By
From the * Lymphocyte Biology Unit, Swiss Institute for Experimental Cancer Research, CH-1066
Epalinges, Switzerland; Section de Biologie, Institut Curie, F-91405 Orsay Cédex, France; § Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066
Epalinges, Switzerland; and
Génétique et Microbiologie, Centre Médical Universitaire, Universite de
Genève, 1211 Genève 4, Switzerland
Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the
IL-2R gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R
gene this control is exerted via two regulatable elements, a promoter proximal region, and an
IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of
the IL-2rE in the rodent thymic lymphoma-derived, CD4
CD8
cell line PC60 demonstrated
that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for
members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like
protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from
normal CD4
CD8
thymocytes. We have identified this molecule as Elf-1 according to a
number of criteria. The complex possesses an identical electrophoretic mobility to that formed
by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE
probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant
and PC60-derived proteins bind with the same relative affinities to different mutants of site III.
In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we
provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of
the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require
direct interactions between the proteins binding these three elements.
IL-2 is a T cell-derived cytokine implicated in the regulation of growth and differentiation of a variety of cells expressing IL-2 receptors. The high affinity IL-2 receptor
(IL-2R)1 consists of three distinct subunits, the IL-2R The rodent thymic lymphoma-derived cell line PC60
is growth factor independent and resembles early thymic T
cell precursors in that it expresses neither CD4 nor CD8 antigens. This line was the first in which induction of IL-2R
Table 1.
IL-2rE Mutants
, -
,
and -
chains, which are assembled into a signaling complex after their interaction with IL-2 (for reviews see references 1). Lymphocytes control their level of IL-2 responsiveness by regulating the expression level of IL-2R.
This is achieved via transcriptional regulation of the IL-2R
gene, and in certain cells the IL-2R
gene; the IL-2R
subunit is constitutively expressed (4). Resting lymphocytes do not express the IL-2R
gene. Transcription of the
gene is induced by signals from the antigen receptor, but in
the absence of any other stimuli, these signals only trigger a
transient wave of IL-2R
synthesis in T lymphocytes. Maximal and sustained IL-2R
transcription depends on IL-2
itself, which thus acts as a positive feedback regulator of
IL-2R expression and IL-2 responsiveness. The level of
IL-2R
gene expression is also modulated by other extracellular stimuli, notably IL-1 and TNF (7). In CD4
CD8
thymocytes, IL-1 has a similar effect as antigen in
mature T cells in that it acts synergistically with IL-2 to increase IL-2R
transcription (6, 10).
expression by IL-2 was reported (11) and is a well-characterized model system in which to analyze the IL-2 responsiveness of this gene (6, 12). As in normal CD4
CD8
thymocytes, IL-1 and IL-2 act synergistically to induce IL-2R
transcription (9). In a manner similar to antigenic stimulation in mature T cells, in PC60 IL-1 induces only a transient wave of IL-2R
expression, and it primes cells to become IL-2 responsive. Transcriptional control of the IL-2R
gene is exerted by two separate regulatory regions in the 5
flanking region, one of which is promoter proximal and required for IL-1 inducibility and the other a more distal IL-2-
responsive enhancer (IL-2rE) (6). Promoter-proximal positive regulatory regions (PRRs in the terminology proposed by John et al. [13]), located between positions
54 to
584 in the mouse, and
244 to
276 (PRRI) and
137
to
64 (PRRII) in the human IL-2R
genes, respectively,
are required for the rapid appearance of IL-2R
mRNA
after IL-1 exposure of PC60 or EL4 (14) cells, or PMA
treatment of human T cell leukemias (12, 13, 15). Several
DNA-binding factors were implicated in exerting effects on
transcription via these elements, including NF-kB, Elf-1,
and HMG-I(Y) (12, 16). Transcriptional stimulation by
IL-2, on the other hand, operates via IL-2rE. The position of this enhancer in the mouse gene was mapped, by transient transfection experiments, to a 48-nucleotide stretch,
1.3 kb upstream of the transcriptional start site (12). It corresponds to a DNase I hypersensitive site (DH2), that appears
in the chromatin of normal mouse T cells upon stimulation
with Con A and IL-2 (10), and can increase transcription in
response to IL-2 stimulation both in its normal context of
2.5 kb of IL-2R
5
flanking region as well as when inserted upstream of a non-orthologous promoter. Recently,
the human homologue of the mouse IL-2rE has been
identified in a region ~4 kb upstream of the transcription
start site of the human IL-2R
gene (17, 18). The mouse
IL-2rE contains three separable cis-acting elements, named
sites I, II, and III; mutations in any one of these three elements abolishes IL-2 responsiveness of reporter constructs
containing the IL-2rE, demonstrating functional cooperativity between the sites (12) (see Fig. 1 A for summary). By
identifying the proteins which bind to these DNA elements
it will be possible to characterize the nature of some of the
downstream transducers of the IL-2 signaling pathway.
