactive, whereas in stimulated cells it is functional in promoting transcription and now resides in the nucleus. Thus, the regulation of the gene coding for the main T cell growth factor changes irreversibly after the first encounter of T cells with antigen. It is most likely that the presence of a silencer contributes to the more stringent activation requirements of naive CD4+ T cells.

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“…18 Briefly, cells were swollen in a hypotonic buffer, briefly sonicated, and salt was adjusted to 300 mM KCl to extract proteins from chromatin. After 20 min on ice, the lysate was spun (100 000 g for 1 h at 41C) and the supernatant collected on a buffer containing 20 mM HEPES, 0.02 mM EDTA, pH 7.6, 0.5 mM 1,4 dithio-DL-theitol (KIB).…”

Section: Cellsmentioning

confidence: 99%

“…Attempts to prove functionally the involvement of PRRE and NFAT sites in HIV-1 activation gave contrasting results, 16,17 indicative of a dual negative and positive role. Transactivation studies of the IL-2 promoter in the Xenopus oocyte system 18 with protein extracts from ex vivo isolated T lymphocytes revealed several novel aspects of IL-2 regulation. The IL-2 promoter is repressed by proteins from resting naïve CD4 cells.…”

Section: Introductionmentioning

confidence: 99%

“…Following T-cell activation, the repressor disappears and an activator is de novo synthesized. [18][19][20] These findings prompted the reinvestigation 21 of HIV-1 LTR regulation by functional transactivation assays using proteins from primary T cells (isolated from cord blood or peripheral blood) proving that the HIV-1 LTR is regulated in a similar manner to the IL-2 promoter. Since the protein binding to the aforementioned repressor sequence exists only in CD4 resting naive T cells of both human and mouse origin, it is probably a key molecule for the immune system, and one potentially important for the pathogenesis of certain autoimmune diseases.…”

Section: Introductionmentioning

confidence: 99%

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Mining microarray data to identify transcription factors expressed in naïve resting but not activated T lymphocytes

et al. 2004

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Transcriptional repressors controlling the expression of cytokine genes have been implicated in a variety of physiological and pathological phenomena. An unknown repressor that binds to the distal NFAT element of the interleukin-2 (IL-2) gene promoter in naive T-helper lymphocytes has been implicated in autoimmune phenomena and has emerged as a potentially important factor controlling the latency of HIV-1. The aim of this paper was the identification of this repressor. We resorted to public microarray databases looking for DNA-binding proteins that are present in naïve resting T cells but are downregulated when the cells are activated. A Bayesian data mining statistical analysis uncovered 25 candidate factors. Of the 25, NFAT4 and the oncogene ets-2 bind to the common motif AAGGAG found in the HIV-1 LTR and IL-2 probes. Ets-2 binding site contains the three G's that have been shown to be important for binding of the unknown factor; hence, we considered it the likeliest candidate. Electrophoretic mobility shift assays confirmed cross-reactivity between the unknown repressor and anti-ets-2 antibodies, and cotransfection experiments demonstrated the direct involvement of Ets-2 in silencing the IL-2 promoter. Designing experiments for transcription factor analysis using microarrays and Bayesian statistical methodologies provides a novel way toward elucidation of gene control networks.

show abstract

Silencing and trans-activation of the mouse IL-2 gene in Xenopus oocytes by proteins from resting and mitogen-induced primary T-lymphocytes.

Cited by 29 publications

References 27 publications

Multiple Layers of Regulation of Human Heat Shock Transcription Factor 1

Multiple Layers of Regulation of Human Heat Shock Transcription Factor 1

Occurrence of a silencer of the interleukin‐2 gene in naive but not in memory resting T helper lymphocytes

Mining microarray data to identify transcription factors expressed in naïve resting but not activated T lymphocytes

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