From the
We have shown that interleukin-1 (IL-1) and IL-2 control IL-2
receptor
Transient transfection experiments show that several
elements in the promoter-proximal region of the IL-2R
Interleukin-2 (IL-2)
In mature B and T lymphocytes, IL-2R
Regulation of IL-2R
In the human IL-2R
Work in our laboratory has
focused on the identification of regulatory elements in the mouse
IL-2R
Here
we show that the response of the IL-2R
Human recombinant interleukins
were gifts of GLAXO IMB S.A. (Plan-les-Ouates, Geneva, Switzerland).
IL-1
Dithiocarbamate (Sigma, Buchs, Switzerland)
compounds were used at final concentrations of 10 and 100
µM.
RNase protection reactions
were fractionated on sequencing gels. Signals were quantified with a
PhosphorImager, model SF (Molecular Dynamics, Sunnyvale CA 94086).
IL-2R
Human and mouse IL-2R
To introduce fragments C3 or C2+C3 into pG1, these
segments were amplified from pwt with the proofreading Pfu polymerase (Stratagene, La Jolla, CA) according to the
manufacturer's instructions. The primers used contained an
XbaI site at the 5` and a HindIII site at the 3` end.
The PCR products were digested with these enzymes and ligated with
HindIII- XbaI-digested pG1. The resulting plasmids
contain single copies of segment -1635 to -1301
(pG1.C2+C3) or segment -1403 to -1301 (pG1.C3) of the
IL-2R
For the mapping of
interleukin response elements in the IL-2R
We have mapped the elements in segment C
required for IL-2 responsiveness in three stages (Fig. 5). In the
first step, we introduced a series of contiguous deletions of
160-250 bp into segment C. Comparing the IL-2 responsiveness of
these plasmids in IL-1-primed PC60 cells (Fig. 5 A) we
found that deletion of the segment between bp -1389 and
-1228 (C3) completely abolished IL-2 responsiveness. The plasmid
with this deletion (pR
To delimit more precisely the sequences
within segment C3 that were required for IL-2 responsiveness, we
introduced six contiguous deletions within this segment into plasmid
pwt that contains the entire IL-2R
To test whether the segment
defined by deletions
Finally, we mapped the elements required for IL-2
responsiveness of the IL-2R
Fig. 6B compares the
constitutive expression and IL-1 responsiveness of plasmids missing
various segments of the region between position -585 and
-54. Note that the ratio between reference plasmid and IL-2R
PC60 Is a Model for Normal
CD4
Stimulation of PC60 cells with IL-1 + IL-2
induces a biphasic increase in IL-2R
Previous work has shown that the interleukin induced
rise in IL-2R
In spite of the strong homology between the promoter
proximal regions of the mouse and the human IL-2R
Using IL-1 primed cells, we have shown that
the elements in segment C that are strictly required for IL-2
responsiveness of the IL-2R
Stimulation of IL-2R
IL-2 has the
characteristics of a progression factor, in that it stimulates the
proliferation of ``competent'', IL-2-responsive lymphocytes
(55, 56, 57) . In PC60 cells, the effect on
IL-2R
The nucleotide
sequence reported in this paper has been entered into the
GenBank
We thank GLAXO for the generous gifts of interleukin 1
and 2. We thank Tasuku Honjo for the IL-2R
Note Added
in Proof Recently it has been shown
(66) that an
Elf-1 binding site around bp 92 in the human IL-2R
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(IL-2R
) gene transcription in
CD4
CD8
murine T lymphocyte
precursors. Here we map the cis-acting elements that mediate
interleukin responsiveness of the mouse IL-2R
gene using a thymic
lymphoma-derived hybridoma (PC60). The transcriptional response of the
IL-2R
gene to stimulation by IL-1 + IL-2 is biphasic. IL-1
induces a rapid, protein synthesis-independent appearance of IL-2R
mRNA that is blocked by inhibitors of NF-
B activation. It also
primes cells to become IL-2 responsive and thereby prepares the second
phase, in which IL-2 induces a 100-fold further increase in IL-2R
transcripts.
gene
contribute to IL-1 responsiveness, most importantly an NF-
B site
conserved in the human and mouse gene. IL-2 responsiveness, on the
other hand, depends on a 78-nucleotide segment 1.3 kilobases upstream
of the major transcription start site. This segment functions as an
IL-2-inducible enhancer and lies within a region that becomes DNase I
hypersensitive in normal T cells in which IL-2R
expression has
been induced. IL-2 responsiveness requires three distinct elements
within the enhancer. Two of these are potential binding sites for STAT
proteins.
(
)
is a growth factor
for antigen-activated T lymphocytes. IL-2 drives T cell proliferation
by binding to a high affinity receptor that consists of three different
transmembrane proteins, IL-2 receptors (IL-2R)
,
, and
(1) . IL-2R
expression is tightly regulated by
extracellular stimuli (for review, see Ref. 2). It thereby controls, in
part, IL-2 responsiveness.
synthesis is triggered by signals from the antigen receptor. In
antigen-activated T cells, IL-2 itself is required for prolonged
IL-2R
expression
(3, 4, 5, 6, 7, 8, 9) .
In early CD4
CD8
thymic T cell
precursors that have been activated with phorbol 12-myristate
13-acetate and a calcium ionophore, IL-1 synergizes with IL-2 to
increase IL-2R
mRNA levels and surface expression
(10) .
expression occurs mostly through changes in
the rate of IL-2R
gene transcription. This has been shown by
run-on transcription experiments for stimulation by phytohemagglutinin,
IL-2 and IL-1
(8, 9, 11, 12) , and
confirmed by an analysis of mice carrying a mouse IL-2R
5`-flanking region/chloramphenicol acetyltransferase transgene
(65) .(
)
gene,
cis-acting regulatory elements have been identified by
transient transfection of leukemia cell lines in which, however, the
regulation of the resident gene had not been well characterized
(13, 14, 15, 16, 17, 18, 19, 20) .
