©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mouse Interleukin-2 Receptor Gene Expression
DELIMITATION OF cis-ACTING REGULATORY ELEMENTS IN TRANSGENIC MICE AND BY MAPPING OF DNase-I HYPERSENSITIVE SITES (*)

Elisabetta Soldaini (1)(§), Maria Pla (1)(§)(¶), Friedrich Beermann (1), Enric Espel (1)(**), Patricia Corthésy (1), Sonia Barangé (1), Gary A. Waanders (2), H. Robson MacDonald (2), Markus Nabholz (1)(§§)

From the (1) Swiss Institute for Experimental Cancer Research (ISREC) CH-1066 Epalinges, Switzerland and the (2) Ludwig Institute for Cancer Research CH-1066 Epalinges, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 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.


INTRODUCTION

Interleukin-2 receptors (IL-2R)() 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.

In mature T cells, the primary signal inducing IL-2R 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 TcRCD3 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) .

Among the T cell precursors, IL-2R 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 (CD4CD8 thymocytes) (15, 16) . Upon activation of CD4CD8 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) .

Regulation of the IL-2R 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 CD4CD8 T cell line in which IL-1 and IL-2 have a similar synergistic effect on IL-2R expression as in normal CD4CD8 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.

To assess if the 5`-flanking region of the mouse IL-2R 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) .

In a complementary approach, we have screened a 11.9-kb region around the promoter of the IL-2R 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) .


MATERIALS AND METHODS

Generation of Transgenic Mice

The mouse IL-2R-CAT fusion gene was isolated from plasmid pmIL-2RPrCAT1 (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) . CD4CD8 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).


RESULTS

Generation of IL-2R-CAT Transgenic Mice

We have previously shown that the mouse IL-2R 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-CAT Transgene Expression

To determine in which tissues the IL-2R-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-CAT Transgene Is Inducible in Lymphocytes of Four Transgenic Lines

Since mature T cells express IL-2R 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 ().

When we compared the time course of transgene expression to that of the endogenous IL-2R 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-CAT Transgene Is Expressed in Anti-CD3-stimulated T Cells But Not in LPS-activated B Cells

Anti-CD3 antibodies induce T lymphocytes to express the IL-2R 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-CAT Transgene Is Up-regulated by IL-2 in Activated T Cells

Once T cells have been induced to display IL-2R 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-CAT Transgene Responds to IL-1 and IL-2 in Early Thymic T Cell Precursors

Early thymic T cell precursors express neither CD4 nor CD8 at their surface (CD4CD8 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 CD4CD8 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. CD4CD8 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 Gene

Fig. 6A shows a schematic representation of the mouse IL-2R 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) .

Fig. 6C shows the strategy for detecting DHS in the 11.9-kb segment of the IL-2R 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.

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).

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).

To compare the pattern of DHS in activated T cells that transcribe the IL-2R 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 5`-Flanking Region

By comparing the effect of different DNase-I concentrations on the full-length genomic IL-2R 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.


DISCUSSION

In this paper, we have examined the control of mouse IL-2R 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.

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 CD4CD8 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 CD4CD8 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).

In lymphocytes from four out of five lines, the transgene response to stimulation correlated very well with that of IL-2R. 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 CD4CD8 thymic lymphocyte precursors shows that these responses are also regulated by changes in the rate of IL-2R gene transcription.

Our data demonstrate that the segment between bp -2539 and +93 of the mouse IL-2R 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.

The IL-2R-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).

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 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) .

In summary, the experiments described here delimit the region of the mouse IL-2R 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

CAT activity in 100 µg of protein extracted from different organs. Dashes indicate percentages of chloramphenicol conversion 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.


  
Table: -


FOOTNOTES

*
This work was supported in part by grants from the Swiss Cancer League, Krebsforschung Schweiz, and the Swiss National Science Foundation (to M. N.) and the Generalitat de Catalunya (CIRIT) (to M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to the work described in this paper.

Present address: Dept. Biologia Cellular, Universitat de Girona, E-17071 Girona, Spain.

**
Present address: Dept. Bioquimica i Fisiologia, Universitat de Barcelona, E-08028 Barcelona, Spain.

§§
To whom correspondence should be addressed: ISREC, CH-1066 Epalinges, Switzerland. Tel.: 21 692 58 58; Fax: 21 652 69 33; E-mail Markus.Nabholz@isrec.unil.ch.

The abbreviations used are: IL-2R, interleukin-2 receptor; DHS, DNase-I hypersensitive site; IL, interleukin; TcR, T cell receptor; CAT, chloramphenicol acetyltransferase; kb, kilobase(s); bp, base pair(s); PMA, phorbol 12-myristate 13-acetate; LPS, lipopolysaccharide; mAb, monoclonal antibody; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PE, phycoerythrin; FACS, fluorescence-activated cell sorter.

J. Di Santo and A. Wilson, personal communication.

The numbering of bp positions has been modified with respect to previous publications (21, 58). +1 corresponds to the major start site of IL-2R transcripts in T cell lines (59) as well as normal mature T cells and thymocytes (S.-M. Wang and M. N., unpublished results).

E. Espel, unpublished results.


ACKNOWLEDGEMENTS

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.


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