©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interactions of a Transcriptional Activator in the env Gene of the Mouse Mammary Tumor Virus with Activation-dependent, T Cell-specific Transacting Factors (*)

(Received for publication, October 19, 1995; and in revised form, January 22, 1996)

Damaraju Sambasivarao Verner Paetkau (§)

From the Department of Biochemistry, Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mouse mammary tumor virus env gene contains a transcriptional activator (META) that can control transcription of the adjacent long terminal repeat region. Transcriptional control by META parallels that of several lymphokine genes, being specific to T cells, dependent on their activation, and inhibited by the immunosuppressive drug cyclosporine (CsA). DNase I footprinting indicated that nuclear factors from activated T lymphocytes bound a promoter-proximal site, META(P), and a promoter-distal site, META(D+), within the 400-base pair META region. Nuclear factors from unstimulated, but not from activated cells, bound a site, META(D-), adjacent to META(D+). META(D+) directed transcription of a linked luciferase gene, and gel shift analysis revealed binding of inducible, CsA-sensitive T cell factors, in parallel with transfection results. Authentic NFAT and NF-kappaB targets did not compete for the META(D+) binding factor(s). The SV40 core sequence competed for META(D+) binding factors, but META(D+) failed to compete for the complexes obtained with the SV40 probe. Our results, taken together, indicate that META(D+) is a novel transcriptional enhancer element that is similar in its cell-type specificity, activation dependence, and CsA sensitivity to the NFAT element. It may be relevant to the role of MMTV in expression of Mls antigens or the induction of T cell lymphomas.


INTRODUCTION

Mouse mammary tumor virus (MMTV) (^1)is involved in at least three different biological processes: induction of mammary adenocarcinoma, generation of thymic lymphoma, and expression of minor histocompatibility superantigens of the Mls type. The relatively large and complex long terminal repeat (LTR) is implicated in all three responses. Enhancers in the LTR activate cellular proto-oncogenes to induce tumorigenesis in mammary tumors(1) . On the other hand, MMTV variants that induce T lymphomas rather than mammary adenocarcinomas carry large deletions of LTR sequences(2) , whose absence or modification is necessary for T cell tumors to develop(3) . And in the case of the minor Mls antigens, the LTR encodes the protein antigen in one of several forms(4, 5) . In each of these three systems, there is a requirement for MMTV-related transcriptional activity in lymphocytes, although the molecular mechanisms regulating transcription control may differ between them. The requirement for expression in lymphocytes includes the induction of tumors by the mammotropic strains of MMTV, as part of the mechanism of transmission to the mammary epithelium. This participation requires expression of the virally encoded Mls antigen(6) . The LTR contains strong, corticosteroid-inducible transcriptional activator elements(7) , but it is not clear what their role is, if any, in transcriptional control in lymphocytes.

The LTR of MMTV resembles that of other retroviruses in that it contains the transcriptional control elements and start site for viral RNA synthesis. We have described a second, novel promoter located in the env gene of MMTV, which is activated in certain T lymphoma cell lines (8, 9) (Fig. 1). This promoter generates transcripts of the LTR found in the mouse EL4.E1 cell line(8, 14) , in which the amplified genomic copies of MMTV provirus contain a large deletion typical of these lymphomas. The starting point of the transcript lies at position 7247 in the MMTV map, within the env gene (8, 9) (Fig. 1a). The relevant promoter is apparently controlled by the sequence immediately upstream of it in the env gene. The transcriptional activity of a 411-bp env gene segment (Fig. 1b), which we term META (for MMTV env transcriptional activator) was established by transient transfection experiments(9) , and it was further shown that this activity resembled closely the activity of the interleukin 2 (IL2) promoter in the same cells. It was restricted to T helper lymphocytes, it was dependent on cellular activation through antigen receptors or their mimics, and its induction was inhibited by the drug cyclosporine (CsA). META was active in mouse and human T lymphocyte cell lines that resemble T helper cells by being inducible for lymphokine synthesis, including mouse EL4.E1 cells, a mouse T hybridoma cell line, and the human Jurkat cell line. It was not active in HeLa cells, or, under the conditions studied, in a mouse B cell line or a cytotoxic T lymphocyte cell line. The env gene was shown to contain both a transcriptional activator and a start site for mRNA synthesis. In this paper, we have examined META for interactions with activation-induced DNA-binding factors and have identified an element that can account for the transcriptional control properties of the env gene itself.


Figure 1: Structure of META. The 9.9-kb MMTV genome is represented in a, with the META segment located in the env gene. The LTRs (1328 bp) contain the conventional MMTV promoter, responsible for the synthesis of MMTV RNA, which originates near the 3` end of the 5` LTR. The MMTV provirus from which META was first isolated, derived from EL4.E1 T lymphoma cells, carried a 494-bp deletion within the LTR, indicated by the shaded box in the figure. META is responsible for the activation-dependent, CsA-suppressible transcription described earlier(8, 9) , which originates within the env gene and copies the 3` LTR. b, pBLCAT2-C30 is a plasmid in which a META sequence lacking the transcriptional start site was cloned as a BamHI insert upstream of the herpes simplex virus tk promoter and the CAT coding region(9) . It represents segment 6814-7212 of MMTV. c, the nucleotide sequence of the META-BamHI fragment in pBLCAT2-C30, and derived plasmids used in this study. Sequence numbering here is with respect to the transcription initiation site within META, which occurs at position 7246 of the milk-borne virus sequence(10) . Nucleotide positions at the ends of the fragment that were altered to generate restriction enzyme sites are shown in lowercase. The underlined segments represent restriction enzyme sites (thin line) and sequence motifs that are candidates for transcription factor binding sites (heavy underline). The potential binding sequences, labeled ``M-'' (for motif), were identified based on their similarity to the consensus binding sequences for the transcription factors indicated(11) : AP2 and AP3 (activating proteins 2 and 3, respectively); MAF, mammary cell activating factor(12) ; TCEp, proximal T cell element of the IL2 gene(13) ; Pu box, proximal purine box of mouse or human IL2 enhancer. Regions identified by DNase I footprinting are delineated by boxes (META D segments, META P; see Fig. 2and text). The sequence shown here is derived from the MMTV provirus (clone C30) used in this and previous work(8, 9) , and differs slightly from that of the milk-borne virus(10) .




