From the
The
Interleukin-2 receptors (IL-2R)
In mature T
cells, the primary signal inducing IL-2R
Among the T cell precursors,
IL-2R
Regulation of the
IL-2R
To assess if the
5`-flanking region of the mouse IL-2R
In a complementary
approach, we have screened a 11.9-kb region around the promoter of the
IL-2R
When we compared the time course of transgene expression to that of
the endogenous IL-2R
Fig. 6C shows the strategy for detecting DHS in the
11.9-kb segment of the IL-2R
In most previous studies isolated nuclei were used
for chromatin analysis by nuclease cleavage. In this study we used,
instead, cells permeabilized with lysolecithin
(55) . This
method is much simpler than isolation of nuclei and may, in addition,
prevent removal of chromatin proteins and perturbation of chromatin
structure that can occur during the preparation of nuclei
(56, 57) . To control the efficiency of
permeabilization, we stained cells with trypan blue before and after
lysolecithin treatment. In all preparations, more than 95% of treated
cells took up the dye. As a further check, aliquots of genomic DNA from
cells treated with different concentrations of DNase-I were
fractionated on 0.8% agarose gels and stained with ethidium bromide.
This routine control showed that the overall sensitivity of chromatin
to DNase-I was similar in all cell types tested (data not shown).
To compare the pattern of DHS in activated T cells
that transcribe the IL-2R
In this paper, we have examined the control of mouse
IL-2R
Transgene expression was restricted to lymphoid organs, where
endogenous gene expression is also detectable. Constitutive CAT
activity was found in the thymus of four out of five lines.
Surprisingly, fractionation of thymic tissue showed that the bulk of
CAT activity was contained in the stroma and not in the thymic
lymphocytes. Constitutive CAT activity was detectable in
CD4
In lymphocytes
from four out of five lines, the transgene response to stimulation
correlated very well with that of IL-2R
Our data demonstrate that the segment between bp -2539 and
+93 of the mouse IL-2R
The IL-2R
An additional
lymphocyte-specific constitutive DHS, DH1, maps to a region between bp
-100 and +1, in a segment with strong homology to the
promoter region of the human IL-2R
In summary, the experiments described here delimit the
region of the mouse IL-2R
CAT activity in 100 µg of protein extracted from
different organs. Dashes indicate percentages of chloramphenicol
conversion
We thank Richard Boyd for help in the
immunohistochemical analysis of thymic sections, Michel Braun for
setting up the kidney epithelial cell cultures, Anne Wilson and Jovan
Mirkovitch for valuable discussion of the work and advice concerning
the manuscript. We also thank GLAXO, Geneva, for generous gifts of
recombinant IL-1 and IL-2; Pierre Zaech and Christian Knabenhans for
operating the FACS; and Claudine Ravussin, Pierre Dubied, and Marcel
Allegrini for help in the preparation of the manuscript and the
figures.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
chain of the interleukin-2 receptor (IL-2R
) is a
key regulator of lymphocyte proliferation. To analyze the mechanisms
controlling its expression in normal cells, we used the 5`-flanking
region (base pairs -2539/+93) of the mouse gene to drive
chloramphenicol acetyltransferase expression in four transgenic mouse
lines. Constitutive transgene activity was restricted to lymphoid
organs. In mature T lymphocytes, transgene and endogenous IL-2R
gene expression was stimulated by concanavalin A and up-regulated by
IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and
IL-2 cooperatively induced transgene and IL-2R
gene expression.
These results show that regulation of the endogenous IL-2R
gene
occurs mainly at the transcriptional level. They demonstrate that
cis-acting elements in the 5`-flanking region present in the
transgene confer correct tissue specificity and inducible expression in
mature T cells and their precursors in response to antigen, IL-1, and
IL-2. In a complementary approach, we screened the 5` end of the
endogenous IL-2R
gene for DNase-I hypersensitive sites. We found
three lymphocyte specific DNase-I hypersensitive sites. Two, at
-0.05 and -5.3 kilobase pairs, are present in resting T
cells. A third site appears at -1.35 kilobase pairs in activated
T cells. It co-localizes with IL-2-responsive elements identified by
transient transfection experiments.
(
)
are composed of three distinct transmembrane proteins,
called IL-2R
, IL-2R
, and IL-2R
or
chain (for review, see Refs. 1 and 2). These chains can combine
to form different classes of IL-2R.
and
heterodimers form
intermediate affinity receptors that are required for signal
transduction
(3, 4) . The
chain by itself forms
low affinity receptors and is devoid of signaling ability. It
associates with the other two chains to form high affinity IL-2R, which
are required for IL-2-driven T cell proliferation
(5) . While
all mature T cells and their thymic precursors constitutively express
the
chain
(6)
(
)
and
some resting T cells express IL-2R
(7) , IL-2R
is
undetectable in resting T cells but is efficiently induced upon T cell
activation
(8) . This makes IL-2R
a key regulator of IL-2
responsiveness and has prompted a number of studies aimed at
understanding the control of IL-2R
expression.
expression is triggered
by antigen through the T cell receptor (TcR)
CD3 complex.