Fig. 1.
Identification of a DNA-binding protein that recognizes a critical element in the IL-2rE of the mouse IL-2R gene. A shows a summary of
the published results (12) of the functional analysis of the mouse IL-2rE. The histogram shows the effect of mutations in the IL-2rE on the IL-2 inducibility of constructs containing 2.5 kb of the IL-2R
5
flanking region. Mutations are either substitutions (M) or deletions (
) (see Table 1). The presence of the three separate elements, site I, II, and III, in the enhancer is required for IL-2 responsiveness. The Ets-like consensus site that overlaps with site III
and the STAT consensus binding motifs in sites I and II are underlined. (B). Bandshift competition analysis of the complexes formed between a probe
containing the entire IL-2rE (nucleotides
1384 to
1290) and nuclear extracts isolated from IL-1-primed, IL-2-induced PC60 cells. The competitors
are unlabeled IL-2rE fragments used at a 200-fold molar excess which contain the mutations indicated in A. Competitors containing the mutations defining site III do not compete for the formation of complex 1 which is marked with a filled arrowhead. Empty arrowheads point to complexes 2 and 3, which are competed by all competitors. F, free probe. (C) DNase I footprint analysis of the IL-2rE in vitro. DNase I protection analysis of the IL-2rE
was performed with nuclear extracts isolated from either untreated or IL-1-primed and IL-2-induced PC60 cells as indicated. The reactions were performed either in the presence or absence of unlabeled IL-2rE probe (wt) as detailed. The positions of the three sites (sites I, II and III) delineated in A
were determined by fractionating a sequencing reaction on the same gel (data not shown). (D and E) A site III probe (SIII) is sufficient to allow formation
of the IL-2rE-specific complex corresponding to complex 1 in B from PC60 (D) and CD4
CD8
thymocytes (E). The radiolabeled probe SIII spanning
nucleotides
1336 to
1302 of the IL-2rE forms a specific complex, indicated with a filled arrowhead, with nuclear extracts isolated from IL-1-primed, IL-2-induced PC60 cells. The SIII-specific complex was subjected to competition analysis with the following unlabeled probes: SIII, unlabeled SIII; NS,
a nonspecific oligonucleotide of the same length as SIII; C1, a mutant form of SIII (see Table 1) which contains a mutation in the core of the Ets-like
binding site; M9 to M15, mutant forms of the IL-2rE (see A and Table 1).
[View Larger Versions of these Images (74 + 26 + 96 + 62K GIF file)]
Sites I and II of the IL-2rE contain sequence elements potentially recognized by signal transducers and activators of transcription (STAT) proteins (19), some of which have recently been shown to be activated by IL-2 (20, 21). In PC60, IL-2rE function most likely also depends on the binding of IL-2 activated STAT5 to both sites I and II (Meyer, M., P. Reichenbach, V. Schindler, E. Soldaini, M. Nabholz, manuscript submitted for publication). Site III contains a consensus binding site for members of the Etslike family of transcription factors, distinguished by a conserved DNA binding domain which recognizes sequences containing a GGAA/T core (22). Ets-like proteins have been shown to be involved in the regulation of genes important for several different biological processes including lymphocyte differentiation, T cell activation, growth control, viral infection cycles, and transformation (22). Ets-like proteins often interact with other transcription factors and accessory proteins forming complexes both in the presence and absence of DNA (23).