Regulatory elements conferring responsiveness to a number of stimuli
including phorbol 12-myristate 13-acetate and IL-1 were found in the
promoter proximal segment of the 5`-flanking region (5`-FR) of the gene
(3` of position -390, see below). The elements required for IL-2
responsiveness have not yet been mapped.
gene. We have shown
(65) that in transgenic mice the
segment between bp -2539 and +93 of the mouse IL-2R
gene confers on a reporter gene correct cell type specificity and
inducible expression in T cells and their early thymic precursors. In
the experiments described in this paper, we have used a rodent thymic
lymphoma-derived cell line, PC60
(21, 22) to define the
regions in the 5`-FR that are required for IL-1- and IL-2-inducible
expression. PC60 cells express neither CD4 nor CD8 surface markers
(22) , and, like normal CD4
CD8
thymocytes
(10) , they respond to IL-1 and IL-2 by a
strong increase in IL-2R
gene transcription. Previous work had
shown that in this cell line the 2.5-kb 5`-FR of the mouse IL-2R
gene conferred a response pattern on a reporter gene that was very
similar to that of the genomic IL-2R
genes
(11) .
Interleukin responsiveness clearly depended on sequences upstream of
the promoter proximal region analyzed in the human gene. Experiments
using other cell lines, including an antigen specific IL-2-dependent T
cell clone confirmed that the upstream segment was crucial for correct
expression of mouse IL-2R
gene recombinants.
gene to IL-1 + IL-2 is
biphasic, and that this reflects a bipartite distribution of the
interleukin response elements. IL-1 responsiveness is mediated by
several elements in the promoter proximal region, which includes an
NF-
B site conserved in both human and mouse genes, whereas IL-2
responsiveness depends on three separable 8-12-nucleotide
elements within 48 nucleotides of an IL-2-responsive enhancer 1.3 kb
upstream of the transcription start site. This segment overlaps with a
DNase I hypersensitive site detected only in normal T cells in which
IL-2R
expression has been induced
(65) .
Cell Lines and Culture Conditions: Cytokines and
Inhibitors
The PC60.21.14 cell line (here referred to as PC60)
has been described in previous publications
(11, 21, 22, 23) . PC60(G3) are stable
transfectants that constitutively express a cDNA clone encoding the
human IL-2R chain
(24) . PC60 has inherited from its
lymphoma parent the CD4
CD8
phenotype
(22) and the capacity to grow independently of
IL-2. The culture conditions used, as well as the stimulation with IL-1
and IL-2, have been described previously
(11, 23, 25) .
(IL-1) was used at a final concentration of 1 ng/ml; IL-2 was
used at 100 units/ml.
RNA and Nuclear Extract Preparations: RNase Protection
and Band Shift Assays
RNA was extracted according to the method
of Gough
(26) . Nuclear extracts were prepared by a modification
of the same method
(27) . Methods used for RNase protection and
band shift assays are modifications of techniques developed by others
and have been described in previous publications
(23, 28) . The probes used have been described in detail
elsewhere
(10) . The mouse IL-2R probe corresponds to a
PstI cDNA segment (bp 89-503). Experiments with rat T
cell blast RNA showed that this probe also protects defined fragments
of rat IL-2R
mRNA (data not shown).
signals were normalized to the
-actin signal, which was
reduced to comparable levels by labeling the
-actin probe with
[
-
P]CTP of 10-fold lower specific activity.
Sequence Analysis
Parts of the sequence of the
5`-FR of the mouse IL-2R gene have been published previously
(29) . The sequence from bp -2539 to +93 has been
determined on both strands by the dideoxy method and entered into the
Genebank/EMBL data bases as modification of the original sequence (EMBL
accession M16398, version 10).
5`-flanking sequences were aligned with the program BestFit from the
GCG sequence analysis software package
(30) using the default
scoring system (match = 1.0, mismatch = -0.9, gap
weight = 5.0 + 0.3
gap length). TATA box
(31) and GC box
(31) motifs were identified using
program EukProm from PC/Gene (release 6.7 (1992), IntelliGenetics Inc.,
Mountain View, CA). Consensus recognition sequences for transcription
factors were located with the GCG program FindPattern
(30) .
Searches for local sequence similarities were performed using the BLAST
server at the National Institute for Biotechnology Information in
Bethesda, MD
(32) . Optimal local alignments between selected
sequence regions were generated with the aid of GCG program BestFit
(30) using the default scoring system (match = 1.0,
mismatch = -0.9, gap weight = 5.0 + 0.3
gap length). Statistical significance of optimal alignment
scores was assessed by calculating Z scores based on 100 shuffled
sequence pairs. Type and orientation of repetitive elements in the
mouse IL-2R
5`-FR were identified by computing optimal alignment
scores against a representative set of mammalian LINE and SINE
sequences, including the sequences with the following EMBL accession
numbers: X52235 (version 10), M30839 (version 1), M30839 (version 4),
J00628 (version 1, position 93-222), M87890 (version 2), K02592
(version 2, position 250-660), J01879 (version 1, position
441-522), X02215 (version 3, position 587-849). The number
and approximate location of elements sharing significant sequence
similarities with one of the promoter regions was determined by matrix
comparisons using GCG programs Compare and DotPlot
(30) . The
exact delineation of the repetitive regions as shown (see Fig. 4)
is based on optimal local sequence alignments between the regions
highlighted by the DotPlot method and the corresponding repetitive
element sequence. Internal direct repeats were searched for by the
DotPlot method and by generating multiple locally optimal sequence
alignments with the aid of program LAlign from the FastA package
(33) . The scoring system for multiple local sequence alignment
was as follows: match = 4, mismatch = -3, gap
weight = 8 + 4
gap length. Statistical significance
of direct repeats was assessed by Z score calculations using program
RSS from the same package.
Figure 4:
The IL-2R 5`-FR controls
transcriptional responses to IL-1 and IL-2. A,mouse
IL-2R
5`-FR and IL-2R
/rabbit
-globin reporter gene
recombinants. Note that nucleotide numbering is changed with respect to
previous publications (11, 29) to start at the major cap site (+1)
of IL-2R
transcripts in T cell lines (61) as well as normal mature
T cells and thymocytes. In the top representation are also indicated
the positions of repetitive elements. Plasmid pwt contains the
-2539/+93 ( SphI ( S) to PstI
( P)) segment of the IL-2R
gene. The segment is subdivided
into regions A, B, C and D, separated by BglII sites
( B). Plasmid pR is identical to pwt except for restriction
sites that have been inserted into the BglII sites
( H, HindIII; X, XbaI; see
``Materials and Methods''). In pR
C, the entire C segment
is deleted. B, interleukin response pattern of
IL-2R
/rabbit
-globin reporter gene recombinants. PC60 cells
were cultured in normal medium or in the presence of IL-1 for 3 days
prior to transfection. Nonstimulated or IL-1-primed cells were
transfected with a mixture of the indicated IL-2R
construct, and a
reference plasmid carrying a modified version of the same reporter gene
joined to the constitutive rabbit
-actin promoter
(pG
Ac
GlD or ref). Transfected cells were split into aliquots
that were cultured in the presence or the absence of the indicated
interleukins for 48 h. Reporter gene transcripts were revealed by PCR
amplification of
-globin cDNA according to Ref. 34. The ratio of
IL-2R
( i) to reference signal ( r), determined
with a PhosphorImager, is a measure of the promoter activity of the
corresponding IL-2R
construct. By comparing this normalized
promoter activity in unstimulated and interleukin-stimulated cells, one
can quantify the interleukin response of the IL-2R
recombinant.