Antigenic stimulation can be mimicked by mitogenic lectins, such as
ConA or phytohemagglutinin
(9) , by antibodies directed against
components of the TcR
CD3 complex
(10) or by a combination
of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator,
and ionomycin, a calcium ionophore
(11) . These stimuli also
induce IL-2 production, which not only drives T cell proliferation but
also up-regulates IL-2R
expression
(12, 13, 14) .
gene expression is restricted to an early stage of thymic T
cell differentiation. IL-2R
cells belong to a
heterogeneous subset of thymocytes that expresses neither CD4 nor CD8
(CD4
CD8
thymocytes)
(15, 16) . Upon activation of
CD4
CD8
thymocytes in vitro with PMA and ionomycin, IL-2R
expression declines to barely
detectable levels. However, addition of IL-1 or IL-2 to such cultures
induces a strong increase in IL-2R
mRNA and cell surface protein.
IL-1 and IL-2 can enhance IL-2R
expression through independent
mechanisms
(17, 18, 19) .
gene by antigen, IL-1, and IL-2 has been shown to occur at
least in part by changes in its transcription
(20, 21, 22) . Several groups have analyzed the
regulation of expression of the human IL-2R
gene by transient
transfection of plasmid constructs containing the human IL-2R
gene
5`-flanking region linked to the reporter gene chloramphenicol
acetyltransferase (CAT). Experiments with the leukemic T cell line
Jurkat and the natural killer-like cell line YT have identified a
series of elements regulating responses to PMA, phytohemagglutinin,
tumor necrosis factor-
, IL-1, and human T cell leukemia virus-type
I tax protein, which are clustered between base pair (bp) -390
and -236
(23, 24, 25, 26, 27, 28, 29, 30, 31) .
In the human gene only the region up to -1249 bp has been
analyzed, whereas in the mouse gene, we have shown that a region
further upstream plays a major role in the transcriptional regulation
of the IL-2R
gene. Transient transfection experiments in PC60, a
rodent CD4
CD8
T cell line in which
IL-1 and IL-2 have a similar synergistic effect on IL-2R
expression as in normal CD4
CD8
thymocytes
(19) , have shown that the 5`-flanking region
of the mouse IL-2R
gene (bp -2539 to +93) fused to a
reporter gene
(
)
confers a response pattern to
IL-1 and IL-2 that is very similar to that of the endogenous IL-2R
gene. In agreement with the studies on the human gene, we have mapped
weak IL-1 responsive elements between bp -585 and -54.
However, the most important regulatory elements in the mouse gene are
contained in the segment between bp -1835 and -802
(21) , and we have shown that three distinct elements near
position -1.3 kb are required for IL-2 responsiveness
(69) . Together with the data obtained from the analysis of the
human gene, our results suggest that the IL-2R
promoter proximal
region mediates early responses to antigen or IL-1, while previously
unrecognized elements further upstream are required for the later
IL-2-induced rise in IL-2R
transcription.
gene that we have analyzed
by transient transfection of the cell line PC60 contains the
cis-acting elements required for correct tissue-specific and
inducible expression of IL-2R
gene in normal cells, we have
generated transgenic mice in which this region controls the expression
of the bacterial CAT gene. We chose CAT as a reporter gene, despite the
fact that it does not lend itself to analysis at the single-cell level
(unless its expression is driven by very strong promoters), because
previous reports had shown that CAT expression could be detected in
normal lymphoid tissues
(32, 33, 34) . In
contrast, lacZ, a reporter gene that would have appeared more
suitable for this study, is not expressed or is poorly expressed in
normal lymphocytes
(35, 36) .
gene for DNase-I hypersensitive sites (DHS). DHS have been
associated with sequences that control the transcriptional activity of
neighboring genes, whereas general sensitivity to nuclease attack has
been used to define open chromatin domains around genes that are
transcriptionally active or can be activated upon stimulation or
differentiation
(37, 38) . DHS can be ubiquitous or cell
type-specific. The latter class includes DHS that are present in cells
in which the associated locus is silent but will be transcribed upon
stimulation or differentiation, as well as DHS whose appearance
correlates with gene expression. Generally, tissue-specific DHS map
within or close to, cis-acting regulatory elements that behave
as cell type-specific enhancers or locus control regions. Recently, it
has been demonstrated that ubiquitous DHS may mark the boundary of an
active chromatin segment by isolating other transcription units from
the effect of enhancer elements
(40) .
Generation of Transgenic Mice
The mouse
IL-2R-CAT fusion gene was isolated from plasmid pmIL-2R
PrCAT1
(21) as a 3.9-kb fragment (see Fig. 1) and used to
generate transgenic mice according to published procedures
(41, 42) . The construct was isolated by agarose gel
electrophoresis, purified using glass powder (Geneclean, Bio 101 Inc.,
La Jolla, CA), and dissolved in injection buffer (10 mM Tris,
pH 7.4; 0.1 mM EDTA). One to two pl of DNA at 3 µg/ml were
injected into pronuclei of fertilized oocytes derived from NMRI mice
(IFFA-Credo, Les Oncins, France). Injected oocytes were transferred to
foster mothers. The presence of the transgene in the offspring was
assessed by Southern blot analysis (see below) of genomic DNA isolated
from mouse tails
(42) and digested with TaqI. A
fragment of 2.6 kb indicated the presence of the transgene, while a
fragment of around 6.0 kb was derived from the endogenous IL-2R
gene. Comparison of the intensity of these two bands, evaluated with a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA), allowed estimation
of transgene copy number. Transgenic founders were crossed to NMRI
mice. 6-12-week-old transgenic mice or nontransgenic littermates
of either sex were used for the experiments.