The aim of this study is to elucidate which, if any, of the
Ets-like proteins bind to site III and form part of the complex of proteins generating the response of the IL-2R
gene to IL-2. Using several experimental approaches, we
demonstrate that site III specifically binds the Elf-1 transcription factor. A direct correlation between the effect of
site III point mutations on Elf-1 binding in vitro and on the
in vivo activity of the corresponding IL-2rE forms, strongly
implies Elf-1 as the transcription factor involved in the IL-2
inducibility of the IL-2R
gene. Experiments designed to
elucidate the molecular basis of the cooperativity observed
between the three sites making up the IL-2rE suggest that
this phenomenon does not rely on the formation of direct interactions between Elf-1 and the factors binding to sites I and II.
Cells and Culture Conditions.
The PC60.21.14 cell line, further referred to as PC60, and its culture conditions have been described previously (9, 28, 29). Human recombinant interleukins
were the kind gifts of Glaxo IMB S.A. (Geneva, Switzerland).
IL-1, further referred to as IL-1, was used at final concentrations
of 1 ng/ml; IL-2 was used at 100 units/ml. Spleenic T cells were
prepared by fractionation on nylon wool columns according to
Current Protocols in Immunology (30). CD4
CD8
thymocytes
were prepared and cultured as described previously (31). The purity of CD4
CD8
thymocytes, determined by flow cytometry,
was 98%.
Plasmids and Preparation of Mutant Constructs.
The reference plasmid for transient transfection experiments, pGAc
G1D, the
wild-type IL-2R
/reporter construct, pwt1
G1, in which the
segment from nt
2539 to +93 of the IL-2R
5
flanking region
is fused to a
-globin reporter gene, and the mutants used for the
experiments described in Fig. 1, B and D have been described previously (12, 32). Mutations within the IL-2rE sequence were
introduced into the pwt1
G1 plasmid using the Chameleon Mutagenesis Kit (Stratagene, Zürich, Switzerland). Plasmids were
prepared using Qiagen Plasmid kits, and the validity of all the
constructs was confirmed by DNA sequencing using a T7 polymerase sequencing kit (Amersham Rahn, Zürich, Switzerland).
Transfection of PC60 Cells and Reporter Gene Assays.
PC60 cells
were cultured in the presence of IL-1 for 3 d and then transiently
transfected with DNA constructs using the DEAE-dextran method (33). Each transfection included a defined ratio between the reference plasmid and a IL-2R/reporter construct. After transfection, cells were split into two equal aliquots. One aliquot was
grown for an additional two days in the presence of IL-1 and
IL-2, while the other was cultured in the presence of IL-1 alone.
The transcripts from the IL-2R
/reporter gene and from the reference plasmid were then measured by RT PCR as described
previously (12, 32). Results from this assay were quantified using
a PhosphorImager and ImageQuant software (Molecular Dynamics,
Sunny Vale, CA). Signals due to the IL-2R
/reporter gene constructs were normalized by comparing them to signals generated
from the reference plasmid. To determine the level of IL-2 induction, transcriptional activity generated from the reporter construct in the presence of both IL-1 and IL-2 was divided by the
activity observed in cells cultured in the presence of IL-1 alone.
Electrophoretic Mobility Shift Assays (Bandshifts).
Nuclear extracts
were prepared essentially as described by Schreiber et al. (34): 1 × 106 cells were rinsed in PBS and lysed in 200 µl of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM DTT, 2% NP-40). The lysate was centrifuged, the
supernatant discarded, and the proteins extracted from the nuclear
pellet in buffer B (high salt buffer: 20 mM Hepes, pH 7.9, 0.4 M
KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT). Both buffers A
and B contained protease (1 µg/ml aprotinin, 1 µg/ml leupeptin,
1 mM PMSF) and phosphatase inhibitors (10 mM NaF, 1 mM
Na3VO3). Binding reactions were performed in a final volume of
20 µl in binding buffer (10 mM Tris, pH 7.5, 100 mM KCl, 10%
glycerol, 1 mM DTT, 1 µg/ml BSA, 1 µg dIdC, 0.5 µg sonicated salmon sperm DNA) containing 1 µg of cell nuclear extract
or 1 µg of recombinant protein, 1 × 106 cpm of an end-labeled
PCR fragment (spanning bases 1384 to
1290 of the 5
flanking region of the IL-2R
gene, Fig. 1 B) or SIII oligonucleotide
probe (see Table 1). Reactions were incubated on ice for 20 min
and then separated on 4.5% non-denaturing polyacrylamide gels
in 0.3× TBE. For competition experiments, unlabeled PCR
fragments or oligonucleotides were premixed with the radiolabeled probe before the addition of the proteins; for antibody supershift experiments the antibodies were preincubated with the
nuclear proteins for the specified time before the addition of the
radiolabeled probe.
