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.

Identification of the core-histone-binding domains of HMG1 and HMG2.

High mobility group (HMG) nonhistone chromosomal proteins are a group of abundant, conservative and highly charged nuclear proteins whose physiological role in chromatin is still unknown. To gain insight into the interactions of HMG1 and HMG2 with the fundamental components of chromatin we have introduced the methodology of photochemical crosslinking. This technique has allowed us to study the interaction of HMG1 and HMG2 with the core histones, in the form of an H2A X H2B dimer and an (H3 X H4)2 tetramer, for an effective time of crosslinking of less than 1 ms and under very mild conditions. This is achieved by using flash photolysis. With this procedure we found that both HMG1 and HMG2 interact with H2A X H2B and also with (H3 X H4)2. In the second case, they seem to do this through histone H3. To obtain more information about the interactions, we split HMG1 and HMG2 into their peptides using staphylococcal proteinase. The peptides obtained, which reflect the domain distribution of these proteins, were then used along with the histone oligomers to elucidate their interactions by means of photochemical crosslinking. Results obtained indicate that the domain of HMG1 and HMG2 involved in the interaction with H2A X H2B histones is the highly acidic C-terminal, whereas the N-terminal is involved in the interactions with (H3 X H4)2 histones. In all cases, the interactions found appear appreciably strong. Along with other data published in the literature, these proteins appear to have at least one binding site per domain for the chromatin components.

Phosphorylation of high-mobility-group protein 14 by two specific kinases modifies its interaction with histone oligomers in free solution.

Chromosomal protein HMG14 can be specifically phosphorylated by the cyclic AMP-dependent protein kinase at the N-terminus and by casein kinase 2 at the acidic C-terminus. Under the same conditions used for HMG14, HMG17 is not significantly phosphorylated by either of the two kinases. Further, we have studied the effect of phosphorylation by these kinases on the interaction of HMG14 with histone oligomers, using chemical cross-linking. Our results indicate that the phosphorylation of HMG14 by casein kinase 2 enhances its interaction with histone oligomers in free solution, whereas a minor effect was observed by phosphorylation with cyclic AMP-dependent protein kinase. In contrast, HMG17 does not interact at all with any histone oligomer in free solution under the conditions used. To gain insight into the possible effect that phosphorylation may play in vivo, the pattern of distribution among different chromatin fractions was analysed. It was found that, although phosphorylation of HMG14 by both kinases allowed reconstitution of HMG14 to chromatin, the patterns obtained showed some slight differences.

Activity and interleukin 1 responsiveness of SV40 enhancer motifs in a rodent immature T cell line.

We have analysed the enhancer activity and the interleukin 1 (IL1) responsiveness of individual motifs of the SV40 enhancer in an immature rodent T cell line, PC60. Transient transfection assays showed that tetramers of GT-I plus GT-IIC motifs, the TC-II or the P motif have significant enhancer activity in PC60, while neither Octamer nor SphI+II motifs have a detectable effect on promoter strength. Two motifs, TC-II and P, strongly respond to stimulation by IL1. DNase I and methylation protection experiments with nuclear extracts show specific footprints in the TC-II region of the SV40 enhancer. Exposure of PC60 cells to IL1 increases their intensity. The TC-II sequence forms several complexes detected in band shift assays. The molecules involved all have similar sequence specificity as NF-kappa B. Surprisingly, band shifts with extracts from control or IL1 treated cells differ only slightly. However, if GTP is added to the binding reactions the intensity of bands formed by extracts from control cells is strongly reduced, whereas extracts from IL1 treated cells form a single retarded complex that co-migrates with NF-kappa B from a pre-B cell line. The results suggest that in PC60 IL1 induces NF-kappa B activity by activating molecules that are already in the nucleus.

Epitope-specific engagement of the protein tyrosine phosphatase CD45 induces tumor necrosis factor-alpha gene expression via transcriptional mechanisms.