Figure 2: DNase I footprinting analysis of META. The lower (A) or the upper (B) strand of META was end-labeled and subjected to DNase I digestion in the presence of nuclear extracts from uninduced Jurkat cells(-), or cells stimulated (+) with PMA plus ionomycin (A) or PMA plus A23187 (B). The positions of segments differentially protected in induced and uninduced extracts are indicated as ``D'' (distal) or ``P'' (proximal) on the line representation of the sequence on the left, which also shows the location of the label (*). Protected regions META(D-) and META(D+) were observed with the uninduced and induced nuclear extracts, respectively, and region P was found to be weakly protected with induced extracts. N, no nuclear extract; G, G/A, and T/C indicate Maxam-Gilbert chemical sequencing reactions.



The induction of transcription from the META promoter resembles the activation of IL2 transcription in the same cells. Expression of the IL2 gene in T helper lymphocytes is controlled primarily by the sequence lying within 300 bp upstream of the transcriptional start site (15, 16) . Several transcription factors interact with this region, including AP1, AP3, Oct-1, NF-kappaB, and NFAT(13, 17, 18) . The NF-kappaB and NFAT sites show activation-dependent binding with their relevant factors in vivo(19) . NFAT is the most sensitive of these sites to CsA, as its binding factor is completely blocked by the drug. Both NF-kappaB and AP3 are somewhat less sensitive(20) . The META region contains several transcription factor binding motifs, but none with the exact NFAT sequence of the human IL2 gene (Fig. 1c). Thus, it was of interest to determine whether an element could be identified that accounted for the characteristic transcriptional activating properties of META. This paper describes in detail such an element, which we term META(D+).


MATERIALS AND METHODS

Cells

Jurkat (human T cell leukemia), EL4.E1 (mouse T-lymphoma), S194 (mouse myeloma), and HeLa (endothelial carcinoma) cell lines were maintained in RHFM medium comprising RPMI 1640, 20 mM HEPES (pH 7.4), 100 µM 2-mercaptoethanol, antibiotics, and 10% fetal bovine serum(9) .

Nuclear Extracts

Jurkat cells were stimulated for 4-6 h with 15 ng/ml 12-phorbol 13-myristate acetate (PMA) plus 1.5 µM ionomycin, or with PMA plus 0.5 µM A23187. IL2 was measured by bioassay(21) . CsA was present at 100 ng/ml where indicated. Nuclear extracts from unstimulated and stimulated cells were prepared by the lysolecithin lysis method(22) . Nuclear extracts were aliquoted, frozen in liquid nitrogen, and stored at -70 °C. Protein concentrations in cell extracts were determined with a Coomassie protein assay kit (Bio-Rad).

DNA Methodology and Reagents

Restriction enzyme digestions, DNA polymerase reactions, ligations, end labeling, agarose or polyacrylamide gel electrophoresis (PAGE), gel elution of DNA fragments, and Sephadex G-50 spin column chromatography were carried out by standard protocols(23) . Oligonucleotides were obtained from the DNA synthesis facilities of either the Departments of Microbiology or Biochemistry at the University of Alberta. Molecular biological reagents and enzymes were obtained from Life Technologies, Inc. (Burlington, ON), the luciferase reporter vector pGL2 from Promega (Madison, WI), Maxam-Gilbert chemical sequencing kit from DuPont NEN, and the Maxi Plasmid DNA purification kit from Qiagen Inc. (Charlesworth, CA). PMA, ionomycin, A23187, luciferin, acetyl coenzyme A, and DNase I enzyme were purchased from Sigma. CsA was a gift from Sandoz Canada Inc. (Dorval, Quebec).

DNA sequences were amplified by the polymerase chain reaction (PCR) using primers described in Table 1, carrying the prefixes S (for sense orientation) and A (for antisense). Primers and templates were heat denatured and annealed(23) . Typical conditions for the PCR were: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01% gelatin, 3.0 mM MgCl(2), 0.2 mM dNTPs, 80 nM primers, 6.25 ng of plasmid template, and 1.25 units of Taq DNA polymerase, in a total volume of 50 µl. The Hybaid thermal reactor (Bio/Can Scientific, Toronto, ON) was programmed for the following conditions: 96 °C for 5 min, then two cycles of 94 °C for 1 min, 45 °C for 1 min and 72 °C for 1 min; followed by 25 cycles of 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min; and ending with an extension reaction at 72 °C for 3 min. PCR products were separated by PAGE in 1 times TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA).