DNaseI Footprint Analysis.
The probe for in vitro footprinting
assays was obtained by subcloning the 188-bp SauIIIA-HindII fragment of the IL-2R 5
flanking region into Sma I-BamHI-
digested pUC18. To label the non-coding strand this plasmid was
linearized with EcoRI and the resulting sticky end filled in with a
nucleotide mixture containing [32P]ATP. After digestion with
PstI the labeled IL-2R
fragment was purified on a non-denaturing 8% polyacrylamide gel and recovered by electroelution. For
each footprint reaction 2 ng of probe and, where indicated, a
100-fold excess of competitor was incubated with 35 µg of nuclear extract in 27 µl of buffer (33 mM Hepes, pH 6, 0.1 mM
EDTA, 50 mM KCl, 0.6 mM DTT, 4 mM MgCl2, 0.4 mM 2-ME, 0.1 mg/ml poly-dIdC, containing several protease inhibitors, listed above) for 30 min on ice. The samples were prewarmed for 1 min at 20°C and 0.1 unit of Dnase I (Pharmacia, Duebendorf, Switzerland) in 2 µl 25 mM CaCl2 and 10 mM Hepes, pH 7.6, was
added. After 1 min at 20°C the reaction was stopped and the DNA
extracted, denatured, and fractionated on a 6% sequencing gel.
Recombinant Proteins and Antibodies. Recombinant AcNPV baculoviruses expressing rElf-1 and rEts-1 proteins (35, 36), and anti- Ets-1/2 (no. 8 [37]), anti-Pu-1 (no. 65 [38]) and anti-Fli-1 (no. 61 [39]) antisera have been described previously. Anti-Elf-1 polyclonal antibodies (C-20) were purchased from Santa Cruz Biotechnology (CA), and HRP-coupled goat anti-rabbit antibodies from Biorad Laboratories (CA).
Affinity Precipitation of IL-2rE-bound Proteins.
Biotinylated IL-2rE
probes were obtained as follows: (a) wild type and probes carrying mutations that abolish the activity of a single enhancer site
were made by amplifying the corresponding reporter plasmid
(M4 for site I, M9 for site II, M12 for III, see Table 1 and reference 12 for sequences) with primers spanning the segment between nucleotides 1402 (oligonucleotide A) and
1286 (biotinylated oligonucleotide B) of the IL-2R
5
flanking region. (b)
Probes in which more than one site was inactivated were obtained in three steps essentially as described in Ho et al. (40) and
explained here for the example of a probe in which all three enhancer sites are destroyed. (i) The plasmid carrying the mutation
in site I (M4) was amplified with oligonucleotide A and a 3
oligonucleotide C
, covering site II with the changes destroying this
site. Simultaneously the plasmid with a mutation in site III (M12)
was amplified with a 5
primer (C) complementary to oligonucleotide C
and oligonucleotide B. (ii) The resulting PCR products
were gel-purified, denatured, and annealed with each other. (c) The
annealing mixture was used as template for an extension reaction,
and the resulting full-length IL-2rE fragments amplified with
primers A and B. PCR fragments were purified and quantified by
densitometric analysis of the corresponding bands on agarose gels.
To identify the proteins that
bind to the IL-2rE, and may therefore be implicated in the
regulation of IL-2rE activity, we performed bandshift assays
with a probe containing the entire IL-2R enhancer (nucleotides
1384 to
1290). This probe forms a number of
complexes with proteins extracted from IL-1-primed and
IL-2-induced PC60 nuclei (Fig. 1 B). To determine which
of these complexes is due to specific DNA-binding proteins recognizing functionally important sites within the
IL-2rE, we used a DNA competition-based assay. In this
assay DNA fragments harboring specific mutations defining
the sites required for IL-2rE enhancer activity are used as
competitors to disrupt the formation of the IL-2rE complexes (Fig. 1 B). Three of the IL-2rE complexes were inhibited by the addition of an excess of unlabeled DNA, but
only the complex with the lowest mobility (complex 1) results from an interaction with a protein specifically binding
a discrete region of the IL-2rE. Mutations M12 and M13
strongly impair the capacity of the corresponding DNA
fragments to compete for the formation of this specific
complex. The flanking mutations M11 and M14 result in a
relatively lower, but still significant, reduction in the competitive efficiency of the IL-2rE DNA. M12 and M13
abolish IL-2rE activity and define site III; the striking correlation of this in vivo effect with the effect on competitor
efficiency in vitro suggests that the specific bandshift complex is due to one or several proteins participating in the
control of the IL-2rE activity.