The common leukocyte antigen CD45 plays a central role in T cell activation in coupling the T cell receptor (TCR) to the phosphatidylinositol pathway via interactions with TCR-associated protein tyrosine kinases lck and fyn. We here demonstrate that engagement of CD45 by monoclonal antibodies (mAb) on activated T cells induces tumor necrosis factor (TNF)-alpha as well as TNF-beta, interleukin (IL)-2 and IL-3 gene expression. When human alloreactive T cells are stimulated with mAb 4B2, which recognizes a determinant common to all CD45 isoforms, a vigorous production of TNF-alpha mRNA was detected, which peaked 2 h later. Anti-CD45 mAb cross-linking was required. In contrast, neither mAb 10G10, which recognizes an epitope distinct from the one recognized by mAb 4B2, nor mAb UCHL-1, a CD45RO-specific antibody, induced any significant increase in TNF-alpha transcription. Nuclear run-on transcription assays demonstrated that CD45 cross-linking caused transcriptional activation of the TNF-alpha gene. De novo protein synthesis was not required, since incubation with cycloheximide (CHX) did not block transcriptional activation. CHX in contrast up-regulated TNF-alpha gene expression and increased transcript half-life, an effect that was under control of post-transcriptional mechanisms. Engagement of CD45 by itself did not affect transcript stability. CD45 ligation resulted in TNF-alpha secretion. These results indicate that in addition to its role in TCR/CD3-mediated T cell activation, CD45, in an epitope-specific manner, may act as a primary signaling molecule, leading to the transcriptional regulation and secretion of a major pro-inflammatory cytokine.

Binding of HMG14 non-histone protein to histones H2A, H2B, H1 and DNA in reconstituted chromatin.

The interaction between calf thymus HMG14 and rat liver chromatin components has been studied via reconstitution and chemical cross-linking. Selective labeling of HMG14 with photoactivable reversible heterobifunctional reagents has allowed a clear identification of the histones interacting with it (histones H2A, H2B and H1). These results are not dependent on whether the chromatin samples used were bulk chromatin, mononucleosomes, or core particles (for H2A and H2B). In addition to histone proteins, DNA also seems to be involved in HMG14 attachment to nucleosome.

Interaction between histone H1 and non-histone HMG14 detected by chemical cross-linking.

The interaction between histone H1 and non-histones HMG14 and HMG17 has been studied by chemical cross-linking. Cross-linking kinetics show the appearance of discrete bands which correspond to the interaction between H1 and HMG14. Interaction between H1 and HMG17 has not been detected.

Detection by chemical cross-linking of interaction between high mobility group protein 1 and histone oligomers in free solution.

Among the more abundant non-histone proteins is the high mobility group (HMG), with an unknown role in chromatin. We have investigated, by chemical cross-linking, the interaction of the protein HMG 1 with the histone dimer H2A X H2B and the histone tetramer (H3 X H4)2 in free solution. Cross-linking with dimethyl suberimidate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the cleavable cross-linker dimethyl-3,3'-dithiobispropionimidate, by two-dimensional electrophoresis reveals the existence of an interaction between HMG 1 and the histone dimer, and also between HMG 1 and the histone tetramer. In the case of the H2A X H2B dimer, the analysis of the patterns of the cross-linking products shows the presence of a trimer, (H2A X H2B) X HMG 1, and of another oligomer of higher molecular weight which also contains H2A X H2B and HMG 1. Non-histone HMG 1 has been found to interact with (H3 X H4)2, both by cross-linking kinetics and also by gel permeation chromatography, displaying a stoichiometry of one HMG 1/histone tetramer. The results have been interpreted as indicating the existence of an interaction between HMG 1 and both oligomers through two different binding sites.

Wortmannin inhibits translation of tumor necrosis factor-alpha in superantigen-activated T cells.