Plasmids

Numbering of META segments is based on the transcriptional start site of the META-driven promoter in MMTV(9) . Construction of the plasmid pBLCAT2-C30 containing META sequences from -29 to -439 (Fig. 1b) has been described(9) . Plasmids pGL2 promoter and control vectors are the luciferase reporter plasmids containing the SV40 promoter, and the SV40 promoter plus enhancer, respectively. Plasmid pDS-tk13 has the thymidine kinase (tk) promoter in place of the SV40 promoter in the pGL2 promoter vector background, and was constructed as follows. Plasmids pBLCAT2-C30 and pGL2 promoter vectors were digested with BglII and HindIII, respectively, and the recessed ends were filled in using the Klenow DNA polymerase enzyme. Linearized, blunt-ended pBLCAT-C30 was cut with BamHI to yield the 411-bp META fragment, a 168-bp tk promoter fragment, and a 4.3-kb vector DNA fragment. Digestion of the linearized, blunt-ended pGL2 promoter vector with BglII yielded a 203-bp fragment containing the SV40 promoter and 5.59-kb vector DNA fragment. The tk promoter fragment generated from pBLCAT-30, and the linearized pGL2 vector segment lacking the SV40 promoter were purified on agarose gels and ligated to yield the pDS-tk13 enhancerless luciferase reporter vector.

A number of double-stranded DNAs were prepared by PCR amplification using primers described in Table 1. META(D+) DNA with protruding 5` AA (S21) and TT (A21) termini were first filled in using Klenow DNA polymerase and then blunt-end ligated for 16 h at 14 °C to generate multimers. These were cloned upstream of the tk promoter in pDS-tk13 to generate recombinant plasmids pDSD61 to pDSD65 (see Fig. 5). The head-to-tail configuration of the META(D+) segments generated a HpaI restriction enzyme site, which was used in conjunction with other methods to determine the orientation and copy number of D(+) enhancer constructs (head-to-head and tail-to-tail configurations lack the HpaI site). Plasmid pDSM25F is a luciferase reporter plasmid derived from pDS-tk13 harboring the META fragment extending from -29 to -429 upstream of the tk promoter. The complete META fragment (411 bp) was liberated from pBLCAT2-C30 by SalI digestion, purified by PAGE, and ligated into XhoI-cut pDS-tk13 to generate pDSM25F. Human genomic IL2 sequences from -98 to -362 or from +45 to -180 relative to the transcription start site were amplified by PCR using primer pairs S17 and A17 (for -98 to -362) or S16 and A16 (for +45 to -180) (Table 1). The hIL2 PCR product for -98 to -362 region was gel-purified, digested with EcoR I and HindIII, and ligated into pGEM-3Z vector cut with EcoRI and HindIII. The ligation mixture was transformed into Escherichia coli DH5alpha, and plated on LB agar-ampicillin indicator plates containing 5-bromo-4-chloro-3-indoyl beta-D-galactoside and isopropyl-1-thio-beta-D-galactopyranoside for blue/white colony selection. White colonies were selected, and the recombinant plasmid pDIL2 thus generated was purified and sequenced. Recombinant constructs were confirmed by both sequencing and/or restriction digestion analysis.


Figure 5: Inducible and CsA-suppressible expression of META(D+)-luciferase constructs in Jurkat cells. The structures of the plasmids used are schematically shown to the left of the graph that depicts their induction by PMA plus A23187. Plasmid pGL-2 control vector contained an SV40 promoter and enhancer. All other plasmids were expressed under the control of the thymidine kinase promoter (tk prm). The compositions of the plasmids were as follows: pDSM25F, complete META region, containing a single copy of META(D+); pDSD 63, pDSD 69, pDSD 61, pDSD 65, plasmids containing the 39-bp META (D+) element in 3-7 copies upstream of the tk promoter, in the orientations indicated by arrows. Rel. fold induction represents the induced/uninduced signals for a given construct, relative to the same ratio for the appropriate enhancerless promoter. The actual values of the luciferase assay are given in the table at the bottom of the figure (light units/10 µg of protein in the assay). pGL-2 and pDS-tk13 are the enhancerless promoters of SV40 and thymidine kinase, respectively. The luciferase expression values for the plasmids induced in the presence of CsA are also given in the table (bottom row), with the expression in CsA relative to the maximum in parentheses. Data are presented from a single transfection experiment, but similar results were observed in at least three independent experiments.



DNase I Footprint Analysis

Plasmid pBLCAT2-C30 was used as a source of META (-439 to -29, Fig. 1b). For upper strand labeling, the plasmid was linearized with XbaI, dephosphorylated, and end-labeled using [-P]ATP and T4 polynucleotide kinase I. Radiolabeled DNA was separated from unincorporated label on a Sephadex G50 spin column. PstI digestion of this DNA resulted in 528-bp labeled META fragment with flanking vector sequences, a 9-bp labeled PstI-XbaI fragment from the multicloning site, and the remaining 4363-bp vector DNA. The lower strand of META was labeled at the XbaI end starting with pBLCAT2-C30 containing META (-29 to -439) in the reverse orientation. HindIII digestion of this DNA resulted in the release of a 406-bp labeled META fragment along with labeled 20-bp XbaI-HindIII fragment from the multicloning site and the 4485-bp of the vector DNA. The labeled META DNA along with the vector DNA fragments obtained by these procedures was used without further fractionation.