On a probe spanning the entire IL-2rE (nucleotides
1472 to
1268), PC60 extracts produce a specific footprint extending from the 5
end of site III to several bases
downstream, essentially covering the Ets-like protein consensus binding motif (Fig. 1 C, lanes 2 and 3). Detection of
this footprint is abolished by the addition of unlabeled probe
(Fig. 1 C, lanes 1 and 4). The protein complex that gives
rise to the footprint is present in nuclear extracts from both
unstimulated and IL-1-primed and IL-2-stimulated cells
(compare Fig. 1 C, lanes 2 and 3). The same is true for the
protein(s) forming the site III-specific complex detected in
bandshift assays (data not shown). (Note that under the
conditions used here we could not detect PC60 proteins
binding specifically to site I or II. However, using other
techniques we have shown that IL-2-induced STAT5 proteins bind to these sites but not to site III [Meyer, M., P. Reichenbach, V. Schindler, E. Soldaini, M. Nabholz, manuscript
submitted for publication].)
SIII, a shorter IL-2rE probe spanning nucleotides 1336
to
1302, contains all the necessary sequence information
to allow the formation of the site III-specific complex with
extracts from PC60 (Fig. 1 D) or normal CD4
CD8
thymocytes (Fig. 1 E). The single, specific SIII complex demonstrates the same sensitivity to mutant IL-2rE fragments in
competition assays as that of the complete IL-2rE probe-
site III-specific complex (compare Fig. 1, B, D, and E).
Site III contains a consensus binding sequence for members of the Ets-like family of transcription factors. The two mutations, M12 and M13, which define site III and abolish competition for the site III-specific complex, destroy this consensus binding site. Mutation M14, which has no significant effect on enhancer activity and reduces competition in band shifts only to a minor extent (compare Fig. 1, A and B), results in a sequence that can still bind some Ets-like proteins. To determine which, if any, of the numerous Ets family members participates in the formation of the site III-specific bandshift complex several approaches were employed.
First, using recombinant Elf-1, an Ets-like protein, and Ets-1 itself in bandshift assays, we found that Elf-1 forms a complex with the SIII probe that migrates with a mobility identical to that of the complex generated in PC60 extracts. Recombinant Ets-1 binds to the SIII probe but results in the formation of a faster migrating complex (Fig. 2 A). The observation that the complex generated by PC60 nuclear extracts comigrates with that formed by recombinant Elf-1 suggests that there is no requirement for additional proteins.
Second, we tested the capacity of antibodies raised
against the Ets-like proteins Elf-1, Ets-1/2, Fli-1 (Fig. 2, B
and C) and Pu-1 to affect the mobility of the site III-specific bandshift complex. The anti-Elf-1 antibodies supershifted the complex formed by non-induced, as well as
primed and IL-2 induced nuclear extracts from PC60 (Fig.
2, B and C; the appearance of the additional nonspecific higher mobility bands in Fig. 2 B results from incubation at
room temperature. These bands are not present when the
incubations are performed on ice as in Fig. 2 C) or with
CD4CD8
thymocytes (Fig. 2 D). The observation that
the proportions of the complexes supershifted varies between experiments suggests that the anti-Elf-1 antiserum
only partially supershifts the site III specific complex as a
result of the relatively low affinity of this reagent. Anti-Elf1 antibodies also supershift the site III specific complex
formed with the complete IL-2rE probe (data not shown).
Antibodies against Ets-1 and Ets-2, Fli-1 (Fig. 2 C), and
Pu-1 (data not shown) do not affect the mobility of the site
III-specific complex.