The values shown represent means of three independent experiments with
ranges below each column. C and D, kinetics
of the IL-2 response of IL-2R
5`-FR/
-globin reporter gene
recombinants. Primed or unstimulated PC60 cells were transfected with
0.4 pmol of the reference plasmid and 2 pmol of IL-2R
5`-FR/
-globin recombinants pwt or pR
C3 (see Fig.
5 A). Aliquots of the transfected cells were cultured either in
normal medium or in the presence of IL-1 for 48 h. IL-2 was added to
individual cultures to be present for the indicated period prior to
harvest and RNA extraction and assay of reporter gene expression. Note
that the IL-2R
mRNA levels were measured in the same samples (see
Fig. 3). The gel shown ( D) represents the results obtained
with primed cells.
Plasmids
Plasmids were prepared according to Ref.
23. Unless mentioned otherwise, plasmid constructions were made in
pGEM3Zf(+) (Promega). The reference plasmid pGAc
GlD
(hereafter referred to as ref) has been described previously
(34) . In plasmid pwt1
Gl (hereafter referred to as pwt, see
Fig. 4A) the SphI- PstI fragment from
pUC18 mIL-2Rp
(29) was joined to the 1664-bp PvuII
rabbit
-globin gene fragment from Z-pCRI/pRChr
G-1
(35) . To facilitate the introduction of deletions in segment B
and C, we constructed plasmid pBC in which B and C, separated by a
PstI site, are cloned between the XbaI and
HindIII sites of pGEM3Zf(+). In plasmids p
BC1
Gl
(p
BC) and p
BC2
Gl, (p
BC2) segments B and C are
replaced by a double-stranded oligonucleotide
(5`-gatcAAGCTTaagTCTA 3`, 3` TTCGAAttcAGATCTag 5`) containing two unique restriction
sites, HindIII (boldface) and XbaI (underlined), in
either one or the other orientation. pR1
Gl (pR) and pRi
Gl
(pRi) were obtained by introducing the XbaI- HindIII
fragment of pBC into p
BC or p
BC2. All constructs were checked
by DNA sequencing.
Mutations in Segment C
Deletions
C1-
C5 (see Fig. 5 A) were introduced into
pBC using existing restriction sites. The modified
XbaI- HindIII fragments were used to replace the
XbaI- HindIII fragment in pR. Deletions and
substitution mutations within segment C3 (see Fig. 5, B and D) were introduced into pBC with the in vitro mutagenesis system described in Ref. 36. The modified
NsiI fragments were used to replace the corresponding fragment
of pwt.
Figure 5:
Mapping of IL-2 responsive elements in
segment C. A, the sequences required for IL-2
responsiveness lie between bp -1389 and -1228. The
indicated IL-2R/
-globin recombinants were transfected into
IL-1 primed PC60 cells together with reference plasmid at a ratio of
5:1. The transfected cells were cultured for 48 h in IL-1 with or
without IL-2. Promoter activity of the IL-2R
constructs is
determined by normalizing the corresponding mRNA signal to that from
the cotransfected reference plasmid. The IL-2 response is the ratio
between promoter activity after culture in IL-1 + IL-2 and the
activity after culture in IL-1 alone. Indicated is the mean of two
independent experiments, with ranges given below each
bar. Base-line promoter activity (in IL-1 only) of the
different IL-2R
constructs, normalized to that of pwt (=1),
varied within the following ranges: p
BC, 1.4-1.5; pR,
0.6-0.8; pRi, 0.3-0.5; pR
C1, 0.2-0.3; pR
C2,
0.3-0.5; pR
C3, 1.6-1.7; pR
C4, 0.4-0.5;
pR
C5, 1.0-1.5. B, IL-2 responsiveness is
controlled by 75 bp in segment C3. IL-2 responses of the different
plasmids were determined as in A. Base-line expressions,
normalized to that of pwt, ranged as follows: pR
C3, 1.5-2.0;
pR
C3.1, 1.1-1.8; pR
C3.2, 0.6-1.0; pR
C3.3,
0.9-1.6; pR
C3.4, 0.9-1.3; pR
C3.5, 1.0;
pR
C3.6, 1.9-3.0. C, segment C3 acts as an IL-2
responsive enhancer. Single copies of different portions of the
IL-2R
5`-flanking region were inserted upstream of the minimal
-globin promoter of pG1. IL-2 responses of the different plasmids
were determined as in A. The results shown are means of two
experiments. The responses varied by less than 20%. Mean base-line
expressions, normalized to that of pG
Ac
GlD (reference
plasmid), were 0.06 ( pG1), 0.06 ( pG1. BC),
0.15 ( pG1. C2+C3) and 0.12
( pG1.C3). D, three separate elements in the enhancer
are required for IL-2 responsiveness. Below the sequence of
segments C3.1, C3.2 and C3.3 are indicated the 14 mutations that were
introduced into pwt ( dash, deletion; lowercaseletter, substitution) and the IL-2 responses of the
corresponding plasmids. The mean of two experiments is given, and the
result of a single experiment is shown below. IL-2 inducibility varied
by less than 20% between experiments. I, II, and
III indicate the separate elements mutations in which abolish
IL-2 inducibility.
Deletions in Segment D
To construct deletions in
region D of pwt, this segment was subcloned as a BglII
(-802) to PstI (+93) fragment. Segments
D1-D6 (see Fig. 6 B) were removed using suitable
restriction sites. The modified D segments were used to replace the
XbaI- BsmI fragment of plasmid pR, to give rise to
plasmids pRD1-pR
D6. From each of these plasmids,
segment C was removed by digestion with PstI and XbaI
to give rise to pR
C
D1, etc. (see Fig. 6 B).