Binding reactions and DNase I digestions were carried out at room temperature as described in the Life Technologies, Inc. DNase I footprinting manual, with minor modifications. Briefly, the assay system contained 10 mM Tris-HCl, pH 7.0, 2 mM CaCl(2), 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 30 mM KCl, 2.5 µg/ml bovine serum, 0.5% glycerol, 100 µg of Jurkat cell nuclear protein, and 2 µg of poly(dI-dC) in a reaction volume of 50 µl. Reactions were incubated for 10 min followed by the addition of 1 ng of DNA probe (20,000 cpm). After an additional incubation of 20 min, 50 µl of DNase I buffer (10 mM HEPES, pH 7.8, 5 mM MgCl(2), and 1 mM CaCl(2)) was added, and 50 ng of DNase I. Digestion was carried out for 1 min, and the reaction was terminated by the addition of 10 µl of stop buffer (10 mM MES, pH 7.0, 15 mM EDTA, 0.5% sodium dodecyl sulfate, 250 µg of salmon sperm DNA, and 10 µg of proteinase K). Proteinase K digestion was continued for 30 min at 37 °C. The reactions were extracted with phenol:chloroform:isoamyl alcohol (25:24:1), precipitated using ethanol and sodium acetate, dissolved in formamide loading buffer, and subjected to 6% acrylamide, 7 M urea PAGE (0.4 mm thick gel) in 1 times TBE buffer using a Life Technologies, Inc. vertical gel apparatus. Dried gels were exposed to Kodak X-Omat AR film at -70 °C with an intensifying screen.

Mobility Shift Assays

Single-stranded oligonucleotides (sense strands) were end-labeled using [-P]ATP and polynucleotide kinase, the kinase was heat-denatured, and the fragments were annealed to complementary antisense oligonucleotides. Unincorporated label was separated on G-50 Sephadex spin columns. Binding reactions were carried out with 0.5 nM probe (5,000-10,000 cpm) in a final reaction volume of 20 µl at room temperature. Reaction mixtures contained 25 mM HEPES, pH 7.8, 75 mM KCl, 1 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 5% glycerol, 3 µg of poly(dI-dC), and 10 µg of Jurkat cell nuclear extracts. Probe was added to the reaction mix after 10 min, or a total of 25 min when cold competitor DNA was included. Binding reactions were then continued for 30 min, analyzed on 4% acrylamide (1.5-mm-thick gels) in 1 times TBE at room temperature. Dried gels were exposed to Kodak X-Omat AR film at -70 °C with an intensifying screen.

Transfections and Measurement of Luciferase Activity

Purified plasmid DNA that was >90% closed circular, as judged by gel analysis and ethidium bromide fluorescence quantitation (27) was used in the DEAE-dextran transfection protocol(9) . A total of 15 µg of DNA was used in a volume of 2 ml. Following transfection, cells were resuspended, divided, and maintained in RHFM medium for 36-42 h. Cells were induced for 6 h by adding PMA plus A23187 with or without CsA. Cells were pelleted, washed twice with phosphate-buffered saline, and resuspended in lysis buffer: 1% Triton X-100, 25 mM glycylglycine, pH 7.8, 15 mM MgSO(4), 4 mM EGTA, and 1 mM DTT (28) . Luciferase activities in whole cell extracts were measured as described in the Promega luciferase assay manual using a Lumat LB 9501 luminometer. The luciferase assay contained 20 mM glycylglycine, pH 7.8, 1 mM Mg(CO(3))(2), 2.7 mM MgSO(4), 0.1 mM EDTA, 33 mM DTT, 0.47 mM luciferin, 0.53 mM ATP, and 0.27 mM coenzyme A in a reaction volume of 100 µl.


RESULTS

DNA Footprint Analysis of the Interaction of Nuclear Factors from Jurkat Cells with the META Sequence

The complete META segment of MMTV, comprising the 411-bp region lying between 29 and 439 bp upstream of the novel env promoter (Fig. 1), has previously been shown to mediate inducible, CsA-suppressible, and T lymphocyte-specific expression of reporter genes(9) . Overall, it responds in parallel with lymphokine enhancers, such as that of IL2, in T cell lines. We therefore carried out DNase I footprinting experiments to look for interactions with nuclear factors from Jurkat cells, a human T cell line that is induced to synthesize IL2 by a combination of PMA and a calcium ionophore. Nuclear extracts from uninduced Jurkat cells, and from cells stimulated with PMA plus ionomycin, were examined. The footprint pattern shown in Fig. 2A revealed two regions of protection with induced extracts, compared to extracts from unstimulated cells. These are referred to as proximal (META(P)) and distal (META(D)). META(P) is located around -116 to -146 relative to the META transcriptional start site. Extracts from induced cells showed partial or faint protection of this region, compared to uninduced cells. In three independent experiments with end-labeled lower strand, partial protection of META(P) was consistently observed. On the other hand, META(D) comprised two strong and discrete footprints with extracts of stimulated cells, one in the -296 to -323 region and the other in the -325 to -349 region.

To further delineate the META(D) footprint, we performed DNase I digestions using the upper strand of META (Fig. 2B). The footprint pattern in the META(D) region showed some differences compared to the lower strand results of Fig. 2A. Specifically, the -296 to -323 region was protected by uninduced nuclear extracts, whereas the -325 to -349 region was protected with induced extracts. We have repeated these experiments three times with each of the lower and upper strands, using either PMA/ionomycin or PMA/A23187 as inducing agents (the two calcium ionophores are similarly effective in inducing IL2 synthesis in Jurkat cells, and showed no difference in footprinting results). The META(D) footprint shown in Fig. 2A was the only case in which both parts of META(D) were protected by nuclear extracts from induced cells only. All of the experiments in which the upper strand was labeled were similar to the result in Fig. 2B. Two out of three experiments with the lower strand labeled also showed this protection pattern, i.e. protection of one element of META(D) in unstimulated cells (META(D-)), and of an adjacent element in stimulated cells (META(D+)). The single anomalous result shown in Fig. 2A, i.e. protection of the complete META(D) region by induced extracts, did not result from either peculiarities of the binding reaction or limited resolution at the top of the gel; the results were reproducible with that extract and were unchanged upon longer electrophoresis. In all experiments, however, META(D) and META(P) always exhibited differential protection, and no protected regions other than these were seen in the 411-bp META sequence.