Third, the nature of the protein(s) which could be precipitated from PC60-derived nuclear extracts by biotinylated IL-2rE oligonucleotide probes was investigated. IL-2rE
probes containing all three sites precipitate a protein recognized by the anti-Elf-1 antiserum and whose molecular
mass, 91 kD, corresponds to that of Elf-1 (Fig. 3 A). The Elf-1
doublet observed on Western blots has been reported previously (41), and the relative levels of the two protein species remain unchanged after IL-1 priming or IL-2 induction
(data not shown). As expected from the bandshift experiments, the presence in PC60 nuclei of Elf-1 active for
DNA binding does not depend on IL-2 stimulation, although sometimes a small increase is seen in extracts from
IL-2-treated cells (Fig. 3, A and C). Experiments with biotinylated IL-2rE probes containing mutations in sites I, II,
or III clearly show that Elf-1 binding to IL-2rE is abolished
by a mutation in site III, whereas mutations in sites I or II
have no effect on the amount of Elf-1 protein precipitated
(Fig. 3, B and C). The human IL-2rE contains an additional Ets-like protein-binding site overlapping a STAT
consensus binding motif in site I (17, 18). Fig. 3 B demonstrates that the murine site I does not bind Elf-1, in agreement with the inability of site I to compete for the site III-
specific complex (Fig. 1 B). Transfection experiments in
human lymphoid cell lines suggest that Elf-1 binding to site I
may produce a negative effect on the activity of the IL-2rE
enhancer (17). If this effect is important in the regulation of
the human IL-2R gene expression in normal lymphocytes, it suggests the presence of subtle differences in the
transcriptional control of the homologous mouse and human genes.
Finally, we compared the effect of point mutations in the core of the site III Ets-like consensus site both on the ability of recombinant Elf-1 to bind and on the formation of the site III-specific complex by PC60 extracts. Binding site selection studies have identified the consensus sequences recognized by human and mouse Elf-1 as being A(A/t)(C/a) CCGGAAGT(A/S) (42) and A(A/T)(A/c)CCGGAAGT (G/T)(G/A) (3) respectively. Both contain the core GGAA (underlined) characteristic of Ets-like binding sites; site III of the IL-2rE (AATCAGGAAGTTG (in italics nucleotides identical in all three motifs) is very similar to these sequences. There is complete concordance in the behavior of recombinant Elf-1-derived and PC60-derived Elf-1 site III complexes in terms of their sensitivities to the various mutations present within the core sequence (Fig. 4, A and B). Note, in particular, the effect of the change GGAA to GGAT (mutant C5), which is not expected to affect binding of Ets-1 but does, as previously described (35, 42), result in a strong reduction of the binding of recombinant Elf-1 as well as of the site III-binding protein in PC60 extracts.
The Effects of Site III Mutations On Elf-1 Binding In Vitro Correlate with Those on IL-2rR Activity In Vivo.
The data presented above demonstrate that the protein in PC60 extracts
binding to site III of the IL-2rE is Elf-1. Although previous
mutational analyses suggest that Elf-1 is required for IL-2rE
inducibility, they do not rule out the possibility that another protein, not detectable in in vitro binding experiments, is responsible for site III function in vivo. To obtain
further evidence that Elf-1 is indeed the protein upon
which site III function depends in vivo, we compared the
effect of mutations in the core of the Elf-1 binding site on
the in vivo IL-2 responsiveness of the IL-2rE. This was
measured using an accurate PCR-based reporter assay described previously (12, 32). Comparison of the results obtained from these assays (Fig. 5) with those obtained in the bandshift competition experiments (Fig. 4, A and B) shows
good agreement between these two parameters. Mutations
C1 and C3 very strongly affect Elf-1 binding in vitro and
reduce the IL-2 responsiveness of the IL-2rE about fivefold.
Mutations C2 and C4 result in a slightly weaker effect on
both Elf-1 binding and IL-2rE activity, and mutation C5,
which competes for Elf-1 binding more strongly than the
other core mutants, reduces enhancer inducibility twofold. As mentioned above this mutation reduces the binding of
Elf-1 but should have little effect on the binding of Ets-1 or
Ets-2. This change therefore results in a core element still
recognized by Elf-1 but at a much reduced efficiency. Two
additional mutations, S1 and S2, which alter the nucleotides flanking the Ets-like core sequence, almost completely abolish IL-2rE activity. Note that S2 overlaps with
M14 which itself has little effect on Elf-1 binding and enhancer inducibility. Together these data lend strong support to the hypothesis that Elf-1 participates in the IL-2 response of the mouse IL-2rE. In the recently identified human IL2rE the individual sites required for enhancer activity, including site III, are conserved (17, 18), and John et al. (18)
has provided evidence suggesting that the human gene is
also regulated by Elf-1.