Figure 6:
Mapping of IL-1 responsive elements in
segment D. A, sequence comparison of the promoter-proximal
segments of human and mouse IL-2R genes. The sequences upstream of
positions -339 in the human (EMBL accession number M15864,
version 2) and -335 in the mouse gene do not share significant
similarities according to shuffling tests (see ``Materials and
Methods''). Cap sites are indicated by arrows. Apart from
minor differences transcription start sites in normal mouse lymphocyte
(S. M. Wang and M. Nabholz, unpublished results) correspond to the
sites determined by Froussard et al. (61). TATA boxes and
consensus binding sites for transcription factors were identified by
computer analysis. TATA boxes were searched for on the + strand
only. TATA and GC boxes conform to the weight matrix descriptions by
Bucher (31). The score (-8.80) of the TATA box at position
-30 in the human gene falls slightly below the recommended
cut-off value of -8.16. A SRF binding site is defined as a
perfect match with the sequence CCWWWWWWGG (62). An NF-
B site is
defined as occurrence of the consensus sequence GGGRNNNYYCC (63) with
one mismatch allowed in the YYC segment. An AP-1 site is defined as the
consensus sequence TGASTCA (62), with one mismatch allowed in any
position. Studies on the human gene have provided evidence for a
regulatory role of negative regulatory elements (NRE)-1 (64) and -2
(17) and for NF IL-2RA or UE-1 (19, 44), as well as for the SRF (19),
Sp1 (GC-box) (43, 44), and NF-
B (15, 16, 18) binding sites
upstream of bp -230. B, delineation of IL-1
response elements. Nonstimulated PC60 cells were transfected with a
mixture of the reference plasmid, and the indicated IL-2R
5`-FR/
-globin recombinants, which lack region C as well as a
portion of region D.
B1 and
B2 indicate the positions of the
two NF-
B sites shown in Fig. 6 A. After transfection cells
were cultured for 48 h with or without IL-1. IL-1 responses are plotted
as fold stimulation of expression over base-line expression. For each
plasmid, the mean and range of values obtained in three independent
transfection experiments are indicated. C, amplified mRNA
signals from one transfection experiment. The ratios between IL-2R
constructs and reference plasmid were 10:1 (pR
C, pR
C
D2,
pR
C
D4); 2.5:1 (pR
C
D1); 25:1 (pR
C
D3,
pR
C
D6); and 100:1 (pR
C
D5). i, mRNA signals
from IL-2R
5`-flanking region/
-globin reporter gene
recombinants; r, mRNA signals from reference
plasmid.
pG1 Recombinants
pG1.BC was obtained by subcloning
the XbaI- HindIII fragment of pBC into pG1
(37) . The resulting plasmid contains a single copy of segment
B+C of the IL-2R 5`-flanking region in the reverse
orientation.
5`-flanking region in their normal orientation.
Reporter Gene Assay
Cells were transiently
transfected using DEAE-dextran according to Ref. 38. Reporter gene
expression was measured as described previously
(34) . Briefly,
cells were transfected with a defined ratio of an IL-2R/rabbit
-globin gene construct and the reference plasmid. This contains a
rabbit
-globin gene with a 40-bp deletion in the second exon and
is transcribed under the control of the constitutive chicken
-actin promoter
(39) . Cytoplasmic RNA of the transfected
cells was treated with DNase I. After cDNA synthesis from a poly(dT)
primer with avian myoblastosis virus reverse transcriptase,
-globin cDNA was amplified in the presence of
[
-
P]dATP with primers spanning the deletion
in the reference plasmid and the first intron. In some experiments we
used, instead of the previously described 3` primer
(34) , the
oligonucleotide 5` ACGTTGCCCAGGAG
GAAGT 3`, which spans the second
intron of the
-globin gene (
marks exon boundaries). As
expected, this reduces signals due to residual plasmid contaminating
mRNA preparations. PCR products were denatured and separated on a
sequencing gel. mRNA derived signals were quantified by PhosphorImager.
Induction of IL-2R
PC60 is a somatic cell hybrid between the mouse
cytolytic T lymphocyte line B6.1 and the rat thymic lymphoma C58
(21) . It has inherited from its lymphoma parent the
CD4 Expression in PC60 Occurs in
Two Phases
CD8
phenotype
(22) and
the capacity to grow independently of IL-2. In unstimulated PC60 cells,
IL-2R
mRNA is barely detectable. IL-1 and IL-2 induce appearance
of IL-2R
mRNA and synergize to control its level
(11) . The
interleukin response pattern of the rat IL-2R
gene in the parental
C58 lymphoma and in PC60 is identical to that of the mouse gene in PC60
(data not shown). This excludes the possibility that the interleukin
response of the mouse IL-2R
gene is an artifact due to a
rearrangement of the mouse gene in the parental mouse T cell line or in
PC60 itself. A very similar response is observed in normal
CD4
CD8
thymocytes
(10) .
Previously reported run-on experiments showed that IL-1 and IL-2
regulate IL-2R
gene expression in PC60 cells by changing its rate
of transcription, and we have shown
(65) that the same is true
for normal thymocytes. Detailed analysis by RNase protection
experiments (Fig. 1) reveals that the interleukin-induced
increase in IL-2R
mRNA levels in PC60 cells is biphasic.
Stimulation with IL-1 + IL-2 induces detectable transcripts within
1 h. The rate of increase in transcript levels slows down between 4 and
5 h after interleukin addition, but accelerates again between 10 and 18
h. This biphasic increase in IL-2R
mRNA has been observed in three
other experiments. Maximal transcript levels, more than 1000-fold above
background, are reached by about 48 h (Ref. 11 and data not shown).
Figure 1:
Kinetics of IL-1 + IL-2-induced
IL-2R mRNA increase in PC60. A, PC60 cells were cultured
for the indicated times in saturating concentrations of IL-1 and IL-2.
Total cytoplasmic RNA was assayed by RNase protection with a mixture of
antisense RNA probes for mouse IL-2R
and
-actin ( emptyarrowheads on left side of gel, see
``Material and Methods''). Note that the specific activity of
the
-actin probe is 10 times lower than that of the IL-2R
probe. B, mouse IL-2R
signals were quantified by
PhosphorImager and normalized to the mouse
-actin signal in the
same lane.