Figure 1:
Schematic
representation of the IL-2R-CAT transgene and Southern blot
analysis of founder mice. A, at the top, the
endogenous mouse IL-2R
gene is represented. The first exon is in
black, and the first intron is in gray. The segment
of 5`-flanking region present in the transgene is hatched.
Eg is the 6.0-kb TaqI ( T) genomic
restriction fragment detected in Southern blots. The transgene
( bottom) contains the mouse IL-2R
5`-flanking region from
kb -2.539 ( SphI, S) to +0.093
( PstI, P) fused to the CAT gene ( stripedbox). Tg is the 2.6-kb TaqI restriction
fragment seen in Southern blots of transgenic DNA. The probe used for
identification of transgenic mice was the IL-2R
genomic fragment D
(see Fig. 6 B). B, founder mice were identified by
Southern blot analysis of tail DNA digested with TaqI. DNA
from a nontransgenic mouse ( ntg) was also analyzed. Transgene
copy numbers are indicated below the
blot.
Cell Preparation and Culture
For the DNase-I
hypersensitivity experiments, we used 2-3-month-old C57Bl/6 mice
(IFFA-Credo). Lymph node and spleen cells were prepared by gentle
homogenization. Spleen cells were sedimented onto Ficoll (Pharmacia
Biotech Inc.). These cells were used either directly (not cultured) or
cultured at 1 10
cells/ml with ConA (2.5 µg/ml,
Sigma) for different times. Where specified, IL-2 was added at the
indicated concentrations. T cells were purified by incubating cell
suspensions from lymph nodes or spleen either with anti-CD4 (GK1.5) and
anti-CD8 (53-6.7) mAb or with an anti-Thy1.2 (A15) mAb coupled to
magnetic microbeads (MACS® Miltenyi Biotec GmbH, Bergisch-Gladbach,
Germany) followed by two rounds of magnetic sorting with a magnetic
cell separation device (MACS Miltenyi Biotec GmbH)
(43) . B
cells were recovered in the flow-through. Cells were used either
directly (not cultured) or after 80 h of culture at 1
10
cells/ml with LPS (30 µg/ml; Escherichia coli 0127:B8, Life Technologies, Inc.) or with plastic-bound anti-CD3
mAb (145-C11), as described previously
(44) .
CD4
CD8
thymocytes were purified
from thymi of 6-week-old mice, using a combination of
complement-mediated cytotoxicity and magnetic bead depletion with goat
anti-rat Ig-coated Dynabeads, as described previously
(19) .
Cells were cultured at 4
10
/ml with PMA (3 ng/ml)
and ionomycin (62.5 ng/ml) (Calbiochem, San Diego, CA) for 48 h in
96-well flat bottomed plates (Costar, Cambridge, MA). Where indicated
IL-1
at 1 ng/ml and/or IL-2 at 200 units/ml were added. Anti-mouse
IL-2 mAb S4.B6.1
(45) was added to all the cultures, while
anti-mouse IL-2R
mAb 5A2
(46) was added only to cells
cultured without IL-2. Bone marrow was prepared by flushing femur and
hip bones with ice-cold PBS through a 26-gauge needle. Thymic stroma
and lymphocytes were separated by gently squeezing total thymus in a
loose-fitting glass homogenizer. Mouse kidney epithelial cells were
prepared as described elsewhere
(47) . Culture medium was
Dulbecco's modified Eagle's medium containing 5% fetal calf
serum
(44) . IL-1 and IL-2 were human recombinant proteins,
kindly provided by GLAXO (Geneva, Switzerland).
Flow Cytometry
For immunofluorescence analysis of
IL-2R surface expression, the rat anti-mouse IL-2R
mAb PC61
(48) was used, either conjugated with FITC, biotinylated, or as
culture supernatant. PE-conjugated streptavidin or FITC-conjugated goat
anti-rat Ig were used as second-step reagents (Caltag Laboratories, San
Francisco, CA). The purity of MACS-purified cells before and after
culture was assessed by staining cells with PE-conjugated anti-CD4
(Becton Dickinson, Mountain View, CA) plus PE-conjugated anti-CD8
(Boehringer Mannheim) and FITC-conjugated F(ab`)
sheep
anti-mouse Ig (Silenus Laboratories, Hawthorn, Australia). Samples were
analyzed with a FACScan flow cytometer (Becton Dickinson) using the
LYSYS II program.
Preparation of Extracts and CAT Assays
Protein
extracts from fresh and cultured cells were prepared, and CAT activity
was measured as described in Ref. 21, except that extracts were heated
at 60 °C for 10 min before the assay. Tissues were flash frozen in
dry ice and ethanol and stored at -80 °C. Samples were
homogenized in 250 mM Tris-HCl, pH 7.8, freeze-thawed 3 times,
and centrifuged for 15 min at 10,000 rpm. Supernatants were heated at
60 °C for 10 min and centrifuged for another 10 min at the same
speed. Protein concentration was determined by the Bio-Rad protein
assay (Bio-Rad). CAT reactions were run at 37 °C overnight. CAT
activity was expressed as percentage of
[C]chloramphenicol converted to acetylated
forms, as evaluated by PhosphorImager scanning.