To summarize, the META(D+) element was consistently protected by extracts from induced cells, relative to uninduced extracts. In most cases, META (D-) was protected by extracts from uninduced cells (the exception being the result of Fig. 2A). META(P) was protected weakly by extracts of induced cells. META(D+) protection was the most strongly correlated with conditions of transcriptional induction of META itself (9) and of lymphokines, and it was therefore selected for further analysis.

Binding of Jurkat Nuclear Factors to the META(D) Elements

The META(D+) and META(D-) elements are delineated in Fig. 1C. Oligonucleotides containing these sequences (Table 1) were used to look for binding factors by gel shift analysis with nuclear extracts from Jurkat cells (Fig. 3). The D(+) probe (lanes 1-4) demonstrated one very strong complex (arrow 1), and one that was barely detectable (arrow 2) with extracts from unstimulated cells. Under conditions of transcriptional activation, a very strong additional band of intermediate mobility (arrow 3) was seen, and a greatly enhanced amount of complex in the region of the slower constitutive band (arrow 2). Formation of these complexes was almost completely abolished by CsA, which blocks activation of META transcription(8, 9) . The META(D-) probe (lanes 5-8) showed a strong band with uninduced cell extracts, which was almost completely abolished under conditions of cellular induction, and was partially restored when CsA was present (arrow 4).


Figure 3: Binding of nuclear factors to the META(D) region. Mobility shift assays were carried out using META(D+) or META(D-) probes at 0.5 nM. The probe for META(D+) consisted of oligonucleotides S21 + A21, and the probe for META(D-), of oligonucleotides S22 + A22 (Table 1). Lanes 1-4, META(D+) probe; lanes 5-8, META(D-) probe. NE, nuclear extract; FP, free probe; -, nuclear extract from uninduced Jurkat cells; +, nuclear extract from Jurkat cells stimulated with PMA and A23187; CsA, nuclear extract from induced plus CsA-treated cells. Arrow 3 on the left points to the complex found only with induced extract, and almost completely suppressed by CsA. Arrow 1 identifies a constitutive complex, while arrow 2 points to a complex barely visible in unstimulated cells, and strongly induced by PMA plus A23187. The complex observed with the META(D-) probe (arrow 4) was seen with uninduced extracts and with extracts induced in the presence of CsA, and was barely visible with extracts from induced cells. A slower migrating complex (arrow 5) was just detectable with uninduced and CsA-treated cell extracts.



The results of gel shift analysis are consistent overall with the results from DNase footprinting, in that a specific complex was found under conditions of transcriptional activation when META(D+) was used as the probe, whereas the reciprocal pattern was seen with META(D-) as probe.

Specificity of the META(D+) Inducible Complex

The binding of nuclear factor(s) to the META(D+) probe was investigated by using unlabeled homologous and heterologous competing oligonucleotides (Fig. 4). The signal due to binding of both constitutive and induction-dependent factors to META(D+) was titrated out by unlabeled META(D+) probe, but not by a nonspecific oligonucleotide derived from a nearby, but unrelated, META sequence of similar length. (The nonspecific competitor contains an element similar to the AP3/TCEp motif of the upstream control region of IL2(13) ).


Figure 4: Specificity and saturability of the META(D+) binding site. A gel shift experiment was carried out as in Fig. 3, using META(D+) as the probe. Lanes 3-5 show that the binding of the induction-dependent nuclear factor (arrow) could be titrated away with the unlabeled ``self'' probe due to saturation. An unrelated oligonucleotide, corresponding to positions -278 to -253 of META (Fig. 1) did not compete (NS, lanes 6 and 7). Amount (x) indicates the molar ratio of unlabeled competitors to labeled probe.



The 41-bp META(D+) probe contains an RsaI site between positions -334 and -333 ( Fig. 1and Table 1). When the probe was treated with RsaI, no retarded band was seen with any extracts (data not shown). These results suggest that the central region of META(D+) must be intact for binding of nuclear factors. This segment contains an AP3/NF-kappaB motif (see Fig. 7and ``Discussion'').


Figure 7: Comparison of the META(D+) sequence to various enhancer domains. The META(D+) region defined by DNase I protection, from -349 to -325, is indicated, as is a short inverted repeat sequence. The sequences shown here correspond to the oligonucleotides (see Table 1) used in the mobility shift experiments (Fig. 8Fig. 9). The binding motifs are indicated. The NF-kappaB motif in META(D+) is on the opposite strand (dashed underline). Numbers in parentheses indicate the number of matches to the reported consensus binding sequence(11) : AP2 CCC(A/C)N(G/C)(G/C)(G/C); AP3, TGTGG(A/T)(A/T)(A/T)GT; NF-kappaB, GGGA(A/C)TN(T/C)CC. Mismatches of the SV40, NF-kappaB, and NFAT motifs with META(D+) are shown in lowercase. The GGAAA motif characteristic of the NFAT family of proteins is underlined.




Figure 8: Competition by AP3 and NF-kappaB binding sequences for the META(D+)-binding proteins. Gel mobility shift experiments were carried out using labeled META(D+) probe and unlabeled competitor oligonucleotides, either an SV40-derived AP3 site (sequences SAP3 and AAP3, Table 1) or hIL2-derived NF-kappaB (S09 and A10, Table 1). Lanes 1 and 2, no competitor; lanes 3-6, AP3/SV40 competitor; lanes 7-11, hIL2 NF-kappaB competitor; lanes 12-14, nonspecific oligonucleotide competitor, corresponding to positions -125 to -96 of META (sequences S20 plus A20, Table 1). Other symbols and conditions are as in Fig. 3and Fig. 4. The region of free probe is not shown in this autoradiogram.