The Initial, IL-1-induced Phase of IL-2R
We
previously showed that the early response of the IL-2R Induction
Can Be Blocked by Inhibitors of NF-
B Activation
gene can be
induced by IL-1 alone and is protein synthesis-independent
(11) . IL-2 has no or very little effect during this stage. The
immediate responses of many genes to IL-1 are due to activation of
NF-
B
(40) . In PC60 cells, IL-1 induces appearance of
NF-
B in the nucleus and activates transcription from promoters
with NF-
B binding sites
(23, 41) . We therefore
tested whether inhibitors of NF-
B activation
(42) block
the early response of the IL-2R
gene to IL-1. Fig. 2shows
that the addition of 100 µM pyrrolidinedithiocarbamate
(PDTC) prior to and during a 3-h stimulation with IL-1 inhibits
appearance of NF-
B in the nucleus and completely blocks the rise
in IL-2R
mRNA. 10 µM PDTC has no inhibitory effect.
Longer incubation at inhibitory doses results in significant loss of
viability. Diethyl-DTC has the same effects (results not shown). The
similar sensitivity of NF-
B activation and IL-2R
mRNA
appearance to PDTC suggests that the two phenomena are linked and that
the early phase of IL-2R
gene induction by IL-1 is mediated by
NF-
B.
Figure 2:
Effect of an inhibitor of NF-B
activation on the early response of the IL-2R
gene to IL-1. PC60
cells were cultured with or without IL-1, in the presence of the
indicated concentrations of PDTC. The inhibitor was added 30 min prior
to IL-1. Three h after IL-1 addition, cytoplasmic RNA and nuclear
extracts were prepared. A, IL-2R
and
-actin mRNA
levels were determined by RNase protection. Emptyarrowheads on the left, undigested probes;
filledarrowheads on the right, mRNA
signals. The signals just below the band corresponding to the
IL-2R
probe are due to a interferon-
probe also present in
the reaction. B, NF-
B was detected by band shift assay
with a probe containing the TC-II element of the SV40 enhancer. The
identity of the protein giving rise to the complex labeled as NF-
B
has been verified previously (41). F, free
probe.
IL-1 Primes PC60 Cells to Become IL-2
Responsive
Stimulation of PC60 with IL-1 alone induces, beyond
the first protein synthesis-independent phase of the response, a
significant further increase in IL-2R expression
(11) .
This is due to the fact that IL-1 induces PC60 cells to secrete IL-2,
so that treatment with IL-1 alone amounts to stimulation with both
interleukins. IL-2 secretion peaks around 24 h, and after 72 h IL-2 is
no longer detectable in culture supernatants. This explains why
IL-2R
mRNA levels remain maximal in cells stimulated for 72 h with
both IL-1 and exogenously supplied IL-2
(11) , while they have
returned to very low levels in cells stimulated with IL-1 only.
However, the addition of IL-2 to such IL-1-prestimulated cells induces
a rapid rise in IL-2R
mRNA levels, whereas in cells that have not
been exposed to IL-1, IL-2 induces a barely detectable increase
(Fig. 3). The initial kinetics of the response of cells
precultured in IL-1 do not depend on the continued presence of IL-1
(data not shown). Thus, preculture in IL-1 increases the capacity of
PC60 cells to respond to IL-2. We refer to this effect as IL-1 priming.
Priming of PC60 cells presumably depends on a low level of IL-2R
expression that persists after 72 h in IL-1, necessary for IL-2
responsiveness. This is consistent with the finding that the IL-2
response of primed PC60(G3) cells, which constitutively express a human
IL-2R
cDNA clone
(24) , is much stronger than that of PC60
itself. But the fact that priming can be observed also in PC60(G3)
indicates that it requires other IL-1-induced changes. PC60 cells
express the IL-2R
gene constitutively but prolonged exposure to
IL-1 induces a 10-30-fold increase in IL-2R
mRNA and a
10-fold increase in high affinity IL-2 receptors (data not shown). This
suggests that priming also depends on increased IL-2R
chain
expression. There may yet be other factors that control responsiveness.
This might explain the considerable variation in the maximal inducible
level of IL-2R
mRNA in primed PC60 cells, which is 3-10
times weaker than that in unprimed cells stimulated with IL-1 +
IL-2 in the same experiment (data not shown).
Figure 3:
Priming with IL-1 increases the
responsiveness of the IL-2R gene to stimulation with IL-2. PC60
cells were cultured for 3 days in normal medium or in the presence of
IL-1. Both IL-1-primed and nonprimed cells were aliquoted into fresh
cultures (0 h), and cells were harvested 48 h later. During this time,
IL-2 was added to individual cultures to be present for the indicated
period prior to RNA extraction. IL-2R
mRNA was detected by RNase
protection. Signals were quantified and normalized to
-actin mRNA
signals (A). The gel shown in B shows the
results obtained with primed cells.
Thus, IL-1 has two
effects on PC60 cells. First, it rapidly induces IL-2R gene
transcription, at least in part via protein synthesis-independent
pathways that lead to activation of NF-
B. Second, it increases the
capacity of PC60 cells to respond to IL-2, partly by increasing
IL-2R
and IL-2R
expression. This priming effect permitted us
to use PC60 cells for the mapping of the cis-acting elements
that control IL-2 responsiveness of the IL-2R
gene.
Mapping of Interleukin Responsive cis-Acting Elements in
the IL-2R
Previously, we had shown that in PC60 cells
a segment of the mouse IL-2R Gene
gene comprising the promoter, the
transcription start site, and a 2.5-kb 5`-FR (position -2539 to
+93, segment A-D, see Fig. 4 A) conferred on a
chloramphenicol acetyltransferase reporter gene a response pattern to
IL-1 and IL-2 that resembled closely that of the genomic IL-2R
gene
(11) . In normal CD4
CD8
thymocytes of transgenic mice carrying the same construct,
chloramphenicol acetyltransferase expression responds to IL-1 and IL-2
with the same pattern as in PC60
(65) .