RNase Protection Assay
RNA was prepared by
guanidinium thiocyanate extraction
(49) . RNase protection
assays were performed as described previously using mouse IL-2R
and
-actin probes, which, when hybridized with mRNA, generate
RNase-resistant fragments of 414 and 138 nucleotides, respectively
(19) .
DNase-I Treatment of Cells and Isolation of Genomic
DNA
To make cells permeable to DNase-I, we treated them with
lysolecithin as described by Miller et al.(50) . Cells
were washed with PBS and resuspended to 10/ml in buffer A
(150 mM sucrose, 80 mM KCl, 35 mM Hepes, pH
7.4, 5 mM potassium phosphate, pH 7.4, 5 mM
MgCl
, 0.5 mM CaCl
). One-third volume
of a solution of 1 mg/ml of lysolecithin (Fluka Chemie AG, Buchs,
Switzerland) in buffer A was added, and cells were incubated at 37
°C for 70 s. Permeabilization was stopped by adding 20 volumes of
ice-cold PBS. Cells were pelleted and resuspended to 10
/ml
in ice-cold buffer A containing 2 mM CaCl
.
Aliquots of 10
cells were incubated with different amounts
of DNase-I (Pharmacia) for 1 min at 20 °C. Nuclease digestion was
stopped by the addition of 1 volume of stop solution (20 mM
EDTA, 1% SDS, 0.5 mg/ml proteinase K). Samples were incubated for at
least 2 h at 37 °C before extraction with
phenol/chloroform/isoamylalcohol. Nucleic acids were
ethanol-precipitated from the aqueous phase and redissolved in Tris
EDTA buffer. Their concentration was estimated by measuring
A
. An aliquot was fractionated on a 0.8% agarose
gel to check the quality of the DNA and the effect of DNase-I
digestion.
Southern Blot Analysis
60-80 µg of DNA
from each sample were digested to completion with the indicated
restriction enzymes and electrophoresed in 0.8-1% agarose gels in
Tris borate buffer at 1-1.5 V/cm. DNA was transferred to
polyvinylidene difluoride-based membranes (Immobilon transfer
membranes, Millipore Corp., Bedford, MA) according to the indications
of the manufacturer. To screen for DHS, we prepared probes using
IL-2R
genomic fragments C or D (see Fig. 6) as templates,
with the random priming kit supplied by Boehringer Mannheim. Membranes
were hybridized with Quickhyb
solution (Stratagene, La Jolla,
CA) according to the protocol supplied by the manufacturer at 65
°C. Filters were autoradiographed for 1-7 days at -70
°C with intensifying screens.
Figure 6:
Mapping
of DNase-I hypersensitive sites in the 5` end of the mouse IL-2R
gene. Filledarrowheads indicate the DHS defined in
this paper. A, schematic representation of the IL-2R
gene
5` end. The first exon is in black, and the first intron is in
gray. The segment of 5`-flanking region present in the
transgene is hatched. Restriction sites shown are as follows:
P ( PstI), S ( SphI), or B ( BglII). DHS are mapped to an accuracy of about 100 bp.
B, magnified segment of the 5`-flanking and promoter region,
which controls CAT expression in transgenic mice. This region has been
arbitrarily divided into fragments A, B, C, and D, that are separated
by BglII sites. C, fragments detected by indirect
end-labeling with probe D after complete digestion of genomic DNA from
DNase-I treated cells with PstI or
SphI.
To detect TcR rearrangements,
blots were hybridized with an antisense RNA probe (86T1) specific for
the constant region of the TcR gene
(51) labeled by
in vitro transcription with Riboprobe® Systems (Promega,
Madison, WI).
Generation of IL-2R
We
have previously shown that the mouse IL-2R-CAT Transgenic Mice
gene 5`-flanking region
from bp -2539 to +93 confers a pattern of expression on a
linked bacterial CAT gene that closely resembles that of the endogenous
IL-2R
gene in the rodent T cell line PC60
(21) . We
therefore used this construct, referred to as IL-2R
-CAT, to
generate transgenic mice. Five transgenic mice (out of 40 live births)
were identified by Southern blot analysis. They carried between two and
29 copies of the transgene (Fig. 1). All founders transmitted the
transgene to offspring at a constant copy number. As expected, the
transgenic mice appeared normal with regard to behavior, lifespan, and
physiology. They contained normal numbers of lymphocytes with normal
subset distributions (data not shown). The responses of the lymphocytes
in in vitro culture conditions were indistinguishable from
those of cells from nontransgenic littermates.
Tissue Distribution of IL-2R
To determine in which tissues the IL-2R-CAT Transgene
Expression
-CAT
transgene was expressed, organs were taken from mice of each transgenic
line and analyzed. CAT activity was undetectable in extracts
(100-500 µg) of liver, kidney, heart, brain, and ovary of any
transgenic line (data not shown). Lack of transgene expression in these
tissues correlates with the absence of IL-2R
cells observed by Takacs et al.(52) by
immunohistochemistry. No constitutive CAT activity was detected in
lymph nodes of any line, whereas one line (1028) expressed activity in
the spleen. The transgene was also expressed in the bone marrow of two
lines (1028 and 1041) and in the thymus of four out of the five
transgenic lines (). Levels of CAT activity in thymi of
different transgenic lines correlated with transgene copy number
( r
= 0.93; see ). Unexpectedly,
when thymic lymphocytes and stroma were separated, constitutive CAT
activity was found in the stroma fraction but not in the lymphocytes
(data not shown, see ``Discussion'').