The superantigen toxic shock syndrome toxin (TSST)-1 can induce tumor necrosis factor (TNF)-alpha expression in T cells and monocytes, through different signaling pathways. We have stimulated peripheral blood mononuclear cells with TSST-1 and found that the major cell producers of TNF-alpha as detected by cytofluorimetry and immunocytochemistry were CD4(+) T lymphocytes. The expression of TNF-alpha by CD4(+) T cells can be inhibited by either, wortmannin (WN) or LY 294002, two phosphatidylinositol 3-kinase (PI 3-K) inhibitors. The inhibitory effect is not transcriptional as WN does not change the mRNA steady state of TNF-alpha at any of the concentrations tested and LY 294002 when preincubated with mononuclear cells at its median inhibitory concentration (IC(50) = 1. 4 microM) significantly inhibited the expression of TNF-alpha but not its mRNA. Immunoprecipitation of pulse-labeled intracellular TNF-alpha showed a specific decrease in the synthesis of this cytokine on cells treated with PI 3-K inhibitors. The p38 mitogen-activated protein kinase (MAPK) is involved in control of TNF-alpha translation in human macrophages. In T cells, we have found that the p38 MAPK inhibitor SB 203580 significantly decreased the secretion of TNF-alpha but not its mRNA. In addition, the combined use of WN and SB 203580 had an additive inhibitory effect on secretion of TNF-alpha. Therefore, both PI 3-K and p38 MAPK signaling pathways control TNF-alpha production in T cells.

Narrow A/T-rich zones present at the distal 5'-flanking sequences of the zein genes Zc1 and Zc2 bind a unique 30 kDa HMG-like protein.

Nuclear extracts from maize endosperm were used to investigate protein-DNA interactions in the 5'-upstream region of the Zc1 and Zc2 genes. These genes encode for zeins of apparent molecular mass (MWapp) 16 and 28 kDa, respectively, which accumulate in the endosperm during seed maturation. Binding assays revealed specific binding of a nuclear protein to three A/T-rich elements, 0.9-1.0 kbp upstream from the initiation codon. One of these elements (41 bp, 88% A/T), present in Zc1, contained a 13 nucleotide duplication. The other two (28 bp, 86% A/T; 42 bp alternating A-T) are consecutive elements in Zc2. Competition experiments strongly suggest that the three elements bind to the same protein. Protein-DNA interaction was detected in endosperm nuclear extracts of 8 to 21 days after pollination (DAP), as well as in 25 DAP embryos and in different tissues from plantlets. The protein factor has an MWapp of ca. 30 kDa. This factor has properties suggesting it is an HMG-like protein. These results are consistent with a growing accumulation of data for a number of genes indicating that A/T-rich elements, located at distal and proximal zones of the 5'-flanking sequences, interact with HMG-like proteins.

Glucocorticoid hormones upregulate interleukin 2 receptor alpha gene expression.

We present evidence that glucocorticoid hormones increase expression of IL-2Rec alpha chain on T cells by regulating IL-2Rec alpha gene transcription. We have previously reported that glucocorticoids can upregulate IL-2Rec alpha mRNA and protein expression in some T cell hybrids. In the present study we show that the glucocorticoid analogue dexamethasone increases mRNA levels of the endogenous IL-2Rec alpha gene and the expression of plasmids containing 5'-flanking sequences of the IL-2Rec alpha gene linked to CAT reporter genes transiently transfected into different cell lines. We show that the dexamethasone effect depends on cis-acting regulatory elements in a segment (-1835/-802) of the mouse gene that also contains cytokine response elements and a inducible DNase I-hypersensitive site. Dexamethasone responses of IL-2Rec alpha-CAT reporter gene constructs were observed in a CTL line, in an IL-3-dependent bone marrow-derived cell line, and in COS7 monkey kidney cells. In the latter the response depended on cotransfection of a glucocorticoid receptor expression vector. The biological relevance of the glucocorticoid-mediated upregulation of the IL-2Rec alpha gene is discussed.

Integrating signals from T-cell receptor and serum by T cells enhance translation of tumour necrosis factor-alpha.