5`-FR, we switched from
the chloramphenicol acetyltransferase to a rabbit
-globin reporter
gene in order to be able to measure reporter gene mRNA levels with a
previously described PCR-based assay
(34) . This method allows
very accurate quantification of promoter responses to stimulation,
e.g. with interleukins
(41) . Fig. 4 B shows that the interleukin responses of plasmid pR, which contains
segments A-D of the IL-2R
5`-FR, are the same as those of the
previously described equivalent chloramphenicol acetyltransferase
construct (pmIL-2R
PrCAT1, see Ref. 11). When plasmid pR is
transfected into unprimed PC60 cells, subsequent stimulation with IL-1
alone induces, on average, a 5-fold increase in reporter gene
expression. As pointed out above, under these conditions, stimulation
with IL-1 results in transient IL-2 secretion. IL-2 stimulates a small
but reproducible response, whereas both cytokines together stimulate a
12-fold increase in reporter gene mRNA. When the same plasmid is
transfected into PC60 cells that have been primed by culture in IL-1
for 72 h, the pattern of interleukin responsiveness changes. IL-2 alone
induces a response that is almost as strong as that to IL-1 +
IL-2. Comparison of these results with the interleukin response of the
genomic IL-2R
gene in primed and unprimed PC60 cells shows a
strikingly similar pattern, although the responses of the genomic
IL-2R
gene are stronger than that of the reporter gene constructs.
Such differences are observed in most transient transfection studies of
promoters. There is considerable variability in the magnitude of the
IL-2 response of pwt in primed cells (see e.g.Fig. 7
),
which may be related to similar variation in the inducibility of the
genomic IL-2R
gene, but the response pattern is completely
reproducible. The interleukin inducibility of plasmid pwt (see
Fig. 4A) does not differ significantly from that of pR,
indicating that the insertion of restriction sites in the latter
construct had no effect on interleukin responsiveness. Comparing the
time course of IL-2-induced accumulation of IL-2R
transcripts
(Fig. 3) with that of plasmid pwt mRNA in unprimed and IL-1
primed cells (Fig. 4 C), we found that both responded
without a measurable lag and with similar kinetics.
Figure 7:
IL-2
responsiveness does not require promoter-proximal elements. IL-1 primed
PC60 cells were transfected with reference plasmid and plasmids
containing an intact region C but deletions in segment D (pRD1 to
pR
D6, see Fig. 6 B). Aliquots of the transfected cells
were cultured for 48 h in the presence of the indicated interleukins.
Reporter gene expression is compared with that in cells cultured in
normal medium after transfection (=1). Each construct was tested
at least three times. The figure shows the mean of two different
experiments in which the entire series of plasmids was transfected into
the same batch of primed cells, with the range of values obtained
below each column.
These results
show that many if not all of the cis-acting elements that
control the complex response of the IL-2R gene to IL-1 and IL-2
are contained in segments A-D.
IL-2 Response Elements
In previous experiments, we
had found that segment C conferred IL-2 responsiveness on a
heterologous viral (HSV-tk) promoter
(11) . This is consistent
with the results shown in Fig. 4, demonstrating that a plasmid in
which segment C has been deleted (pRC) can still respond to IL-1
in unprimed PC60 cells but completely fails to respond to IL-2 in
either primed or unprimed cells. Note that deletion of segment C (or
B+C, Fig. 5) does not significantly alter constitutive
promoter activity. The orientation of segment C in the 5`-FR can be
inverted (in plasmid pRi) without affecting the response to IL-2
(Fig. 5 A).
C3) failed to respond in cells stimulated
with IL-2 for times between 1 and 48 h (Fig. 4, C and
D). Deletion of segment C2, immediately upstream of C3,
reproducibly reduced but did not abolish the IL-2 response. Deletion of
other parts of segment C did not significantly affect IL-2
responsiveness. Note that the changes in IL-2 responsiveness do not
correlate with variations in constitutive promoter activity (see legend
to Fig. 5 A).
5`-FR. Two of these deletions,
C3.1 and
C3.3, abolished IL-2 responsiveness, and removal of
the intervening segment, C3.2, strongly reduced IL-2 responsiveness
(Fig. 5 B), whereas deletions in the remaining part of
segment C3 (
C3.4,
C3.5 and
C3.6) had no significant
effect on the capacity of the corresponding plasmids to be stimulated
by IL-2. Again, there was no correlation between constitutive promoter
activity and IL-2 responsiveness among the plasmids of this series (see
the legend to Fig. 5 B).
C3.1,
C3.2, and
C3.3 was sufficient
to confer IL-2-inducible enhancer activity on a heterologous promoter,
we inserted the fragment between nucleotides -1406 and
-1301 upstream of the minimal
-globin promoter of pG1
(37) that does not respond to stimulation by IL-2. The resulting
plasmid, pG1.C3, responds to IL-2 as well as pG1.BC, which contains the
entire segment B+C (Fig. 5 C). Addition of segment
C2, in plasmid pG1.C2+C3, does not significantly improve IL-2
inducibility.
gene by scanning the segments C3.1,
C3.2, and C.3.3 with a series of mutations involving 4-9
nucleotides each. The results (Fig. 5 D) show that IL-2
responsiveness of pwt depends on three separate stretches that are
between 6 and 12 nucleotides long. These sites are defined by mutations
M4, M5, and M6 (site I), mutation M9 (site II) and mutations M12 and
M13 (site III). Each of them presumably corresponds to the binding site
of a transcription factor required for IL-2 responsiveness (see
``Discussion'').
IL-1 Response Elements
The results shown in
Fig. 4B demonstrate that removal of segment C does not
abolish IL-1 responsiveness of the IL-2R 5`-FR. The data presented
in Fig. 2indicate that the early protein synthesis-independent
response of the genomic IL-2R
gene to IL-1 requires activation of
NF-
B, and studies on the human promoter proximal region have shown
that the IL-1 response mediated by this segment depended on a NF-
B
site
(18) , which is conserved in the mouse gene
(Fig. 6 A,
B1 in Fig. 6 B).
This led us to investigate the effect of deletions in the conserved
promoter proximal region on the IL-1 responsiveness of the mouse gene.
Since stimulation of PC60 with IL-1 induces secretion of IL-2, we used
for these experiments recombinants that had been made IL-2 unresponsive
by the removal of region C.
recombinant is not the same in all the transfections (see legend to
Fig. 6C). It should also be pointed out that the
normalization of signals derived from IL-2R
recombinant
transcripts ( i) to the signals derived from reference plasmid
mRNA ( r) is very precise and allows reliable determination of
2-fold inductions. The expression of the plasmid containing an intact D
region (pR
C) is increased about 5-fold by stimulation with IL-1.