Expression of the IL-2R
Since mature T cells
express IL-2R-CAT Transgene Is Inducible
in Lymphocytes of Four Transgenic Lines
only after activation
(8) , we analyzed the
inducibility of the IL-2R
-CAT transgene in lymph node and spleen
cells from mice of the different transgenic lines before and after
stimulation with ConA and IL-2. The effectiveness of the stimulation
was checked by FACS analysis of IL-2R
cell surface expression (see
footnote to ). More than 85% of the stimulated cells from
spleen and lymph nodes were IL-2R
and
CD4
or CD8
(data not shown). As shown
in , CAT activity was induced in both cell populations in
four transgenic lines. Levels of expression did not correlate with
transgene copy number, suggesting that the site at which the
integration of the transgene occurred affects its transcriptional
efficiency. The only line (1018) in which transgene expression was not
inducible was the one without detectable constitutive CAT activity in
the thymus (see ). Its transgene may have been integrated
into transcriptionally inactive chromatin. This line was not further
analyzed. Cells taken fresh () or after culture without
stimuli (data not shown) did not have detectable CAT activity, with the
exception of spleen cells from mice of line 1028, which also displayed
constitutive CAT activity in total spleen extracts ().
gene, we found a very similar pattern
(Fig. 2). CAT activity and surface IL-2R
expression were
detectable after 16 h of culture and increased until 72 h. By 6 days of
culture, both activities had fallen to substantially lower levels.
Figure 2:
Time
course of IL-2R-CAT transgene expression in activated lymphocytes.
Spleen cells of line 1045 were either used fresh (time 0) or cultured
for the indicated times with ConA and IL-2 (50 units/ml). A,
at each time point, protein extracts were prepared, and CAT assays were
performed with extracts from 3
10
cells.
B, IL-2R
expression was monitored by FACS analysis of
cells stained with PC61 mAb and FITC-conjugated goat anti-rat Ig, and
expressed as mean fluorescence of the total population. These results
were confirmed with cells from line 1041.
The IL-2R
Anti-CD3 antibodies induce T lymphocytes to express the
IL-2R-CAT Transgene Is Expressed in
Anti-CD3-stimulated T Cells But Not in LPS-activated B
Cells
gene and to proliferate, whereas LPS is a B cell mitogen but
does not stimulate IL-2R
expression
(53) . To determine
whether expression of the IL-2R
-CAT transgene showed the same
specificity, we purified T and B cells from lymph nodes and cultured
them on plastic-bound anti-CD3 mAb or with LPS, respectively. After 80
h, both T and B cell cultures contained a large fraction of activated
cells, as indicated by cell size distribution and percentage of cycling
cells. Anti-CD3 activated cultures contained >95% CD4
and/or CD8
cells, whereas LPS-stimulated
cultures contained >90% Ig
cells (data not shown).
Activated T but not B cells expressed surface IL-2R
(Fig. 3 B) as well as IL-2R
mRNA (data not shown),
and this correlated with CAT activity, which was detectable only in
stimulated T cells (Fig. 3 A). This demonstrates that
induction of transgene expression is not a nonspecific consequence of
lymphocyte activation.
Figure 3:
The IL-2R-CAT transgene is expressed
in activated T but not B cells. T and B cells were purified from the
lymph nodes of transgenic mice of line 1045 or from nontransgenic
littermates with anti-CD4 and anti-CD8 mAb coupled to magnetic beads.
The cells retained by the magnetic device were >95% CD4
and/or CD8
and <1% Ig
. They
were designated as T cells. The cells in the flow-through were >90%
Ig
and <1% CD4
and/or
CD8
. They were designated as B cells. Cells were used
either directly (not cultured) or after 80 h of culture at 1
10
cells/ml with LPS (30 µg/ml) or with plastic-bound
anti-CD3 mAb (145-2C11, 10 µg/ml). A, CAT assays
were performed with extracts from 2
10
cells;
B, IL-2R
surface expression on activated T and B cells
was monitored by FACS analysis. Open profiles correspond to cells
stained with PC61 mAb and FITC-conjugated goat anti-rat Ig. Shadedprofiles correspond to cells stained with the anti-Ig
reagent alone. These results were confirmed with cells from line
1028.
Expression of the IL-2R
Once T cells have been
induced to display IL-2R-CAT Transgene Is
Up-regulated by IL-2 in Activated T Cells
at their surface by signaling through the
TcR, IL-2 can up-regulate IL-2R
expression
(12, 13, 14) . Since in normal T cells antigenic
stimulation leads also to IL-2 expression, it is necessary to
neutralize the endogenously produced IL-2 in order to show the role of
IL-2 in the induction of IL-2R
expression. This can be achieved by
including an anti-mouse IL-2 mAb (S4.B6.1) in the cultures. Under these
conditions, IL-2 concentration can be controlled by the addition of
human IL-2, which does not cross-react with the mAb but is recognized
by the mouse high affinity IL-2R
(19) . Using this approach, we
have investigated the effect of IL-2 on transgene expression in
ConA-activated lymphocytes. As shown in Fig. 4, spleen cells from
transgenic mice stimulated with ConA alone, in the presence of
anti-IL-2 mAb, contained significant amounts of CAT activity after 44 h
of culture. These levels decreased by 68 h. When human IL-2 was added
to such cultures, CAT levels after 44 h were comparable with those
attained in the absence of IL-2. But after 68 h, cells cultured with
IL-2 contained significantly more CAT activity, which remained high
until 96 h. This pattern paralleled very closely the surface expression
of IL-2R
in the same cells (Fig. 4 A). The effect of
IL-2 on the endogenous gene was evident also when we analyzed
IL-2R
mRNA levels (Fig. 4 B). These results show
that IL-2 is important to maintain high levels of expression of the
transgene as well as of the IL-2R
gene. They also show that IL-2
controls the rate of IL-2R
gene transcription in normal T cells
through cis-acting elements, which are contained in the
transgene.