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine produced by several cell types, including T cells upon antigen stimulation. Its production is crucial for the development of an early defence against many pathogens, but its beneficial effects are dependent on the strength and duration of its expression. In this paper we present evidence indicating that serum increases translational efficiency of TNF-alpha in human peripheral blood mononuclear cells stimulated with superantigen. The increase in translation of TNF-alpha due to serum could be inhibited by the phosphatidylinositol (PI) 3-K inhibitors, wortmannin and LY294002, suggesting that PI 3-K is involved in the translational control of TNF-alpha by serum. Similarly to primary T cells, stimulation of Jurkat T cells with superantigen led to TNF-alpha secretion and this was up-regulated by serum. Transfection of Jurkat cells with a constitutively active form of PI 3-Kalpha increased the production of TNF-alpha in cells stimulated with superantigen. Additionally, we used the specific inhibitors targeting ERK kinase and p38 mitogen-activated protein kinase (MAPK), potentially downstream of PI 3-kinase, PD98059 and SB203580. Differently from with PI 3-K inhibitors, the accumulation of TNF-alpha mRNA was inhibited by PD98059 or SB203580. These results suggest that, in T cells, activation of PI 3-K is an important step in controlling TNF-alpha protein synthesis in response to growth factors.

Transcriptional and translational control of TNF-alpha gene expression in human monocytes by major histocompatibility complex class II ligands.

While non-stimulated primary human monocytes exhibit very low levels of tumor necrosis factor (TNF)-alpha mRNA, direct binding of the staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) to major histocompatibility complex (MHC) class II molecules results in a fast (peak 1 h after stimulation), transient induction (sevenfold) of TNF-alpha mRNA. This induction correlates with a fourfold increase in transcription rates of the TNF-alpha gene, as detected by run-on assays, and does not require de novo protein synthesis. Mapping of DNase-I hypersensitive sites (DHS) discloses two constitutive DHS, one located far upstream (within the TNF-beta promoter) and the other centered at -39 +/- 40 bp relative to the major TNF-alpha transcription start site, suggesting that the TNF-alpha gene was transcriptionally competent even prior to MHC class II engagement. Furthermore, stimulation of human monocytes with either TSST-1 or lipopolysaccharide increases the translational efficiency of TNF-alpha mRNA, as shown by a shift in the distribution of this mRNA species in polysome gradients and the translation rates of TNF-alpha measured by immunoprecipitation from cells pulsed with [35S] methionine. The increase in translation efficiency of TNF-alpha mRNA is independent of the half-life of TNF-alpha transcripts, which under the conditions used is unchanged. Taken together, our data indicate that TNF-alpha expression is tightly regulated by MHC class II ligands, both at the transcriptional and translational levels.

Partial overlapping of binding sequences for steroid hormone receptors and DNaseI hypersensitive sites in the rabbit uteroglobin gene region.

Four DNaseI hypersensitive (HS) chromatin regions were found in the uteroglobin locus located at -3.7, -2.4, -0.1 and +4.1 kb with respect to the transcription start site of the gene. The three sites upstream of the gene are only detected in the hormonally stimulated endometrium and disappear after hormone withdrawal, whereas the site at +4.1 is also found in tissues that do not express uteroglobin. In the -2.4 HS region, which is strictly dependent on progesterone treatment, three DNaseI sites are clustered within a 240 bp DNA segment that contains 20 imperfect repeats of an octanucleotide motif. Upstream of the uteroglobin gene there are three regions containing binding sites for the glucocorticoid and the progesterone receptors, located at -3.7, -2.6/-2.7 and -2.4. The -2.4 region contains two binding sites for the hormone receptors flanking the central HS site. In footprinting experiments with naked DNA binding of the receptor also renders this site more susceptible towards digestion with DNaseI. The -2.6/-2.7 region contains three binding sites for the hormone receptors located 140 bp upstream of the HS -2.4. While the -3.7 HS is also located within a receptor binding fragment, there is no binding of the hormone receptors to the promoter region. Thus, interaction of the receptor with DNA sequences far upstream from the promoter alters the chromatin conformation of neighbouring sequences and results in transcriptional activation.

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.