Deletion
D3 completely abolishes this response, indicating that
all IL-1-responsive elements in pR
C are contained in the segment
between positions -585 and -54. Note that the constitutive
expression of plasmid pR
C
D3 is not significantly different
from that of pR
C. Deletion
D4, which removes 23 bp,
containing the NF-
B site (
B1) conserved in the mouse and the
human genes, reduces IL-1 responsiveness to 2-3-fold. Removal of
the segment 5` of this stretch (
D1) reduces responsiveness to the
same extent as
D4, indicating that elements upstream of the
conserved NF-
B site are required for a complete IL-1 response.
Deletion
D6 does not reduce IL-1 responsiveness, confirming that
elements between position -585 and -246 contribute most of
the response. However, the plasmid with the complementary deletion,
D2, still responds reproducibly, albeit weakly, to IL-1,
indicating that elements downstream of
B1 may also contribute to
the IL-1 response of the IL-2R
gene. The increased base-line
expression of pR
C
D1 suggests a role for the conserved
negative regulatory element-2 (see Fig. 6 A) that reduces
expression of human IL-2R
constructs in phorbol 12-myristate
13-acetate-stimulated Jurkat cells
(17) , but in this case,
pR
C
D2 would be expected to show a similarly increased
constitutive activity.
Promoter Proximal Elements Are Not Essential for IL-2
Responsiveness
Testing the effect of deletions
D1-
D6 on the IL-2 response of plasmids containing an
intact C region in IL-1-primed cells we found that none of these
deletions abolish the response (Fig. 7), although some markedly
reduce it. Thus, the function of the IL-2 response elements in segment
C does not depend on sequences between position -585 and
-54. This is in agreement with the finding that segment C3 can
confer IL-2 responsiveness on a heterologous promoter
(Fig. 5 C). The observation that
D2 does not
significantly alter IL-2 responsiveness argues against any role for
NF-
B in this response since computer searches have not revealed
any NF-
B consensus site in the mouse IL-2R
5`-FR apart from
B1 and
B2 (see legend to Fig. 6 A for
definition of NF-
B consensus site).
CD8
Thymocytes-We have
recently shown that IL-1 and IL-2 synergize to induce IL-2R
mRNA
levels and surface expression in normal
CD4
CD8
thymocytes. Analysis of
transgenic mice lines
(65) demonstrated that this regulation
depends on the 5`-FR of the IL-2R
gene. In this paper we have used
a CD4
CD8
cell line whose response
to IL-1 and IL-2 is very similar to that of normal cells with the same
phenotype to analyze more closely the mechanisms that mediate
interleukin responsiveness of the IL-2R
gene and to localize the
cis-acting elements controlling it. Since these cells do not
depend on interleukins for growth or survival, the responses analyzed
are not the indirect consequences of the growth factor activity of
these interleukins.
mRNA levels. The first phase
is a protein synthesis-independent response to IL-1 that can be blocked
by inhibitors of NF-
B activation. The second phase is due mainly
to IL-2 and starts at about 10 h. This delay is due to the fact that
unstimulated cells respond very slowly to IL-2 alone, whereas
stimulation with IL-1 induces a strong increase in IL-2 responsiveness.
This priming effect reflects, most likely, a rise in IL-2R
and
IL-2R
expression that results in an increased number of high
affinity IL-2R.
mRNA is due mainly to an increase in IL-2R
gene
transcription. The experiments described in this paper have allowed us
to map the cis-acting elements that control both phases of
mRNA increase.
IL-1-responsive Elements Map to the Conserved Promoter Proximal
Region
The elements that account for the direct response to IL-1
are contained within the promoter proximal segment (position -585
to -54) of the IL-2R gene. Between bp -335 and the
transcription start site the mouse gene is 78% homologous to the human
gene (Fig. 6 A). Within this segment, a conserved motif
that was recognized as a NF-
B consensus binding site (
B1)
appears to play a major role as an IL-1 response element. This is
consistent with the finding that inhibitors of NF-
B activation
prevent the early response of the endogenous IL-2R
gene in PC60.
It is also in agreement with a previous study in which the human 5`-FR
was analyzed in murine EL4 cells
(18) . However, sequences
upstream of the conserved NF-
B site are required to obtain a full
response. The contribution of this segment (D1) may be due to a second
NF-
B site (
B2). But this motif is not conserved in the human
gene, and it seems more likely that an AP-1 site present in both
species around position -335 contributes to the IL-1 response
(Fig. 6 A). We have previously shown that AP-1 elements
can mediate IL-1 responsiveness in PC60. AP-1-dependent mRNA increases
are slower than those mediated by NF-
B and are not yet detectable
after 4 h of IL-1 treatment
(23) . Thus, NF-
B may mediate
the very early response of the endogenous IL-2R
gene to IL-1,
which is completely blocked by PDTC, while AP-1 may act in a later
response phase.
genes the
binding sites for SRF and Sp1 (GC-box) that are immediately downstream
of the NF-
B site in the human gene and that have been implicated
in its phorbol 12-myristate 13-acetate response
(19, 43, 44) , are not conserved in the mouse
gene. The sequence called NF IL-2RA or UE-1
(19, 44) is
partially conserved, but the specificity of the protein(s) binding to
this element has not been sufficiently well characterized to determine
whether their binding site is present in the mouse gene.