Figure 4:
IL-2R-CAT expression is up-regulated
by IL-2 in activated T cells. Spleen cells from line 1045 were either
used as fresh cells (time 0) or cultured for the indicated times with
ConA with or without human IL-2 (200 units/ml). Anti-mouse IL-2 mAb was
added to all cultures. At the indicated times, cells were harvested,
and a fraction of the cells were washed and recultured under the same
conditions. The remainder of the cells were used for assays of CAT
activity, IL-2R
surface expression and mRNA levels. A,
CAT assays were performed with extracts from 3
10
cells. IL-2R
expression was monitored by FACS analysis of
cells stained with biotinylated PC61 mAb and PE-conjugated streptavidin
and expressed as mean fluorescence of the total population. B,
IL-2R
mRNA was detected by RNase protection. As an internal
control, we measured
-actin mRNA in the same samples. The results
of this experiment were confirmed with cells from line
1041.
The IL-2R
Early thymic T cell precursors
express neither CD4 nor CD8 at their surface
(CD4-CAT Transgene Responds to IL-1 and IL-2 in
Early Thymic T Cell Precursors
CD8
thymocytes) and do not
carry functional TcR. They proliferate upon stimulation with PMA and
ionomycin. Proliferation depends on IL-2 and is potentiated by IL-1
(54) . In these cells IL-2R
cell surface expression is
strongly enhanced by IL-1 and IL-2
(17) . Recently, we have
reported that this reflects an increase in IL-2R
mRNA levels and
have shown that IL-1 and IL-2 can augment IL-2R
expression through
different mechanisms
(19) . Fig. 5shows an experiment in
which we compare the effect of IL-1 and IL-2 on IL-2R
and
transgene expression in the CD4
CD8
thymocyte subset. IL-1 alone increases transgene and, as
previously reported, IL-2R
expression, even when autocrine and
paracrine IL-2 effects are blocked with anti-IL-2 and anti-IL-2R mAb.
IL-2 alone also enhances transgene activity and IL-2R
expression.
Maximal activity of either gene depends on stimulation with both
interleukins. The close parallel between IL-2R
cell surface levels
and CAT activity in this system indicated that IL-1 and IL-2 control
IL-2R
gene expression by regulating its transcription and that the
elements mediating the IL-1 as well as the IL-2 response in early
thymic T cell precursors are present in the transgene.
Figure 5:
The
IL-2R-CAT transgene responds to IL-1 and IL-2 in early thymic T
cell precursors. CD4
CD8
thymocytes
of line 1045 were cultured in the presence of PMA and ionomycin with or
without IL-1
and human IL-2. Anti-mouse IL-2 mAb was added to all
cultures, and anti-mouse IL-2R
5A2 mAb was added only to cultures
without IL-2. A, CAT assays were performed with 3
10
cells. B, IL-2R
expression was evaluated
by FACS analysis of cells stained with FITC-conjugated PC61 mAb
( openprofiles). To correct for the slight
interference of the 5A2 mAb, present in some cultures, with PC61
binding (46), an excess of the former was added to all reactions.
Shadedprofiles correspond to unstained cells. These
results were confirmed with cells from line 1041 and
1028.
Mapping of DNase-I Hypersensitive Sites in the 5` End of
the Mouse IL-2R
Fig. 6A shows a
schematic representation of the mouse IL-2R Gene
gene segment screened
for DHS. It spans 11.9 kb between a PstI site 7.8 kb upstream
and an SphI site in the first intron, 4.1 kb downstream of the
major start site. Fig. 6 B represents the subsegment
present in the transgene (see Fig. 1) screened for regulatory
elements by transient transfection experiments
(69) .
gene. Hybridization of Southern blots
of PstI-digested genomic DNA with a probe for fragment D
allowed detection of two DNase-I hypersensitive sites, DH2 and DH3.
Their position was confirmed by hybridization of the same blots with a
probe for fragment C and by digestion of genomic DNA with different
restriction enzymes and hybridization with probes for D or C (data not
shown). DH2 can also be detected in blots of SphI-restricted
DNA probed for fragment D or C. DH1 is too close to the PstI
site at bp +93 to be detectable in PstI digests, but it
can be detected in SphI-digested DNA probed with fragment C or
D. We have not seen the expected fragment between DH1 and the
SphI site at +4.1 kb in the latter blots, presumably
because the overlap between this fragment and segment D is too short to
allow efficient hybridization with a D-specific probe under the
conditions used.