There is evidence implicating Elf-1 in the regulation of a
number of lymphocyte specific genes (IL-2 [43], GM-CSF
[24], CD4 [41], IL-3 [23], IgH3 [44] and terminal transferase [45]). The case of the IL-2R
gene is interesting as
this gene contains two (or, in man, three) Elf-1 sites, forming part of both the promoter proximal and distal control
elements. Elf-1 DNA-binding activity in PC60 cells is not
affected by IL-2 (Fig. 3 A). Thus, the simplest model for
the molecular basis of the IL-2rE's IL-2 inducibility is that
enhancer activity depends on the binding of other IL-2-activated proteins (most likely STAT5 [Meyer, W., P. Reichenbach, V. Schindler, E. Soldaini, M. Nabholz, manuscript submitted for publication]) to sites I and II and that Elf-1
remains constitutively present on site III. Constitutive binding of Elf-1 to regulatory elements implicated in stimulation of transcription has been described previously in studies
of the PHA and PMA inducible IL-3 gene (23). Experiments to detect possible posttranslational modifications of
Elf-1 controlling its capacity to contribute to enhancer activity have so far been unsuccessful. Elf-1 activity may also
depend on interactions with other inducible factors binding
in its immediate vicinity. In this context it is interesting
that mutations M11 and
15 flanking the Elf-1 binding site
result in a moderate reduction of IL-2rE activity; these regions contain potential binding sites for NFIL-6 and AP-1.
Note, however, that the site III specific complex formed
by PC60 extracts has the same electrophoretic mobility as
that formed with recombinant Elf-1, indicating that the
PC60 complex does not contain additional proteins.
One of the striking features resulting from the study of the mouse IL-2rE is that mutations in any one of the three sites that make up this enhancer abrogate IL-2 responsiveness. The observation that the distances between the three IL-2rE sites (~20 nucleotides or two helical turns from center to center) are conserved between mouse and man suggests that evolutionary conservation of the relative orientations of proteins binding to them is important, possibly because of a requirement for direct interactions between the factors binding to the three sites. However, the insertion of five nucleotides in mutant I5, which changes the relative orientation of site III with respect to sites I and II, results in only a minor effect on IL-2 inducibility, arguing against direct protein- protein interactions (Fig. 5). Using biotinylated IL-2rE oligonucleotide probes, we demonstrated that the presence or absence of sites I and II has no affect on the amount of Elf-1 protein precipitated from PC60-derived nuclear extracts (Fig. 3 C; probing the same membranes with anti-STAT5 antiserum reveals that STAT5 binds to site I and II [Reichenbach, P., manuscript in preparation]) again arguing against direct protein-protein interactions. However, this possibility needs to be explored further using additional approaches.
The simplest model consistent with our findings is that the different IL-2rE binding proteins cooperate by interacting independently with components of the transcription initiation complex, as has been described previously in in vitro studies of transcriptional regulation by the Drosophila proteins Bicoid and Hunchback (46) and in the control of the c-fos gene (47). But our data do not rule out more complex models involving additional proteins or cofactors needed for an efficient stimulation of transcription by the IL-2rE.
Address correspondence to Markus Nabholz, Lymphocyte Biology Unit, Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland. Dr. Pla's current address is Department Biologia Cellular, Universitat de Girona, Pl. Hospital 6, 171071 Girona, Spain. Dr. Serdobova's current address is Départment de Biologie Moléculaire, Universite de Genève, 1211 Genève 4, Switzerland.
Received for publication 15 October 1996 and in revised form 31 January 1997.
This work was supported, in part, by grants from the Swiss National Science Foundation and the Swiss Cancer League, to M.N, as well as by grants from the Swiss Federal Office of Education and Science awarded in conjunction with projects approved by the Biomed and Human Capital and Mobility programs of the European Union.We thank Marcel Allegrini and Pierre Dubied for preparation of the figures, Antonietta Giugno for excellent technical assistance and Claudine Ravussin for help with the preparation of the manuscript. We are grateful to Wolfram Meyer for letting us mention his unpublished data.
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