IL-2 Responsiveness of the Mouse IL-2R
None of the deletions in segment D abolishes IL-2
responsiveness. Removal of the NF- Gene Depends
on Three Closely Spaced Elements 1.3 kb Upstream of the Transcription
Start Site
B consensus sites
B1 and
B2 in deletion
D2 does not significantly affect IL-2
inducibility. Since a computer search revealed no additional NF-
B
sites in segments A-D (see legend to Fig. 6 A for
search conditions), it is unlikely that NF-
B plays a direct role
in the transcriptional regulation of the IL-2R
gene by IL-2. Even
pR
D3 in which all IL-1 response elements have been removed is
still significantly stimulated by IL-2. This result implies that
IL-1-mediated transcriptional activation of the IL-2R
gene is not
a prerequisite for the activity of the IL-2-responsive elements in
segment C. It is consistent with the observation that segment C3 acts
as an IL-2-responsive enhancer on a heterologous promoter
(Fig. 5 C) that cannot be stimulated with IL-1
(45) . Nevertheless, the reduced IL-2 responsiveness of plasmids
with deletions in the promoter proximal region (particularly p
D5,
p
D3, and p
D6) suggests that maximal activity of the
IL-2-responsive elements in segment C3 does depend on elements in the
promoter region itself.
gene map to a 48-bp segment 1.3 kb
upstream of the transcription start site. Elsewhere
(65) we
show that in normal T lymphocytes, a DNase I-hypersensitive site
appears at this position when cells are stimulated to express the
IL-2R
gene. This provides completely independent evidence for an
important regulatory role of this segment. Our results show that three
separate elements within the 48-bp segment are required for full IL-2
responsiveness. Site I (defined by mutations M4-M6) contains a
nucleotide sequence that is identical to the core of the GAS-1 element
in the tryptophanyl-tRNA synthetase gene. This sequence binds several
cytokine-inducible members of the STAT family of transcription factors
(46, 47, 48) . Since it has recently been shown
that IL-2 can activate STAT proteins that recognize similar motifs
(49, 50) , it is likely that site I is the target of
such a factor. However, band shift and footprinting experiments with
PC60 nuclear extracts have not yet revealed any IL-2-inducible
DNA-binding proteins specific for sequences in C3. The sequence of site
II (defined by mutation M9) is very similar to that of site I and may
also be a target of an IL-2-induced STAT protein. But it includes a
potential binding site for factors of the GATA family, a member of
which has been implicated in the regulation of T cell-specific gene
expression
(51, 52, 53) . Site III (defined by
mutations M12 and M13) forms part of a consensus binding site for
transcription factor Ets-1
(54) . The consensus site extends
into the nucleotides altered in M14, but this mutation is compatible
with Ets-1 binding. Site III overlaps with a DNase I footprint over
position -1331 to -1314 that is obtained with extracts from
induced as well as nonstimulated PC60 cells.
(
)
Further work is required to definitively identify the
transcription factors that confer IL-2 responsiveness to the C3
segment, and to define their role in this response. We also plan to
explore whether the finding that significant IL-2 responsiveness
requires three distinct but closely spaced elements is a reflection of
some form of direct cooperation between the proteins binding to these
sites.
transcription by IL-2 has been
observed in human cells, and it seems likely that the proteins and
sequence elements that mediate this regulation are the same in rodents
and in man. The sequence of the 5`-FR of the human gene until position
-1241 has been published. The two genes share highly homologous
promoter-proximal regions, but apart from homology between repetitive
Alu and B1 elements (see Fig. 4 A for repetitive elements
in the mouse gene) there is no significant similarity upstream of
position -335 in the mouse gene. Recombinants between the
-1241/+93 segment of the human IL-2R
and a
chloramphenicol acetyltransferase reporter gene do not respond to
stimulation with IL-2 in PC60 cells.
(
)
Thus, the
human IL-2 response elements do not seem to be present in the part of
the gene for which sequence data have been published.
expression is uncoupled from the growth factor activity of
IL-2. This suggests that the signal transduction pathway leading from
the IL-2 receptor to the IL-2R
gene is different from the one that
drives cells into S-phase. It may overlap with the pathways that
mediate IL-2 stimulation of granzyme and perforin expression
(28, 58) in T cells. In B cells, IL-2 stimulates
IgJ-chain expression
(59) . IL-2 responsiveness of the J-chain
gene depends on a 28-bp stretch between position -74 and
-41
(60) . This sequence has no significant similarity
with segment C, suggesting that IL-2 stimulates IgJ expression via
different transcription factors.
A General Model for the Regulation of IL-2R
The results presented in this paper are summarized
in the model shown in Fig. 8. They show that the complex
transcriptional response of the IL-2R Gene
Transcription
gene in
CD4
CD8
thymocytes to IL-1 +
IL-2 can be explained by the existence of two sets of
cis-acting elements. Promoter proximal elements, among which a
conserved NF-
B site plays a dominant role, mediate an early
response that is stimulated by IL-1 alone. IL-1 also increases IL-2
responsiveness of the cells by augmenting the number of functional
IL-2R. IL-2 induces the second phase of the IL-2R
gene, which is
mediated by a complex enhancer 1.3 kb upstream of the transcription
start site. Thus, the biphasic response correlates with a bipartite
structure of 5`-FR.
Figure 8:
Model for the transcriptional regulation
of the mouse IL-2R gene by IL-1 (or antigen) and IL-2. The model
postulates that the biphasic response of the IL-2R
gene to
stimulation by IL-1 and IL-2 in CD4
CD8
thymocytes or to antigen and IL-2 in mature T cells is controlled
by two independently functioning sets of regulatory elements. IL-1 or
antigen activate the first phase of transcription via promoter proximal
elements, among them a critical NF-
B site. Simultaneously, these
signals induce cells to become IL-2 responsive, and thus prime them for
the second phase. This is mediated by IL-2-responsive elements 1.3 kb
upstream of the promoter.
We believe that the model presented here also
applies to the antigen-triggered induction of IL-2R expression in
mature lymphocytes and that antigen plays a similar role to IL-1.
Stimulation of T cells with antigen or anti-CD3 induces activation of
NF-
B, and we have found that the early phase of antigen-induced
increase in IL-2R
mRNA in a CD8
T cell clone
shows the same sensitivity to inhibition by PDTC of NF-
B
activation.
(
)
In the same clone, IL-2R
mRNA
levels return to base line 24 h after exposure to antigen unless IL-2
is added, and we have shown that maximal and prolonged expression of
both the normal IL-2R
gene and a IL-2R
5`-FR/chloramphenicol
acetyltransferase transgene in concanavalin A-stimulated peripheral T
cells depends on IL-2
(65) .
/EMBL Data Bank under accession number M16398.
genomic and cDNA clones
and Guido Miescher for the mouse
-actin construct. We also thank
Charles Weissman and Pierre Chambon for plasmids Z-pCRI/pRChr
G-1
and pG1; Claudine Ravussin, Pierre Dubied, and Marcel Allegrini for
preparation of the manuscript and figures; and Bernhard Hirt for useful
suggestions and criticisms concerning the manuscript.
gene is
important for its response to phorbol 12-myristate 13-acetate. This
site is conserved in the mouse gene (see Fig. 6 A).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.