DH2 Is Present in Activated But Not in Resting T
Lymphocytes
As a source of cells in which the IL-2R gene is
fully active, we have used mouse spleen cells cultured for 3 days in
the presence of ConA and IL-2. This population consists of more than
85% T lymphocytes (data not shown) and expresses high levels of
IL-2R
(Fig. 7 A).
Figure 7:
DNase-I hypersensitive sites in the
IL-2R gene of activated and resting T lymphocytes and bone marrow
cells. Activated T cells were prepared by culturing spleen cells with
ConA and IL-2 (100 units/ml) for 3 days. The resulting population
contained >85% T cells, 98% of which were
IL-2R
, as indicated by FACS analysis of cells
stained with PC61 mAb and FITC-conjugated goat anti-rat Ig.
(A, empty histogram. The shadedprofile corresponds to cells stained with the anti-Ig
reagent alone.) Resting T cells were magnetically sorted Thy
1
fresh spleen cells. They consisted of >90%
CD4
or CD8
small cells. Bone marrow
cells were obtained by flushing femur and hip bones with PBS. As
positive control DNA samples from activated T cells (c)
were run on the same gels as bone marrow DNA. Cells were permeabilized
with lysolecithin and treated with the indicated amount (units/ml) of
DNase-I. The extracted DNA was digested with PstI
(B, C, E, G, H) or SphI
(D, F, I). Southern blots were hybridized with probe D
(see Fig. 6 B). Full-length restriction fragments are indicated
by an arrow. The position of fragments generated by DNase-I
cleavage is indicated by filledarrowheads. PanelC shows the same gel as in B, which has been
electrophoresed for a longer time to allow clear separation of the
fragment resulting from DH3 from the full-length restriction fragment.
The results shown here were corroborated by analysis of similarly
activated lymph node cells.
Fig. 7
,
B-D, shows Southern blots of PstI- or
SphI-restricted DNA isolated from such cells after
permeabilization and DNase-I treatment. It documents the presence of
the three DHS described in Fig. 6in this cell population. These
results were confirmed with cells from lymph nodes that were stimulated
with ConA, and IL-2 and expressed similar levels of surface IL-2R
(data not shown).
gene with that in resting T lymphocytes
in which the gene is silent, we purified T cells from the spleen, based
on their expression of the T cell marker Thy1. This population
consisted predominantly of small, resting T cells, as assessed by FACS
analysis (
90% CD4
or CD8
cells,
data not shown). As a further check, we examined Southern blots of DNA
extracted from these cells for T cell-specific gene rearrangements by
hybridization with a probe for the TcR
gene
(51) . In
HindIII-digested DNA, this probe hybridizes to a 3.0-kb
fragment that is not rearranged and serves as internal control, since
it is present in DNA from all cells. The same probe detects a 9.5-kb
fragment that undergoes clonal rearrangements during T cell maturation
and is therefore no longer detectable in polyclonal mature T cell
populations. The absence of the 9.5 kb band in blots of T cell DNA,
even after prolonged exposure, ruled out any significant contamination
with non-T cell DNA (data not shown). Analysis of chromatin of the
resting T cells for DHS in the IL-2R
gene showed bands
corresponding to DH1 and DH3, while DH2 could not be detected
(Fig. 7, E and F).
DH1, DH2, and DH3 Are Lymphocyte-specific
To
determine whether the DHS detected in T cells were cell type-specific,
we analyzed the chromatin of the fibroblast cell line NIH/3T3 and a
primary culture of kidney epithelial cells (data not shown). In neither
cell population could we detect DH1, DH2, or DH3 at any DNase-I
concentration tested (up to 2,000 units/ml; data not shown). To narrow
down the cellular specificity of the DHS described here, we analyzed
bone marrow cells, which are a mixture of the different hematopoietic
cells and their precursors
(58) . Fig. 7, G-I shows that DH1, DH2, and DH3 are not detectable in this cell
population, indicating that they are restricted to the lymphocytic
lineage. The committed precursors of lymphocytes constitute a minor
fraction of total bone marrow cells. Therefore, we cannot exclude that
DH1 and DH3 appear already at this early stage of lymphocyte
differentiation.
Nuclease Sensitivity of the IL-2R
By comparing the effect of different DNase-I
concentrations on the full-length genomic IL-2R 5`-Flanking
Region
genomic fragments
with that on bulk chromatin (as detected by ethidium bromide staining
of DNA fractionated on agarose gels), we could rank the sensitivity of
IL-2R
chromatin in the different cell types as follows: activated
T cells > resting T cells > fibroblasts, kidney epithelial cells,
and bone marrow cells. Fragments due to DH1, DH2, or DH3 can be
detected only over a narrow range of DNase-I concentrations, and their
appearance is not correlated with the disappearance of the full-length
restriction fragments. Therefore, increased DNase-I sensitivity of the
latter in lymphocytes is not simply a consequence of the presence of
DHS, but it reflects a general nuclease sensitivity of promoter
proximal IL-2R
chromatin in cells that are poised to express the
IL-2R
gene. Further experiments are required to map the borders of
this open chromatin domain.
gene transcription by two different experimental approaches,
transgenic mice and DNase-I hypersensitivity. Analysis of five mouse
lines carrying a CAT transgene under the control of the 5`-flanking
region of the mouse IL-2R
gene showed that many, if not all, of
the cis-acting elements controlling tissue specificity and
inducibility of the IL-2R
gene in lymphocytes are contained in the
region between bp -2593 and +93. Analysis of the chromatin
containing the segment between kb -7.8 and +4.1 of the
resident IL-2R
gene for DHS revealed the presence of three
lymphocyte specific DHS. Two of these (DH1, at -0.1 kb and DH3 at
-5.8 kb) are constitutive, whereas DH2 (at -1.35 kb)
appears in T lymphocytes that have been induced to express IL-2R
.