Figure 9: Binding of induced nuclear factors to the AP3 probe. Labeled AP3 probe (see Fig. 8) was used with unlabeled oligonucleotide competitors. Lanes 1-3, no competitor; lanes 4-7, unlabeled AP3/SV40 oligonucleotide (self); lanes 8-12, unlabeled META D+) oligonucleotide; lanes 13-18, unlabeled hIL2 NF-kappaB oligonucleotide; lanes 18 and 19, a nonspecific oligonucleotide corresponding to sequence from -125 to -96 of META (sequences S20 plus A20, Table 1). Other symbols and conditions are as in Fig. 3and Fig. 4. Free probe not shown.



The induced and constitutive complexes of META(D+) differed in sensitivity to the addition of unlabeled homologous probe, with the induced complex (Fig. 4, arrow) showing a greater sensitivity, possibly reflecting a lower concentration of the factor responsible. The results of competition show that the binding was specific and saturable.

The META(D+) Sequence Confers Inducible, CSA-suppressible, Transcriptional Activation

To determine the enhancer function of META(D+) in intact cells, we transfected Jurkat cells with plasmids containing various META(D+)-related constructs, driving the expression of a luciferase reporter gene (Fig. 5). The activities of the enhancer constructs were compared to the relevant promoter constructs lacking enhancers. The pGL-2 plasmid containing an SV40 promoter/enhancer sequence, included as a control, showed increased luciferase gene expression on stimulation with PMA plus A23187, as has been observed by others(13, 24, 29) . The activity observed with the full-length 411-bp META region (pDSM25F) linked to the thymidine kinase promoter was also stimulated by activation with PMA and calcium ionophore, and this activity was CsA-sensitive. This is parallel to the effect of META on the expression of the CAT gene reporter(9) . When individual META(D+) enhancer elements were linked to the tk promoter, luciferase expression was also induced by PMA plus A23187, with the strength of induction being related to the number of elements placed in series. Activation did not require the elements to be oriented in one direction, and in every case was strongly sensitive to CsA. A 7-mer of META(D+) generated more than a 100-fold induction upon activation, compared to the enhancerless promoter construct, with 93% of the induced activity being inhibited by CsA.

The tandem META(D+) arrays were not only induced relatively more by activation than was the pGL-2 SV40 construct, under induced conditions they were higher in absolute level as well, with the most powerful, pDSD 65, being almost 100 times as strongly expressed as the SV40 enhancer-promoter combination (numerical data are given in Fig. 5, bottom panel).

Full Activation of the META(D+) Enhancer Requires Both PMA and Calcium Ionophore and Is T Cell-specific

In an earlier study we showed that the 411-bp META fragment mediates T cell-specific, induction-dependent expression of the CAT reporter gene (9) . In that study, META was activated in both mouse (EL4.E1) and human (Jurkat) T helper cell lines, and in a mouse T helper hybridoma cell line. The dual signal requirement and T cell specificity of the META(D+) enhancer element was therefore investigated using several cell lines, including Jurkat transfected with plasmid pDSD 65 (Fig. 6). Exposure of the transfected cells to calcium ionophore alone had no significant effect on the reporter construct. Stimulation with PMA alone increased the activity to about 37% of the maximum activity generated with both PMA and A23187. CsA inhibited 87% of the fully induced activity, whereas PMA-induced activity was only inhibited by 33%.


Figure 6: Dual signal requirement and T lymphocyte specificity of the META(D+) enhancer. A, Jurkat cells were transfected with the luciferase expression plasmids pDS-tk 13 or pDSD65 (Fig. 5) and stimulated with the agents shown. Data are expressed as relative fold induction, as in Fig. 5. The luciferase activities (light units/10 µg) were as follows: uninduced (UI), 271; A23187 alone, 404; PMA alone, 5,520; PMA + A23187, 79,520; PMA + A23187 + CSA, 10,280. The luciferase activities for the control enhancerless plasmid pDS-tk 13 was 1,870/334 (induced/uninduced). B, relative activities of pDSD65 in different types of cells. S194 (B cell), HeLa (fibroblast), EL4.E1 (mouse T lymphoma), and Jurkat (human T lymphocyte) were transfected with the plasmid and stimulated with PMA plus A23187. Data are expressed as relative fold induction as described above. The luciferase activities (induced/uninduced) were: S194, 189/77; HeLa, 11,560/2,300; EL4.E1, 459,600/12,930; Jurkat, 111,070/460. The luciferase activities for enhancerless plasmid pDS-tk 13 in induced/uninduced conditions were: S194, 194/90; HeLa, 4,070/3,390; EL4.E1, 26,790/14,890; Jurkat, 3,220/431. Data in A and B represent the results from different experiments. These profiles were reproduced in two independent experiments.



The activity of the META(D+) enhancer was dependent on the type of cell used for transfection. The T lymphocyte cell lines EL4.E1 and Jurkat showed high levels of inducible activity compared to the non-T cells S194 and HeLa. It is noteworthy that both S194 and HeLa cells contain active NF-kappaB and AP3 factors under constitutive and/or inducing conditions, yet did not significantly activate META(D+).

