(Received for publication, October 19, 1995; and in revised form, January 22, 1996)
From the
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-B 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.
Mouse mammary tumor virus (MMTV) ()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-B, and NFAT(13, 17, 18) .
The NF-
B 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-
B 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+).
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, 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
TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA).
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 DH5, and plated on LB
agar-ampicillin indicator plates containing 5-bromo-4-chloro-3-indoyl
-D-galactoside and
isopropyl-1-thio-
-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.
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, 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
, and 1 mM CaCl
) 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
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.
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.
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.
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-B 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-B 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-
B, GGGA(A/C)TN(T/C)CC. Mismatches of
the SV40, NF-
B, 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-B 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-
B (S09
and A10, Table 1). Lanes 1 and 2, no
competitor; lanes 3-6, AP3/SV40 competitor; lanes
7-11, hIL2 NF-
B 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-B 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 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).
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-B and AP3 factors under constitutive
and/or inducing conditions, yet did not significantly activate
META(D+).
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-B probe competed moderately well
for the lower mobility SV40 activation-dependent complex, but not for
the other one.
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-B (-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-
B 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
-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-B, 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-B (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-
B 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-
B 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U41642[GenBank].