CD8
thymocytes (data not shown),
about 30-50% of which are IL-2R
(15, 16) , but it was much too low to account for
the activity in total thymus. Furthermore, among the transgenic lines,
CAT activity in CD4
CD8
cells does
not correlate with that in total thymus. This indicates that expression
in lymphocytes and in the CAT-containing cells in the stroma is
regulated differently. In thymic sections, IL-2R
expression has
been observed in cells that may not belong to the lymphoid lineage
(15) . These cells could be thymic dendritic cells which, at
least in vitro, express IL-2R
(59, 60) .
Constitutive transgene expression was detected also in the bone marrow
of two and in the spleen of one transgenic line. Bone marrow does
contain a small fraction of IL-2R
cells (data not
shown), which have recently been identified as pre-B cells
(61, 62) . Neither staining with anti-CAT antibodies nor
in situ hybridization was sufficiently sensitive to test
whether transgene expression in these organs is restricted to
IL-2R
cells (data not shown).
. Both genes were induced
with very similar kinetics by ConA and IL-2 in spleen cells, as well as
in lymph node cells (data not shown). Since anti-CD3-stimulated T
cells, but not LPS-activated B cells, expressed the transgene and the
endogenous IL-2R
gene, transgene expression is not a nonspecific
consequence of lymphocyte activation. Maximal and prolonged expression
of both the transgene and the IL-2R
gene in ConA or
anti-CD3-activated T cells showed a marked dependence on stimulation
with IL-2. These results are in good agreement with a recent report
showing a role for endogenously produced IL-2 in IL-2R
expression
by anti-CD3-stimulated human T cells
(63) . The close
correlation between the induction of the transgene and the IL-2R
gene by IL-1 and IL-2 in CD4
CD8
thymic lymphocyte precursors shows that these responses are also
regulated by changes in the rate of IL-2R
gene transcription.
gene contains cis-acting
elements that control inducibility of IL-2R
expression in normal T
lymphocytes by signals through the TcR and by IL-2 and IL-1. This is in
complete agreement with our analysis of this region by transient
transfection experiments in established cell lines and with the finding
that the only inducible DHS in the 5`-flanking region of the normal,
resident IL-2R
gene is contained in the IL-2R
-CAT transgene.
Indeed, this site, DH2, which appears in T lymphocytes stimulated with
ConA and IL-2 and is found also in the constitutively
IL-2R
cell lines CTLL-2 and B6.1,
(
)
maps to the same position (-1.35 kb) as the
cis-acting elements required for the transcriptional response
of the mouse IL-2R
gene to IL-2
(69) . Note that the
absence of DH2 in exponentially growing fibroblasts and epithelial
cells as well as thymic lymphoma BW5147, a T cell line that does not
express IL-2R
,
indicates that the appearance of this
site is not just the consequence of a cell's entry into the cell
cycle.
-CAT transgene contains elements sufficient to
ensure lymphocyte specific expression. However, the observation that
expression in lymphocytes does not correlate with transgene copy number
suggests the existence of additional regulatory elements not present in
IL-2R
-CAT. The DNase-I hypersensitive site DH3 at -5.8 kb
may be such an element. Its position relative to the promoter and
constitutive presence in cells in which the IL-2R
gene is inactive
but inducible suggests that DH3 may be part of a locus control region
required to confer copy number-dependent integration site-independent
transgene expression (for review, see Ref. 64).
gene
(69) . In the human
and the mouse genes, several regulatory elements have been mapped in
this region, but all are upstream of DH1
(69) (for review, see
Ref. 65). DHS have been found in the promoter of inactive, rapidly
inducible, ubiquitously expressed genes (for review, see Ref. 66). In
tissue-specific genes, they have usually been detected only in cells
that actively transcribe the gene (for review, see Ref. 67). One
exception is the silk fibroin heavy chain gene of Bombyx mori that contains two DHS between bp -200 and +1, which are
detectable in posterior silk gland chromatin when the gene is active
but also during molting stages, when the gene is temporarily inactive
(68) . This is reminiscent of the behavior of the IL-2R
gene, which is transiently expressed in precursors of T cells during a
specific stage of their differentiation into mature lymphocytes
(15) .
gene containing the principal elements
required for lymphocyte-specific expression of this gene in response to
different stimuli. In future experiments, we will test the role of DH3
using a transgene, the expression of which can be assayed in single
cells and allows comparison of transgene and IL-2R
expression in
cell populations that are too small or too difficult to purify to
permit analysis by CAT assay.
Table:
Tissue distribution of IL-2R-CAT transgene
expression
0.5%. Three to seven mice of each line were analyzed.
Numbers represent means with ranges. No CAT activity was found in
100-500 µg of extracts of lymph nodes, liver, kidney, heart,
brain, and ovary from mice of any transgenic line.
transcripts
in T cell lines (59) as well as normal mature T cells and thymocytes
(S.-M. Wang and M. N., unpublished results).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.