Comparison of the META(D) Sequence to Various Cellular and Viral Enhancers

Examination of the META(D) region revealed several potential motifs for binding transcription factors AP1, AP2, AP3, NFAT, and NF-kappaB (Fig. 7). The AP3 binding site in the SV40 enhancer mediates tissue-specific, inducible enhancer functions in a variety of cell types. Several factors interact with SV40 core sequence, including a family of NF-kappaB related proteins (p50, p55, p65, p75, p85) from stimulated Jurkat cells(30) , TCF-1, -2, and -3 from EL4 cells(31) , NP-TCII from unstimulated Jurkat cells(32) , AP3 from HeLa cells, and authentic NF-kappaB from B-lymphocytes(25, 33) . These classes of proteins also bind the NF-kappaB sequence in the human IL2 enhancer(13, 18, 31) . The META(D+) sequence shows similarity to AP3 and NF-kappaB binding sites. Except for the NF-kappaB element of the IL2 enhancer (tGAAA), all others shown in Fig. 7have the conserved motif GGAAA characteristic of the binding regions for AP3, NF-kappaB, NFAT, and other members of the Dorsal/Rel family of transcription factors. The NP-TC II motif in the SV40 enhancer resembles the NF-kappaB binding site, but it binds a different factor(32) . Enhancers from META(D+), SV40, and IL2 have 7-8 nucleotide matches to the consensus NF-kappaB site (GGGA(A/C)TN(T/C)CC)(11) . The NFAT core sequence of the human IL2 enhancer exhibits 6/10 matches to the consensus AP3 binding sequence (TGTGG(A/T)(A/T)(A/T)GT)(11) . We therefore used IL2 enhancer DNA or specific oligonucleotides to compete for binding of the META(D+) element to its factor(s) in nuclear extracts.

The SV40 Oligonucleotide Competes for the META(D+) Binding Factor, but NF-kappaB Does Not

We investigated the binding of factors in activated Jurkat nuclei to the META(D+) probe in the presence of competing SV40 promoter sequences and hIL2 NF-kappaB oligonucleotides. SV40 contains an AP3 motif(33) , and also an element that binds both the inducible factor NF-kappaB (34) and factor NF-TCII(32) , which is expressed constitutively in T and B lymphocyte cell lines. The mobility shift experiments shown in Fig. 8demonstrate that the 22-bp SV40 oligonucleotide (lanes 3-6) competed quite efficiently for the factor(s) binding META(D+), comparable to the META(D+) self-competition (cf. lane 4 of Fig. 4). On the other hand, the hIL2 NF-kappaB element, which shows only a limited similarity to META(D+) (Fig. 7), was a poor competitor; even a 200-fold molar excess produced only a modest diminution of the signal (lanes 7-11). An oligonucleotide containing the mammary cell activating factor-like region of META (-126 to -96, see Fig. 1and Table 1), and which also contains the central GGAAA pentanucleotide characteristic of the AP3 or NFAT sites, did not compete (lanes 12-14), suggesting that sequences flanking the pentanucleotide are essential for META(D+) binding.

The competition by SV40 for the META(D+)-binding factor(s) prompted us to examine the reciprocal situation (Fig. 9). The SV40 probe exhibited a mobility shift with nuclear extracts from both control and induced Jurkat cells, but the two patterns were completely different. Nuclear factors from uninduced cells (lane 1) yielded one very weak band of fairly high mobility, whereas induced extracts (lane 2) yielded two different bands, much stronger and of lower mobility. The two activation-dependent bands were not significantly affected by CsA. META(D+) did not compete effectively for the induced complexes, compared to the SV40 sequence itself. The human IL2-derived NF-kappaB probe competed moderately well for the lower mobility SV40 activation-dependent complex, but not for the other one.

Search for a META(D+) Competitor Sequence in the IL2 Gene

One of the interesting features of the META(D+) complex is that its induction is abolished by the immunosuppressive drug CsA. This is characteristic of very few transcriptional activating elements, chief among these being the NFAT element of the IL2 and other cytokine genes (20) . Although NFAT and META(D+) elements both contain a central GGAAA pentanucleotide, there is no overall similarity between them (Fig. 7). Nevertheless, because of their functional similarity, we tested human IL2 upstream DNA probes, as well as an oligonucleotide carrying the NFAT motif, for their ability to compete with META(D+). DNA from the human IL2 gene representing the regions +45 to -183, -98 to -362, as well as the AP1 and NFAT oligonucleotides were tested as competitors individually. The oligonucleotides corresponding to the various regions in META, namely AP3/TCEp, polypurine element, and mammary cell activating factor, which harbor the GG(A/T)(A/T)(A/T) motifs (Fig. 1c) were also end-labeled and tested individually in mobility shift assays. However, the induction-dependent binding to META(D+) was not affected by any of the IL2 enhancer elements (data not shown). The overall conclusion is that there is a major binding site for inducible, CsA-suppressible nuclear factors in the META(D+) region, and that it is activated in parallel with, but distinct from, the IL2 NFAT site.


DISCUSSION

We have identified an enhancer element in the MMTV env gene, META(D+), located between positions -325 to -350 relative to the start site of the transcript of the 3` LTR described earlier(8, 9) . Previously, the META fragment extending from -431 to -34 was shown to confer orientation-independent, induction-dependent, and CsA-suppressible activity in T helper lymphocytes(9) . The existence of the META(D+) element was suggested by DNase I footprinting analysis and confirmed by gel retardation assays and transient transfection studies. Multimers of the META(D+) element conferred activation-dependent, T lymphocyte-specific, and CsA-suppressible expression on the luciferase reporter gene in transient transfections. The overall enhancer strength was related to the number of copies of the element, but did not require that the individual copies be oriented in the same direction (Fig. 5).

Two other putative control elements, META(P) and META(D-), were indicated by DNase I footprinting analysis. Data in Fig. 2suggest that META(D-) may be a negative control region, since in almost every experiment a binding complex was found under non-transcribing conditions only (either uninduced cells, or cells induced in the presence of CsA). Of the several DNase I footprinting reactions carried out, there was only one instance where the protection with induced nuclear extracts occurred over the entire META(D) region (this exception is shown in Fig. 2A). The mobility shift experiments with META(D-) probe (Fig. 3) are in agreement with its being a negative element (i.e. complexes were formed under non-transcribing conditions only). Examination of the META(D-) sequence indicated a potential AP1 binding site, TGACcAA, in the -314 to -308 region(11) . Similar to the closely spaced (D+) and D(-) elements in META, the NF-kappaB (-208 to -188) and AP1 (-185 to -178) elements in the IL2 enhancer are also adjacent. Footprinting analysis of the IL2 enhancer in vivo has revealed that the AP1 element is protected under uninduced conditions, whereas the NF-kappaB element is protected under conditions of induction(19) . A negative role for the distal AP1 site in mouse IL2 upstream sequence has been suggested, since it showed diminished binding of nuclear factors from stimulated cells compared to the unstimulated ones(35) . In contrast to the strong and reproducible footprints found for META(D+), the proximal site at META(P) was only weakly protected, and we do not know whether it has an enhancer function.

META(D+) contains an inverted repeat overlapping the 5` end of the central binding region (Fig. 7). Such structures can negatively regulate expression of inducible genes(36) , including c-fos(37) and human beta-interferon(38) . The dyad symmetry element encompasses an AP2 motif (7/8), but there was no evidence that it bound an inducible protein.

Given the similarities in the transcription regulatory properties of META and the IL2 upstream region(13) , it was reasonable to suppose that the META(D+) element might be responding to the IL2-related NFAT binding factor(s). However, the human IL2 NFAT probe did not compete for META(D+) in gel shift analysis. A general hypothesis explaining the action of CsA has been proposed, which involves a complex of proteins recognizing NFAT plus one of several ancillary proteins such as AP1, NF-kappaB, or Oct(39, 40) .

The lysolecithin method we used in this paper to extract nuclear binding proteins utilizes 0.6 M KCl. The more conventional methods depend on hypotonic shock and ammonium sulfate precipitation of nuclear proteins(13, 22) . NFAT binding is diminished if the salt concentration used for protein preparation or for binding reactions exceeds 0.3 M KCl(41) . We found that META(D+) probe also bound inducible nuclear factors obtained by conventional methods of extraction, and this binding was also not competed by NFAT oligonucleotide (data not shown).

The human IL2 upstream region (+45 to -362) did not compete for META(D+), nor did its constituent elements NF-kappaB (Fig. 8), NFAT, or AP1 (data not shown). The only strong competition for META(D+) was by the SV40 AP3 element(33) , which encompasses an NF-kappaB site (42) and a site for NP-TCII, which is expressed constitutively in lymphocyte nuclei(32) . However, META(D+) differs from each of these factors in terms of its T helper lymphocyte specificity, its dependence on activation, and its CsA sensitivity, as well as lack of competition for binding. S194 cells (a B-cell line) and HeLa cells (fibroblast) are known to produce NF-kappaB and AP3 proteins, respectively, but META(D+) did not promote transcriptional activity in these cells, either uninduced or exposed to PMA plus A23187 (Fig. 6). In short, its properties cannot be explained by interaction with known binding factors. The nature of the META(D+) binding factor remains uncertain, but it is reasonable to speculate that an NFATp/c family member is involved(43) , perhaps in conjunction with other components that differentiate it from the NF-AT binding factor of the human IL2 gene.

The mammary cell-specific expression of MMTV in mice is brought about by the net actions of positively acting estrogen-dependent elements and negative elements within the LTR. The induction of T cell lymphomas by MMTV has been attributed to the loss of repressor elements in the control regions of the LTR due to deletions and/or rearrangements. In particular, deletion of the repressor elements between -430 and -360 in the LTR promoted transcription of this viral strain in T cells(44) . It is possible that the concerted action of the META(D+) enhancer with known transcriptional regulatory sites in the LTR contributes to the induction of T cell lymphomas. Enhancers of lymphotropic papova virus, Moloney murine leukemia virus, and T lymphomagenic virus SL3-3 also exhibit a high degree of homology to the AP3 binding site in the SV40 enhancer(45, 46, 47) . These enhancers are involved in the generation of hematopoietic malignancies(47) , making META(D+) a candidate enhancer for the observed MMTV-induced T cell lymphomas.

A tantalizing feature of META is that it regulates the expression, at least in certain T lymphoma cell lines, of the LTR transcript, which normally encodes the minor histocompatibility antigens of the Mls family(4, 5) . META, given its lymphoid-specific induction, makes an attractive candidate for the regulator of Mls antigen expression, except for one thing; so far, we have seen expression only in T lymphocytes which, although implicated as Mls presenting cells in the induction of tolerance(48) , are not conventional antigen-presenting cells. We have not observed META activity in B lymphocytes, but this may be because we have not yet looked at the correct B cells in the proper way.


FOOTNOTES

*
This work was supported by a grant from the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U41642[GenBank].

§
To whom correspondence should be addressed. Tel.: 403-492-3357; Fax: 403-492-0886.

(^1)
The abbreviations used are: MMTV, mouse mammary tumor virus; AP1, AP2, and AP3, activating proteins 1, 2, and 3; IL2, interleukin 2; LTR, long terminal repeat; META, MMTV env gene transcriptional activator; NF-kappaB, nuclear factor kappa B; NFAT, nuclear factor of activated T cells; PMA, phorbol myristate acetate; TCEp, proximal T cell element; tk, thymidine kinase; kb, kilobase pair(s); DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; bp, base pair(s); CsA, cyclosporine; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

-We thank Dr. C. L. Miller for discussions and Cliff Gibbs for technical assistance.


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